HK1249179B - Devices and methods for sample analysis - Google Patents

Description

Apparatus and methods for sample analysis

Cross-references to related applications

This application claims the benefit of U.S. Provisional Patent Application No. 62/142,872, filed April 3, 2015; U.S. Provisional Patent Application No. 62/278,303, filed January 13, 2016; and U.S. Provisional Patent Application No. 62/279,488, filed January 15, 2016, all of which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to methods, apparatus and systems for analyzing analytes using nanoporous devices (e.g., operatively coupled to microfluidic devices).

Background Technology

Methods and apparatuses capable of accurately analyzing target analytes in samples are essential for diagnostics, prediction, environmental assessment, food safety, and the detection of chemical or biological warfare agents. Such methods and apparatuses not only need to be accurate, precise, and sensitive, but are also advantageous when it is necessary to analyze small samples rapidly using micro-instruments. Thus, there is interest in methods and apparatuses with improved sample analysis capabilities.

Summary of the Invention

Embodiments of the present invention relate to methods, systems, and apparatus for analyzing analytes in samples. In some embodiments, the sample may be a biological sample.

The method for analyzing an analyte in a sample may include: contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds the analyte; contacting the solid support with a second binding member, wherein the second binding member specifically binds the analyte and wherein the second binding member includes a severable tag attached thereto; removing the second binding member from the analyte not bound to the first binding member; slicing the tag attached to the second binding member, wherein the second binding member binds to the analyte bound to the first binding member; transferring the sliced tag through or across nanopores in a layer; determining the number of tags transferred through the layer; and determining the concentration of the analyte in the sample based on the number of tags transferred through the layer. In some embodiments, the concentration of the analyte can be determined by counting the number of tags transferred through the layer per unit time. In other embodiments, the concentration of the analyte can be determined by determining the time at which the number of tags transferred through the layer reaches a threshold or by setting a time period and counting the accumulated counts over the set time period.

In another embodiment, the method may include: combining a sample containing the target analyte with a known amount of the target analyte or a competing molecule, wherein the target analyte (combined with the sample) or the competing molecule is attached to a tag via a severable connector to generate a tagged analyte or a tagged competing molecule, respectively, and the tagged analyte or the tagged competing molecule competes with the target analyte for binding to a first binding member. The method may further include: contacting the combined sample with the first binding member, wherein the first binding member is immobilized on a solid support, and wherein the first binding member specifically binds the target analyte (and the tagged analyte or tagged competitor molecule); contacting the solid support with a buffer solution for an optional washing step; cleaving a tag attached to the tagged analyte or tagged competitor molecule, the tagged analyte or tagged competitor molecule binding to the first binding member immobilized on the solid support; transferring the cleaved tag through or across nanopores in the layer; determining the number of tags transferred through the layer; and determining the concentration of the analyte in the sample based on the number of tags transferred through the layer. In some embodiments, the concentration of the analyte can be determined by counting the number of tags transferred through the layer per unit time. In other embodiments, the concentration of the analyte can be determined by determining the time at which the number of tags transferred through the layer reaches a threshold or by setting a time period and counting the accumulated counts over the set time period. In this embodiment, the time it takes for the number of tags transferred through the nanopores or through the layer to reach a threshold can be negatively correlated with the concentration of the analyte in the sample. For example, the lower the count or the longer the time it takes to reach the threshold, the higher the concentration of the target analyte in the sample.

In one aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample. The method includes: contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds the analyte; contacting the analyte with a second binding member, wherein the second binding member specifically binds the analyte and wherein the second binding member includes a cleavable tag attached thereto; removing the second binding member from the analyte not bound to the first binding member; cutting the tag attached to the second binding member, wherein the second binding member binds to the analyte bound to the first binding member; transferring the cut tag through or across one or more nanopores in a layer; and evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tags transferred through the layer detects the analyte present in the sample. In some embodiments, the evaluation of the tags transferred through the layer is performed, wherein the number of tags transferred through the layer measures the amount of analyte present in the sample. In some implementations, the label that detects transfer across the layer is evaluated, wherein the label that detects transfer across the layer detects the analyte present in the sample.

In one aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample. The method includes: contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds the analyte; contacting the analyte with a second binding member, wherein the second binding member specifically binds the analyte and wherein the second binding member comprises an aptamer; removing aptamers not bound to the analyte bound to the solid substrate; dissociating the aptamers bound to the analyte and transferring the dissociated aptamers through or across one or more nanopores in a layer; and evaluating the aptamers transferred through the layer, wherein measuring the number of aptamers transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the aptamers transferred through the layer detects the analyte present in the sample. In some embodiments, the evaluation of measuring the aptamers transferred through the layer is performed, wherein the number of aptamers transferred through the layer measures the amount of analyte present in the sample. In some embodiments, the evaluation of detecting the aptamers transferred through the layer is performed, wherein detecting the aptamers transferred through the layer detects the analyte present in the sample.

In one aspect, the present invention relates to an integrated digital microfluidic nanopore device comprising: a bottom substrate including an electrode array; a top substrate spaced apart from the bottom substrate; and a nanopore layer disposed between the bottom substrate and the top substrate. The device includes a proximal portion and a distal portion, and the nanopore layer is disposed in the distal portion. The electrode array in the proximal portion is configured to generate microdroplets. The electrode array is configured to position the microdroplets across the nanopore layer such that the microdroplets are segmented by the nanopore layer into a first portion and a second portion, wherein at least two electrodes of the electrode array are positioned across the nanopore layer, wherein the two electrodes form an anode and a cathode, and operate to drive a current through nanopores in the nanopore layer when the liquid microdroplets are positioned across the nanopore layer.

In one aspect, the present invention relates to an integrated digital microfluidic nanopore device comprising: a bottom substrate including an electrode array; a top substrate spaced apart from the bottom substrate and including electrodes; and a nanopore layer disposed between the bottom substrate and the top substrate. The device includes a proximal portion and a distal portion, and the nanopore layer is disposed in the distal portion. The electrode array and the electrodes in the proximal portion are configured to generate microdroplets. The electrode array and the electrodes are configured to position the microdroplets across the nanopore layer such that the nanopore layer divides the microdroplets into a first portion and a second portion, wherein at least one electrode of the electrode array contacts the first portion of the microdroplet positioned across the nanopore layer, and the electrode in the top substrate is positioned to contact the second portion of the microdroplet positioned across the nanopore layer, wherein the two electrodes form an anode and a cathode and operate to drive current through nanopores in the nanopore layer when the liquid microdroplets are positioned across the nanopore layer.

In one aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample. The method includes: contacting the sample with a binding member, wherein the binding member is immobilized on a solid support and wherein the binding member specifically binds the analyte; contacting the sample with a labeled analyte, the sample possibly containing the analyte bound to the binding member, wherein the labeled analyte is labeled with a severable tag; removing the labeled analyte not bound to the binding member; cutting a tag attached to the labeled analyte bound to the binding member; transferring the cut tag through or across one or more nanopores in a layer; and evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or detecting the tags transferred through the layer detects the analyte present in the sample. In some embodiments, the evaluation of the tags transferred through the layer measures the amount of analyte present in the sample. In some implementations, the label that detects transfer across the layer is evaluated, wherein the label that detects transfer across the layer detects the analyte present in the sample.

In one aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample. The method includes: contacting the sample with a binding member, wherein the binding member is immobilized on a solid support and wherein the binding member specifically binds the analyte; contacting the sample with a labeled analyte, the sample possibly containing the analyte bound to the binding member, wherein the labeled analyte comprises an aptamer; removing the labeled analyte not bound to the binding member; dissociating the aptamer bound to the labeled analyte bound to the binding member and transferring the dissociated aptamer through or across one or more nanopores in a layer; and evaluating the aptamers transferred through the layer, wherein measuring the number of aptamers transferred through the layer measures the amount of analyte present in the sample, or detecting the aptamers transferred through the layer detects the analyte present in the sample. In some embodiments, the evaluation of the aptamers transferred through the layer is performed, wherein the number of aptamers transferred through the layer measures the amount of analyte present in the sample. In some implementations, the aptamer that detects transfer across the layer is evaluated, wherein the tag that detects transfer across the layer detects the analyte present in the sample.

In one aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample. The method includes: contacting the sample with a binding member, wherein the binding member specifically binds the analyte, and the binding member is labeled with a severable tag; contacting the sample with an immobilized analyte, the sample possibly containing the analyte bound to the binding member, wherein the immobilized analyte is immobilized on a solid support; removing binding members not bound to the immobilized analyte; cutting the tag, the tag being attached to a binding member bound to the immobilized analyte; transferring the cut tag through or across one or more nanopores in a layer; and evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or detecting the tags transferred through the layer detects the analyte present in the sample. In some embodiments, evaluating the tags transferred through the layer measures the amount of analyte present in the sample. In some embodiments, evaluating the tags transferred through the layer detects the analyte present in the sample.

In one aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample. The method includes: contacting the sample with a binding member, wherein the binding member specifically binds the analyte, and the binding member comprises an aptamer; contacting the sample with an immobilized analyte, the sample possibly containing the analyte bound to the binding member, wherein the immobilized analyte is immobilized on a solid support; removing binding members not bound to the immobilized analyte; dissociating the aptamer bound to the binding member bound to the immobilized analyte and transferring the dissociated aptamer through or across one or more nanopores in a layer; and evaluating the aptamers transferred through the layer, wherein measuring the number of aptamers transferred through the layer measures the amount of analyte present in the sample, or detecting the aptamers transferred through the layer detects the analyte present in the sample. In some embodiments, the evaluation of the aptamers transferred through the layer is performed, wherein the number of aptamers transferred through the layer measures the amount of analyte present in the sample. In some implementations, the aptamer that detects transfer across the layer is evaluated, wherein the tag that detects transfer across the layer detects the analyte present in the sample.

In some aspects, the tag may be an anionic polymer, a cationic polymer, or nanoparticles. In some cases, the tag may include anionic polymers, such as oligonucleotide polymers. In some cases, the oligonucleotide polymer may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some cases, the oligonucleotide polymer may be a DNA aptamer or an RNA aptamer, wherein the aptamer does not bind the analyte. In an exemplary case, the tag may include nanoparticles, which may be positively charged nanoparticles or negatively charged nanoparticles.

In some embodiments, the tag may be a spherical tag, such as a dendritic polymer, beads, or nanoparticles, such as nanobeads. In some embodiments, the tag may be a non-linear, substantially linear, or elongated shape, such as a polymer of ribose or deoxyribose units, oligonucleotides, and nucleic acids, such as DNA or RNA.

In some cases, the first binding member and the second binding member can be an aptamer, an antibody, or a receptor. For example, the first binding member can be a receptor and the second binding member can be an antibody, or the first binding member can be an antibody and the second binding member can be a receptor. In some cases, the first binding member can be a first antibody and the second binding member can be a second antibody.

In some cases, the tag may be negatively charged, and the transfer may include applying a positive potential across the layer, thereby causing the tag to transfer across the layer.

In some cases, the tag may be positively charged, and the transfer may include applying a negative potential across the layer, thereby causing the tag to transfer across the layer.

In other embodiments, the tag may be a nucleic acid, and the tag may hybridize with an oligonucleotide prior to the transfer, the oligonucleotide including a sequence complementary to the sequence of the tag.

In another embodiment, a method is provided for measuring an analyte present in a biological sample using an aptamer as a second binding member. For example, the method may include: contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds the analyte; contacting the analyte with a second binding member, wherein the second binding member specifically binds the analyte and wherein the second binding member comprises an aptamer; removing aptamers not bound to the analyte bound to the solid substrate; dissociating the aptamer from the analyte bound to the solid substrate and transferring the dissociated aptamer through nanopores in a layer; determining the number of aptamers transferred through the layer; and measuring the analyte in the sample based on the number of aptamers transferred through the layer. In this embodiment, the second binding member is not attached to a label because the second binding member is directly detected by the nanopores.

The aptamer may be a DNA aptamer or an RNA aptamer. The first binding member may be an antibody. In some cases, the analyte may be a ligand, and the first binding member may be a receptor.

This document also discloses methods for simultaneously analyzing multiple different analytes in a sample. For example, the method may include analyzing: a first and a second analyte; a first, a second, and a third analyte; and so on. In some cases, the method for analyzing multiple different analytes in a sample may include contacting the sample with multiple different first binding members, wherein the first binding members of the different first binding members specifically bind a first analyte of the multiple different analytes, the second binding members of the different first binding members specifically bind a second analyte of the multiple different analytes, and so on. The method may also include contacting the different analytes with multiple second binding members, wherein the first binding members of the multiple second binding members bind the first analyte, the second binding members of the multiple second binding members bind the second analyte, and so on. In some cases, each of the multiple different second binding members may include a label that is different from or distinguishable from each other (e.g., each of the multiple different second binding members has a different label). For example, the first binding member of the multiple second binding members may include a first label, the second binding member of the multiple second binding members may include a second label, and so on, wherein the first label and the second label are distinguishable from each other. The labels can be distinguished using any suitable method, for example, based on the properties or unique characteristics of the labels.

The method may further include: removing unbound second binding members; cutting a tag attached to multiple second binding members bound to the analyte; transferring the tag through nanopores in the layer; determining the number of each type of tag transferred through the layer; and measuring multiple different analytes in the sample based on the number of each type of tag transferred through the layer. In some embodiments, the concentration of the analyte can be determined by counting the number of tags transferred through the layer per unit time. In other embodiments, the concentration of the analyte can be determined by determining the time when the number of tags transferred through the layer reaches a threshold. As noted herein, in some cases, the second binding member may be multiple aptamers, and these aptamers are not attached to the tag when the aptamers are counted. In these embodiments, the aptamer may dissociate from the analyte before transfer through or across the nanopores.

In some cases, the different labels (such as the different aptamers) can be distinguished from each other by nanopore force spectroscopy, optical devices, or electrical devices, or a combination thereof.

This document also provides kits, systems, and devices for implementing the disclosed methods. These kits, systems, and devices can be used to perform analyte analysis in an automated or semi-automated manner and may optionally include disposable/consumable components for analyte analysis. Automated and semi-automated devices may utilize microfluidics. Exemplary microfluidics include digital microfluidics (DMF), surface acoustic wave (SAW) microfluidics, droplet-based microfluidic devices, etc. Exemplary microfluidics also include fully integrated DMF and nanopore devices, or fully integrated SAW and nanopore devices. In some cases, the device for implementing the disclosed methods may be a digital microfluidic device used in conjunction with a nanopore device. In other embodiments, the device for implementing the disclosed methods may be an integrated digital microfluidic nanopore device. These devices may be single-use devices or may be reusable (used multiple times for analyte analysis). The digital microfluidic devices and nanopore devices described herein can provide miniaturized, low-cost analyte analysis and can be manufactured using low-cost technologies.

This document also discloses an integrated digital microfluidic nanopore device comprising a microfluidic module and a nanopore module; the microfluidic module comprising an electrode array spaced from a single electrode, the single electrode being sized to overlap at least a portion of the electrode array, wherein the electrode array and the single electrode transport at least one microdroplet of fluid to a transfer electrode in the electrode array, wherein the transfer electrode is positioned at an interface operatively coupled to the microfluidic module and the nanopore module; the nanopore module comprising: a first microchannel positioned on a first surface of a first substrate; and a second microchannel positioned on a first surface of a second substrate; wherein the first surface of the first substrate contacts the first surface of the second substrate, thereby closing the first microchannel and the second microchannel to provide a first capillary channel and a second capillary channel, respectively, wherein at least the first capillary channel extends to the interface between the microfluidic module and the nanopore module and adjacent to the transfer electrode, and is positioned to receive fluid microdroplets positioned on the transfer electrode; wherein the first capillary channel intersects the second capillary channel, wherein the nanopore layer is positioned between the first substrate and the second substrate at the location where the first capillary channel and the second capillary channel intersect.

In some embodiments, the electrode array may include a first transfer electrode and a second transfer electrode, each of the transfer electrodes being configured to position a fluid droplet on the surface of the transfer electrode, wherein a first capillary channel extends to an interface between the microfluidic module and the nanopore module, is adjacent to the first transfer electrode and positioned to receive a fluid droplet located on the first transfer electrode, and wherein a second capillary extends to an interface between the microfluidic module and the nanopore module, is adjacent to the second transfer electrode and positioned to receive a fluid droplet located on the second transfer electrode.

In some embodiments, the second capillary channel may not extend to the interface and may not be connected to the electrodes of the microfluidic module, but may be connected to an outlet or reservoir at one or both ends of the second capillary. In some cases, the second capillary is connected to a first reservoir at one end and a second reservoir at the other end.

In some embodiments, the first reservoir and/or the second reservoir contains fluid positioned opposite the first capillary channel at the intersection, the fluid facilitating the operation of the nanopore layer to drive current through the nanopores of the nanopore layer. In some embodiments, the first capillary channel and/or the second capillary channel vary in cross-sectional width along the length of the capillary, such that the width is reduced at the intersection compared to the width on either side of the intersection.

In some embodiments, the first capillary includes a first pair of electrodes, and the second capillary includes a second pair of electrodes, wherein the first pair of electrodes is positioned in the first capillary channel and laterally connected to nanopores in the nanopore layer, and wherein the second pair of electrodes is positioned in the second capillary channel and laterally connected to nanopores in the nanopore layer. The microdroplet may be a microdroplet containing molecules to be detected and/or counted by transporting them through the nanopores in the nanopore layer.

In some embodiments, the fluid droplets have different compositions and are a first droplet and a second droplet, the first droplet containing molecules to be detected and/or counted by transport across the nanopore layer through the nanopores, and the second droplet containing a conductive fluid lacking the molecules, wherein the conductive fluid facilitates the transport of the molecules across the nanopore layer via the nanopores.

In some embodiments, the first capillary channel includes a first electrode positioned proximal to the nanopore layer, and the second capillary channel includes a second electrode positioned proximal to the nanopore layer, wherein each of the first and second electrodes is exposed in the capillary channel such that it comes into contact with a fluid present in the capillary channel, and wherein when the liquid is positioned to traverse the nanopore layer in the first and second capillary channels, the first and second electrodes operate to drive a current through the nanopores in the nanopore layer.

In some embodiments, the transfer electrode and the first capillary channel are substantially in the same plane, and the fluid droplet is aligned with the opening of the first capillary channel.

In some embodiments, the transfer electrode is located in a plane higher than the first capillary channel, and the device is configured to have a vertical port for transferring the fluid droplets downward to the opening of the first capillary channel.

In one particular embodiment, the first surface of the first substrate includes a first region on which the electrode array is disposed and a second region in which the first microchannel is formed, wherein the electrode array is on a plane that is higher than the plane on which the first microchannel is formed.

In some embodiments, the second substrate includes a groove at a side edge of the interface, wherein the groove is aligned above the first capillary channel and provides a vertical port for transporting a microdroplet located at the transfer electrode to an opening of the first capillary channel.

In some cases, the single electrode extends above the single transfer electrode and is in a biplane configuration with the single transfer electrode, wherein the single electrode and the single transfer electrode operate to move the single fluid droplet toward the single transfer electrode.

In other cases, the single electrode extends over the plurality of transfer electrodes and is in a biplane configuration with the plurality of transfer electrodes, wherein the single electrode and the plurality of transfer electrodes operate to move the plurality of fluid droplets toward the plurality of transfer electrodes.

In some embodiments, the single electrode does not extend over the single transfer electrode and is not in a biplane configuration with the single transfer electrode, wherein the single fluid droplet is moved toward the single transfer electrode by using a coplanar electrode.

In some embodiments, the single electrode does not extend over the plurality of transfer electrodes and is not in a biplane configuration with the plurality of transfer electrodes, wherein the plurality of fluid droplets are moved toward the plurality of transfer electrodes by using coplanar electrodes.

Therefore, using the devices, kits, systems, and methods described herein, analytes present in biological samples can be measured, and patients can be diagnosed.

In another aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample, the method comprising: (a) contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds the analyte; (b) contacting the analyte with a second binding member, wherein the second binding member specifically binds the analyte and wherein the second binding member includes a severable tag attached thereto; (c) removing the second binding member from the analyte not bound to the first binding member; (d) slicing the tag attached to the second binding member, wherein the second binding member binds to the analyte bound to the first binding member; (e) transferring the tag through one or more nanopores in a layer; and (f) evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tags transferred through the layer detects the analyte present in the sample.

In another aspect, the present invention relates to a method for measuring or detecting a target analyte present in a biological sample, the method comprising: (a) contacting the sample with a solid support, a first specific binding member, and a second specific binding member, wherein the solid support comprises an immobilization reagent, the first specific binding member comprises a ligand of the immobilization reagent and specifically binds the target analyte, and the second specific binding member comprises a cleavable tag and specifically binds the target analyte, wherein a solid support/first specific binding member/target analyte/second specific binding member complex is formed. (b) removing the second specific binding member not bound to the solid support/first specific binding member/analyte/second specific binding member complex, (c) cutting the tag attached to the labeled analyte bound to the second specific binding member in the solid support/first specific binding member/target analyte/second specific binding member complex, (d) transferring the tag through one or more nanopores in the layer, and (e) evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tags transferred through the layer detects the amount of analyte present in the sample.

In another aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample, the method comprising: (a) contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds the analyte; (b) contacting the analyte with a second binding member, wherein the second binding member specifically binds the analyte and wherein the second binding member comprises an aptamer; (c) removing aptamers not bound to the analyte bound to the solid substrate; (d) dissociating the aptamers bound to the analyte; (e) transferring the dissociated aptamers through one or more nanopores in a layer; and (f) evaluating the aptamers transferred through the layer, wherein measuring the number of aptamers transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the aptamers transferred through the layer detects the analyte present in the sample.

In one aspect, the present invention relates to an integrated digital microfluidic nanopore device comprising: a first substrate including an electrode array; a second substrate spaced apart from the first substrate; and a nanopore layer disposed between the first and second substrates, wherein the electrode array is configured to position a droplet across the nanopore layer such that the droplet is divided into a first portion and a second portion by the nanopore layer, wherein at least two electrodes of the electrode array are positioned across the nanopore layer, wherein the two electrodes form an anode and a cathode and operate to drive a current through the nanopores in the nanopore layer when the liquid droplet is positioned across the nanopore layer.

In another aspect, the present invention relates to an integrated digital microfluidic nanopore device comprising: a first substrate having an electrode array; a second substrate spaced apart from the first substrate; and a nanopore layer disposed between the first and second substrates, wherein the electrode array is configured to position a droplet across the nanopore layer such that the nanopore layer divides the droplet into a first portion and a second portion, wherein at least one electrode of the electrode array contacts the first portion of the droplet positioned across the nanopore layer, and an electrode in the second substrate is positioned to contact the second portion of the droplet positioned across the nanopore layer, wherein the two electrodes form an anode and a cathode and operate to drive a current through the nanopores in the nanopore layer when the liquid droplet is positioned across the nanopore layer.

In another aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample, the method comprising: (a) contacting the sample with a binding member, wherein the binding member is immobilized on a solid support and wherein the binding member specifically binds the analyte; (b) contacting the sample with a labeled analyte, wherein the labeled analyte is labeled with a severable tag; (c) removing the labeled analyte not bound to the binding member; (d) cutting a tag attached to the labeled analyte bound to the binding member; (e) transferring the tag through one or more nanopores in a layer; and (f) evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tags transferred through the layer detects the analyte present in the sample.

In another aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample, the method comprising: (a) contacting the sample with a binding member, wherein the binding member is immobilized on a solid support and wherein the binding member specifically binds the analyte; (b) contacting the sample with a labeled analyte, wherein the labeled analyte comprises an aptamer; (c) removing the labeled analyte not bound to the binding member; (d) dissociating the aptamer bound to the labeled analyte and transferring the dissociated aptamer through one or more nanopores in a layer; and (e) evaluating the aptamer transferred through the layer, wherein measuring the number of aptamers transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the aptamers transferred through the layer detects the analyte present in the sample.

In another aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample, the method comprising: (a) contacting the sample with a binding member, wherein the binding member specifically binds the analyte and the binding member is labeled with a severable tag; (b) contacting the sample with an immobilized analyte, wherein the immobilized analyte is immobilized on a solid support; (c) removing binding members not bound to the immobilized analyte; (d) slicing a tag attached to a binding member bound to the immobilized analyte; (e) transferring the tag through one or more nanopores in a layer; and (f) evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tags transferred through the layer detects the analyte present in the sample.

In another aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample, the method comprising: (a) contacting the sample with a binding member, wherein the binding member specifically binds the analyte and the binding member comprises an aptamer; (b) contacting the sample with an immobilized analyte, wherein the immobilized analyte is immobilized on a solid support; (c) removing binding members not bound to the immobilized analyte; (d) dissociating the aptamer bound to the binding member bound to the immobilized analyte and transferring the dissociated aptamer through one or more nanopores in a layer; and (e) evaluating the aptamers transferred through the layer, wherein measuring the number of aptamers transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the aptamers transferred through the layer detects the analyte present in the sample.

In another aspect, the present invention relates to an integrated digital microfluidic nanopore device comprising a microfluidic module and a nanopore module; the microfluidic module comprising an electrode array wherein the electrode array transports at least one microdroplet of fluid to a first transfer position in the electrode array, wherein the first transfer position is at an interface between the microfluidic module and the nanopore module; the nanopore module comprising: a first capillary channel; and a second capillary channel; wherein at least the first capillary channel extends to the interface and is adjacent to the first transfer position, and is positioned to receive a fluid microdroplet positioned at the first transfer position; wherein the first capillary channel intersects with the second capillary channel, wherein a nanopore layer is positioned at the location between the first capillary channel and the second capillary channel where the first capillary channel and the second capillary channel intersect.

In another aspect, the present invention relates to a method for measuring an analyte present in a biological sample, the method comprising: (a) contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds the analyte; (b) contacting the analyte with a second binding member, wherein the second binding member specifically binds the analyte and wherein the second binding member includes a cleavable tag attached thereto; (c) removing the second binding member from the analyte not bound to the first binding member; (d) cleaving the tag attached to the second binding member, wherein the second binding member binds to the analyte bound to the first binding member; (e) transferring the tag through one or more nanopores in a layer; and (f) evaluating the tags transferred through the layer, wherein each tag transferred through the layer is a transfer event, wherein the measurement... The number of transfer events measures the amount of analyte present in the sample, wherein the amount of analyte present in the sample is determined as follows: i) counting the number of transfer events within a set time period and correlating the number of transfer events with a control; ii) measuring the time required for the set number of transfer events to occur and correlating it with a control; or iii) measuring the average time between the occurrence of transfer events and correlating it with a control, wherein the control is a reference standard comprising a calibration curve, a standard addition, or a digital polymerase chain reaction, wherein the standard curve in sub-item i) is determined by measuring the number of transfer events for a control concentration of the analyte within the set time period; wherein the standard curve in sub-item ii) is determined by measuring the time required for the set number of transfer events to occur for a control concentration of the analyte; and wherein the standard curve in sub-item iii) is determined by measuring the average time between the occurrence of transfer events for a control concentration of the analyte.

In another aspect, the present invention relates to a method for measuring an analyte present in a biological sample, the method comprising: (a) contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds the analyte; (b) contacting the analyte with a second binding member, wherein the second binding member specifically binds the analyte and wherein the second binding member comprises an aptamer; (c) removing aptamers not bound to the analyte bound to the solid substrate; (d) dissociating the aptamers bound to the analyte; and (e) transferring the dissociated aptamers through one or more nanopores in a layer; and (f) evaluating the aptamers transferred through the layer, wherein each aptamer transferred through the layer is a transfer event, and measuring the number of transfer events measures the presence of analytes in the sample. The amount of analyte, wherein the amount of analyte present in the sample is determined as follows: i) counting the number of transfer events within a set time period and correlating the number of transfer events with a control; ii) measuring the time required for the set number of transfer events to occur and correlating it with a control; or iii) measuring the average time between the occurrence of transfer events and correlating it with a control, wherein the control is a reference standard comprising a calibration curve, a standard addition, or a digital polymerase chain reaction, wherein the standard curve in sub-item i) is determined by measuring the number of transfer events for a control concentration of the analyte within a set time period; wherein the standard curve in sub-item ii) is determined by measuring the time required for the set number of transfer events to occur for a control concentration of the analyte; and wherein the standard curve in sub-item iii) is determined by measuring the average time between the occurrence of transfer events for a control concentration of the analyte.

In another aspect, the present invention relates to a method for measuring an analyte present in a biological sample, the method comprising: (a) contacting the sample with a binding member, wherein the binding member is immobilized on a solid support and wherein the binding member specifically binds the analyte; (b) contacting the sample with a labeled analyte, wherein the labeled analyte is labeled with a severable tag; (c) removing the labeled analyte not bound to the binding member; (d) slicing the tag, the tag being attached to the labeled analyte bound to the binding member; (e) transferring the tag through one or more nanopores in a layer; and (f) evaluating the tags transferred through the layer, wherein each tag transferred through the layer is a transfer event, and wherein measuring the number of transfer events measures the amount of analyte present in the sample. The amount of analyte present in the sample is determined as follows: i) counting the number of transfer events within a set time period and correlating the number of transfer events with a control; ii) measuring the time required for the set number of transfer events to occur and correlating it with a control; or iii) measuring the average time between the occurrence of transfer events and correlating it with a control, wherein the control is a reference standard comprising a calibration curve, a standard addition, or a digital polymerase chain reaction, wherein the standard curve in sub-item i) is determined by measuring the number of transfer events for a control concentration of the analyte within the set time period; wherein the standard curve in sub-item ii) is determined by measuring the time required for the set number of transfer events to occur for a control concentration of the analyte; and wherein the standard curve in sub-item iii) is determined by measuring the average time between the occurrence of transfer events for a control concentration of the analyte.

In another aspect, the present invention relates to a method for measuring an analyte present in a biological sample, the method comprising: (a) contacting the sample with a binding member, wherein the binding member is immobilized on a solid support and wherein the binding member specifically binds the analyte; (b) contacting the sample with a labeled analyte, wherein the labeled analyte comprises an aptamer; (c) removing the labeled analyte not bound to the binding member; (d) dissociating the aptamer bound to the labeled analyte and transferring the dissociated aptamer through one or more nanopores in a layer; and (e) evaluating the aptamer transferred through the layer, wherein each aptamer transferred through the layer is a transfer event, wherein measuring the number of transfer events measures the amount of analyte present in the sample, wherein the presence of analyte is determined as follows: The amount of analyte in the sample: i) counts the number of transfer events within a set time period and correlates the number of transfer events with a control; ii) measures the time required for the set number of transfer events to occur and correlates it with a control; or iii) measures the average time between the occurrence of transfer events and correlates it with a control, wherein the control is a reference standard comprising a calibration curve, a standard addition, or a digital polymerase chain reaction, wherein the standard curve in sub-item i) is determined by measuring the number of transfer events for a control concentration of the analyte within a set time period; wherein the standard curve in sub-item ii) is determined by measuring the time required for the set number of transfer events to occur for a control concentration of the analyte; and wherein the standard curve in sub-item iii) is determined by measuring the average time between the occurrence of transfer events for a control concentration of the analyte.

In another aspect, the present invention relates to a method for measuring an analyte present in a biological sample, the method comprising: (a) contacting the sample with a binding member, wherein the binding member specifically binds the analyte and the binding member is labeled with a severable tag; (b) contacting the sample with an immobilized analyte, wherein the immobilized analyte is immobilized on a solid support; (c) removing binding members not bound to the immobilized analyte; (d) slicing a tag attached to a binding member bound to the immobilized analyte; (e) transferring the tag through one or more nanopores in a layer; and (f) evaluating the tags transferred through the layer, wherein each tag transferred through the layer is a transfer event, and measuring the number of transfer events measures the amount of analyte present in the sample. The amount of analyte present in the sample is determined as follows: i) counting the number of transfer events within a set time period and correlating the number of transfer events with a control; ii) measuring the time required for the set number of transfer events to occur and correlating it with a control; or iii) measuring the average time between the occurrence of transfer events and correlating it with a control, wherein the control is a reference standard comprising a calibration curve, a standard addition, or a digital polymerase chain reaction, wherein the standard curve in sub-item i) is determined by measuring the number of transfer events for a control concentration of the analyte within the set time period; wherein the standard curve in sub-item ii) is determined by measuring the time required for the set number of transfer events to occur for a control concentration of the analyte; and wherein the standard curve in sub-item iii) is determined by measuring the average time between the occurrence of transfer events for a control concentration of the analyte.

In another aspect, the present invention relates to a method for measuring an analyte present in a biological sample, the method comprising: (a) contacting the sample with a binding member, wherein the binding member specifically binds the analyte and the binding member comprises an aptamer; (b) contacting the sample with an immobilized analyte, wherein the immobilized analyte is immobilized on a solid support; (c) removing binding members not bound to the immobilized analyte; (d) dissociating the aptamer, the aptamer binding to the binding member bound to the immobilized analyte, and transferring the dissociated aptamer through one or more nanopores in a layer; and (e) evaluating the aptamer transferred through the layer, wherein each aptamer transferred through the layer is a transfer event, wherein measuring the number of transfer events measures the amount of analyte present in the sample. The amount of analyte present in the sample is determined as follows: i) counting the number of transfer events within a set time period and correlating the number of transfer events with a control; ii) measuring the time required for the set number of transfer events to occur and correlating it with a control; or iii) measuring the average time between the occurrence of transfer events and correlating it with a control, wherein the control is a reference standard comprising a calibration curve, a standard addition, or a digital polymerase chain reaction, wherein the standard curve in sub-item i) is determined by measuring the number of transfer events for a control concentration of the analyte within the set time period; wherein the standard curve in sub-item ii) is determined by measuring the time required for the set number of transfer events to occur for a control concentration of the analyte; and wherein the standard curve in sub-item iii) is determined by measuring the average time between the occurrence of transfer events for a control concentration of the analyte.

In another aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample, the method comprising: (a) contacting the sample with a binding member, wherein the binding member is immobilized on a solid support, the binding member comprising a cleavable tag attached thereto, and the binding member specifically binding the analyte; (b) removing binding members not bound to the analyte; (c) cleaving the tag, the tag being attached to a binding member bound to the analyte; (d) transferring the tag through one or more nanopores in a layer; and (e) evaluating the tags transferred through the layer, wherein each tag transferred through the layer is a transfer event, wherein measuring the number of transfer events measures the amount of analyte present in the sample, wherein the amount of analyte present in the sample is determined as follows: i) counting the number of transfer events over a set time period and correlating the number of transfer events with a control; ii) measuring the time amount at which a set number of transfer events occur and correlating it with a control; or iii) measuring the average time between the occurrence of transfer events and correlating it with a control, wherein the control is a reference comprising a calibration curve, a standard addition, or a digital polymerase chain reaction.

In another aspect, the present invention relates to an integrated digital microfluidic nanopore realization device, comprising: a microfluidic module and a nanopore realization module; the microfluidic module comprising an electrode array spaced from a single electrode, the single electrode being sized to overlap at least a portion of the electrode array, wherein the electrode array and the single electrode transport at least one microdroplet of fluid to a transfer electrode in the electrode array, wherein the transfer electrode is positioned at an interface between the microfluidic module and the nanopore realization module; the nanopore realization module comprising: a first microchannel positioned on a first surface of a first substrate; a second microchannel positioned on a first surface of a second substrate; wherein the first surface of the first substrate contacts the first surface of the second substrate, thereby closing the first microchannel and the second microchannel to respectively provide a first capillary channel and a second microchannel. A second capillary channel, wherein at least the first capillary channel extends to the interface between the microfluidic module and the nanopore realization module and is adjacent to the transfer electrode, and is positioned to receive fluid droplets positioned on the transfer electrode; wherein the first capillary channel intersects with the second capillary channel, wherein a layer is positioned between the first substrate and the second substrate at the intersection of the first capillary channel and the second capillary channel, wherein the layer lacks nanopores and separates the ionic liquid present in the first capillary channel and the second capillary channel, wherein the first capillary channel and the second capillary channel are electrically connected to electrodes for driving a voltage from the first capillary channel to the second capillary channel (or vice versa) to establish nanopores in the layer at the intersection of the first capillary channel and the second capillary channel.

In another aspect, the present invention relates to a method for generating nanopores in an integrated digital microfluidic nanopore realization device, the method comprising: providing an integrated digital microfluidic nanopore realization device as described above; applying a voltage in a first capillary channel and a second capillary channel to drive a current through the layer; measuring the conductance across the layer; and terminating the application of the voltage after detecting conductance indicating the generation of nanopores in the layer.

In another aspect, the present invention relates to an integrated digital microfluidic nanopore device comprising: a first substrate having an electrode array; a second substrate spaced apart from the first substrate; an opening in the first or second substrate, the opening being in fluid communication with a nanopore layer comprising nanopores; and a pair of electrodes configured to apply an electric field through the nanopores, wherein the electrode array is configured to transport at least one microdroplet of fluid to the opening.

In another aspect, the present invention relates to a pair of integrated digital microfluidic nanopore devices comprising: a first integrated digital microfluidic nanopore device as described above, wherein the single electrode is a first single electrode and the capillary channel is a first capillary channel; and a second integrated digital microfluidic nanopore device comprising: a third substrate having a fifth side and a sixth side opposite to the fifth side, wherein the fifth side comprises an electrode array; a fourth substrate spaced from the third substrate, wherein the fourth substrate has a seventh side facing the fifth side of the third substrate and an eighth side opposite to the seventh side, wherein the seventh side comprises a second single electrode, and wherein the nanopore layer is disposed on the eighth side, wherein the fourth substrate includes a second capillary channel extending from the seventh side of the fourth substrate to the eighth side, wherein the nanopore layer is positioned over the opening of the capillary channel, wherein the nanopore layer is interposed between the second substrate and the fourth substrate such that the nanopore provides an electroosmotic conduit between the first capillary channel and the second capillary channel, wherein the detection electrode pair includes a second detection electrode, which is the second single electrode.

In another aspect, the present invention relates to an integrated digital microfluidic nanopore realization device comprising: a first substrate having a first side and a second side opposite to the first side, wherein the first side includes an electrode array; a second substrate spaced from the first substrate, wherein the second substrate includes a third side facing the first side of the first substrate and a fourth side opposite to the third side; a nanopore realization layer lacking nanopores and disposed on an outer surface of the device, wherein the outer side is selected from the second side or the fourth side, wherein one of the first substrate or the second substrate including the outer side includes a capillary channel extending from the first side of the first substrate to the second side or from the third side of the second substrate to the fourth side, wherein the nanopore realization layer is positioned over the opening of the capillary channel; and a pair of electrodes configured to apply an electric field across the nanopore realization layer, wherein the electrode array is configured to transport at least one microdroplet of fluid to the capillary channel.

In another aspect, the present invention relates to a method for generating nanopores in an integrated digital microfluidic nanopore realization device, comprising: providing the integrated digital microfluidic nanopore realization device described above; immersing two sides of the nanopore realization layer in an ionic liquid such that the ionic liquid on each side of the layer makes electrical contact with either of the detection electrode pairs; applying a voltage between the detection electrode pairs to drive a current through the layer; measuring the conductance across the layer; and terminating the application of the voltage after detecting conductance indicating the generation of nanopores in the layer.

In another aspect, the present invention relates to a composition comprising a binding member, a label, and a spacer.

In another aspect, the present invention relates to an integrated digital microfluidic nanopore device comprising: a first substrate having an electrode array; a second substrate spaced apart from the first substrate; and a nanopore layer having a first surface and a second surface disposed between the first substrate and the second substrate, wherein the electrode array is configured to position a first microdroplet at the first surface of the nanopore layer, wherein at least two electrodes of the electrode array are positioned transversely across the nanopore layer, wherein the two electrodes form an anode and a cathode and operate to drive a current through the nanopores in the nanopore layer when the liquid microdroplet is at the first surface of the nanopore layer.

In another aspect, the present invention relates to an integrated digital microfluidic nanopore device comprising a microfluidic module and a nanopore module; the microfluidic module comprising an electrode array wherein the electrode array transports at least one microdroplet of fluid to a transfer location in the electrode array, wherein the transfer location is at an interface between the microfluidic module and the nanopore module; the nanopore module comprising a first capillary channel extending from the transfer location to a nanopore layer.

In another aspect, the present invention relates to an integrated digital microfluidic nanopore device comprising: a first substrate having an array of electrodes; a second substrate spaced apart from the first substrate; a first nanopore layer having one or more nanopores therein; a second nanopore layer having one or more nanopores therein; and at least two electrodes for establishing an electric field to drive a tag through the nanopores in the first and second nanopore layers.

In another aspect, the present invention relates to a kit comprising any of the aforementioned means for use in any of the aforementioned methods.

In another aspect, the present invention relates to a method for using any of the aforementioned devices to measure or detect analytes present in biological samples or for diagnosing patients or screening blood supplies.

In another aspect, the present invention relates to a method for measuring or detecting a target analyte present in a biological sample, the method comprising: (a) contacting the sample with a solid support, a binding member, and a labeled analyte labeled with a cleavable tag, wherein the solid support comprises an immobilization reagent, the binding member comprises a ligand of the immobilization reagent, and the binding member specifically binds the target analyte to form a solid support/binding member/target analyte complex or a solid support/binding member/labeled analyte complex; (b) removing the labeled analyte that is not bound to a binding member in the solid support/binding member/labeled analyte complex; (c) cleaving the tag, the tag being attached to the labeled analyte bound to a binding member in the solid support/binding member/labeled analyte complex; (d) transferring the tag through one or more nanopores in the layer; and (e) evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tags transferred through the layer detects the analyte present in the sample.

In another aspect, the present invention relates to a method for measuring or detecting a target analyte present in a biological sample, the method comprising: (a) contacting the sample with a solid support, a binding member, and an exogenous analyte, wherein the solid support comprises an immobilization reagent, the exogenous analyte comprises a ligand of the immobilized reagent and binds to the solid support to form a solid support/immobilized analyte complex, and the binding member comprises a cleavable tag and specifically binds to the target analyte to form a solid support/target analyte/binding member complex or a solid support/immobilized analyte/binding member complex. (b) Removing binding members not bound to the solid support/immobilized analyte/binding member complex or the solid support/target analyte/binding member complex; (c) Cutting a tag attached to a binding member in the solid support/immobilized analyte/binding member complex; (d) Transferring the tag through one or more nanopores in the layer; and (e) Evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tags transferred through the layer detects the analyte present in the sample.

In another aspect, the present invention relates to a method for measuring or detecting a target analyte present in a biological sample, the method comprising: (a) contacting the sample with a solid support, a binding member, and a labeled analyte labeled with a cleavable tag, wherein the solid support comprises an immobilization reagent, the binding member comprises a ligand of the immobilization reagent, and the binding member specifically binds the target analyte to form a solid support/binding member/target analyte complex or a solid support/binding member/labeled analyte complex; (b) removing the labeled analyte that is not bound to a binding member in the solid support/binding member/labeled analyte complex; (c) cleaving the tag, the tag being attached to the labeled analyte bound to a binding member in the solid support/binding member/labeled analyte complex; (d) transferring the tag through one or more nanopores in the layer; and (e) evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tags transferred through the layer detects the analyte present in the sample.

In another aspect, the present invention relates to a method for measuring or detecting a target analyte present in a biological sample, the method comprising: (a) contacting the sample with a solid support, a binding member, and an exogenous analyte, wherein the solid support comprises an immobilization reagent, the exogenous analyte comprises a ligand of the immobilized reagent and binds to the solid support to form a solid support/immobilized analyte complex, and the binding member comprises a cleavable tag and specifically binds to the target analyte to form a solid support/target analyte/binding member complex or a solid support/immobilized analyte/binding member complex. (a) removing binding members not bound to the solid support/immobilized analyte/binding member complex or the solid support/target analyte/binding member complex; (b) cutting a tag attached to a binding member in the solid support/immobilized analyte/binding member complex; (c) transferring the tag through one or more nanopores in the layer; and (e) evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tags transferred through the layer detects the amount of analyte present in the sample.

In another aspect, the present invention relates to a method for measuring or detecting a target analyte present in a biological sample, the method comprising: (a) contacting the sample with a solid support, a binding member, and a labeled analyte labeled with an aptamer, wherein the solid support comprises an immobilization reagent, the binding member comprises a ligand of the immobilization reagent, and the binding member specifically binds the target analyte to form a solid support/binding member/target analyte complex or a solid support/binding member/labeled analyte complex; and (b) removing any unbound analyte from the solid support. (c) Dissociating the aptamer attached to the labeled analyte bound to the binding member in the solid support/binding member/labeled analyte complex; (d) Transferring the dissociated aptamer through one or more nanopores in the layer; and (e) Evaluating the aptamers transferred through the layer, wherein measuring the number of aptamers transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the aptamers transferred through the layer detects the analyte present in the sample.

In another aspect, the present invention relates to a method for measuring or detecting a target analyte present in a biological sample, the method comprising: (a) contacting the sample with a solid support, a binding member and an exogenous analyte, wherein the solid support comprises an immobilization reagent, the exogenous analyte comprises a ligand of the immobilization reagent and binds to the solid support to form a solid support/immobilized analyte complex, and the binding member comprises an aptamer and specifically binds to the target analyte to form a solid support/target analyte/binding member complex or a solid support/immobilized analyte/binding member complex; (b) Removing binding members not bound to the solid support/immobilized analyte/binding member complex or the solid support/target analyte/binding member complex; (c) Dissociating the aptamer bound to the binding member in the solid support/immobilized analyte/binding member complex; (d) Transferring the tag through one or more nanopores in the layer; and (e) Evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tags transferred through the layer detects the analyte present in the sample.

Attached Figure Description

The details of the subject matter described herein (regarding its structure and operation) can be understood by studying the accompanying drawings, in which the same reference numerals denote the same parts. The parts in the drawings are not necessarily to scale, but rather the emphasis is on explaining the principles of the subject matter. Furthermore, all illustrations are intended to convey concepts, where relative size, shape, and other detailed properties are interpreted schematically, rather than literally or precisely.

Figures 1A and 1B depict a microfluidic device 10 used in conjunction with a nanopore device 15.

Figures 2A and 2B depict schematic diagrams of a reversible integrated device having a microfluidic module 20 combined with a nanopore module 30 via a channel 40. Figures 2C-2L depict schematic diagrams of an exemplary integrated device in which the microfluidic module and the nanopore module are in fluid communication. The nanopore module includes nanopores in a layer that physically separates the two microfluidic channels at locations where they intersect.

Figure 3 illustrates an exemplary integrated device that includes a microfluidic module 300 and a nanopore module 325.

Figure 4 provides an integrated device 400, wherein the digital microfluidic module includes an embedded nanopore module.

Figure 5A shows a top view of an integrated device. Figure 5B shows a side view of the integrated device of Figure 5A.

Figure 6 illustrates an exemplary apparatus and method of the present invention.

Figure 7 illustrates an exemplary apparatus and method of the present invention.

Figure 8 depicts a side view of an exemplary integrated device of the present invention.

Figure 9 depicts an exemplary system of the present invention.

Figure 10 illustrates a schematic diagram of the manufacturing process of a low-cost DMF chip.

Figure 11 depicts a single flexible DMF chip manufactured according to the schematic diagram in Figure 10.

Figure 12 illustrates the driving of microdroplets in a DMF chip according to an embodiment of the present invention.

Figures 13A-13E depict the execution of immunoassays in a DMF chip according to an embodiment of the present invention.

Figures 14A-14C depict the fabrication and design of nanoporous modules according to embodiments of the present invention.

Figure 15A shows a graph of the leakage current measured in real time. Figure 15B depicts the current-voltage (I-V) curves of the nanopore.

Figures 16A-16C illustrate the filling of capillary channels in an integrated DMF-nanopore module device according to an embodiment of the present invention.

Figure 17 shows a schematic diagram of droplet transfer between modules in an integrated DMF-nanopore module device according to an embodiment of the present invention.

Figure 18 shows a schematic diagram of the nanopore module design according to an embodiment of the present invention.

Figure 19 shows a schematic diagram of an integrated DMF-nanopore module device capable of droplet transfer between passive transport execution modules according to an embodiment of the present invention.

Figure 20 shows a schematic diagram of an integrated DMF-nanopore module device capable of droplet transfer between passive transport execution modules according to an embodiment of the present invention.

Figure 21 is a schematic diagram of a silicon microfluidic device containing silicon microchannels according to an embodiment of the present invention, wherein the silicon microchannels allow passive movement of liquid droplets via passive transport.

Figure 22 is an image of a silicon microchannel in a silicon microfluidic device according to an embodiment of the present invention, the silicon microfluidic device allowing passive movement of liquid droplets via passive transport.

Figures 23A and 23B show schematic diagrams of a method for manufacturing an integrated nanopore sensor according to an embodiment of the present invention.

Figures 24A-24C show scatter plots (level duration relative to blocking level) of graphs obtained using data showing transfer events through the following nanopores: (Figure 24A) nanopores containing conventional double-stranded DNA (“dsDNA”); (Figure 24B) nanopores containing DBCO-modified dsDNA; and (Figure 24C) nanopores containing dsDNA stars.

Figure 25 shows a schematic diagram of thiol-mediated chemical cleavage.

Figures 26A and 26B show schematic diagrams of optical cutting experiments performed on magnetic microparticles.

Figure 27 shows a schematic diagram of the reagent arrangement on the DMF chip.

Figure 28 shows a bar graph of the sample relative to the nanopore flux (DMF cut) in ^seconds .

Figure 29 shows the method used to determine the threshold for digital signal counts.

Figures 30A-30C show the current blocking at different time periods for three standards of 94 nM (Figure 30A), 182 nM (Figure 30B), and 266 nM (Figure 30C).

Figure 31 shows the dose-response curves for the number of events over a fixed time period (5 min).

Figure 32 shows the dose-response curves for the time required to complete a fixed number of events.

Figure 33 shows the dose-response curves for events per unit time.

Figure 34 shows the dose-response curves for events per unit time when using Seq31-SS-Biotin.

Figure 35 shows a schematic diagram of the nanopore compartment design in a silicon nanopore module according to an embodiment of the present invention.

Figure 36 shows a table listing the physical parameters used for COMSOL electric field simulation in the nanopore compartments of a silicon nanopore module according to an embodiment of the present invention.

A set of images in Figure 37 shows simulation results of counterion concentration gradients near nanopores in a silicon nanopore module according to an embodiment of the present invention.

Figure 38 illustrates the effect of the diameter of the _SiO2 via prepared on a nanoporous membrane with nanopores on the electroosmotic flow through the nanopores, according to an embodiment of the present invention.

Figure 39 illustrates the effect of the diameter of the _SiO2 via prepared on a nanoporous membrane with nanopores on the electrical conductivity through the nanopores, according to an embodiment of the present invention.

Figure 40 shows a schematic diagram of an integrated DMF-nanopore module device with a nanopore module positioned on one side of a DMF module according to an embodiment of the present invention.

A set of images in Figure 41 illustrates the movement of liquid from the DMF module through pores in the DMF module substrate by capillary force according to an embodiment of the invention.

A set of images in Figure 42 shows an integrated DMF-nanopore module device according to an embodiment of the present invention, having a nanopore module positioned on one side of a DMF module and electrodes fabricated for the nanopores.

Figure 43 is a schematic diagram of an integrated DMF-nanopore module device according to an embodiment of the present invention, the device having a nanopore module positioned on one side of a DMF module.

Figure 44 is a schematic diagram of an integrated DMF-nanopore module device according to an embodiment of the present invention, the device having a nanopore module positioned between two DMF modules.

Figure 45 illustrates an embodiment of the invention, in which nanopores (transmission electron microscopy (TEM) windows) are created in a nanopore membrane by applying a voltage across the membrane, as confirmed by dielectric breakdown.

Figures 46A and 46B show the current-voltage (I-V) curves of the nanopores formed in the membrane before and after the conditioning process, according to an embodiment of the present invention.

Figure 47 shows a scatter plot of the average ratio of the counted marker average diameter and nanopore size relative to the SNR (signal-to-noise ratio).

Invention Details

Embodiments of the present invention relate to methods, systems, and apparatus for analyzing analytes in samples. In some embodiments, the sample may be a biological sample.

1. Definition

Before describing embodiments of the invention, it should be understood that the invention is not limited to the specific embodiments described, as these can certainly be varied. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used herein, the words “comprising,” “including,” “having,” “may,” “containing,” and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. The singular forms “a,” “and,” and “described” include the plural meaning unless the context clearly indicates otherwise. The invention also contemplates “comprising” the embodiments or elements presented herein, “consisting of,” and “substantially constitutes,” whether or not explicitly stated.

With respect to the range of numbers listed in this article, each inserted number in between is explicitly predicted to have the same precision. For example, with respect to the range of 6-9, the numbers 7 and 8 are predicted in addition to 6 and 9, and with respect to the range of 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly predicted.

In this paper, “affinity” and “binding affinity” are used interchangeably to refer to the tendency or strength of binding between a binding member and an analyte. For example, binding affinity can be expressed by the equilibrium dissociation constant ( _KD ), dissociation rate ( _kd ), or binding rate ( _ka ).

As used in this article, "analyte" refers to a molecule with a structure similar to the target molecule (e.g., nucleoside analogs, nucleotide analogs, sugar phosphate analogs, analyte analogs, etc.). An analyte analog is a molecule that is structurally similar to the analyte but whose binding members have different affinities to it.

As used in this article, "aptamer" refers to an oligonucleotide or peptide molecule that can bind to preselected targets (including small molecules, proteins, and peptides) with high affinity and specificity. Aptamers can take on various shapes due to their tendency to form helices and single-stranded loops. Oligonucleotide or nucleic acid aptamers can be single-stranded DNA or RNA (ssDNA or ssRNA) molecules. Peptide aptamers may include short, variable peptide domains that attach to a protein scaffold at both ends.

The terms “beads” and “granules” are used interchangeably in this document and refer to substantially spherical solid supports.

"Component," "multiple components," or "at least one component" generally refers to capture antibodies, detection reagents or conjugates, calibrators, controls, sensitivity test subject groups, containers, buffers, diluents, salts, enzymes, enzyme cofactors, detection reagents, pretreatment reagents/solutions, substrates (e.g., as solutions), stop solutions, etc., which can be included in a kit for the determination of test samples (such as patient urine, serum, whole blood, tissue aspirates, or plasma samples) according to the methods described herein and other methods known in the art. Some components may be in solution or lyophilized to reconstitute for determination.

As used herein, “control” refers to a reference standard for the analyte, such as those known or accepted in the art, or determined empirically using acceptable methods, such as those commonly employed. A “reference standard” is a standardized substance used as the basis for the measurement of similar substances. For example, documented reference standards are disclosed in the U.S. Pharmacopeial Convention (USP-NF), Food Chemicals Codex, and Dietary Supplements Compendium (all available at http://www.usp.org) and other well-known sources. Methods for standardizing references are described in the literature. It is also well known that the amount of an analyte present is quantified by using a calibration curve of the analyte or by comparison with an alternative reference standard. Standard curves can be generated using a series of dilutions or solutions of the analyte at known concentrations using mass spectrometry, specific gravity determination methods, and other techniques known in the art. Alternative reference standards described in the literature include standard addition (also known as the standard addition method) or digital polymerase chain reaction.

In this document, the terms "Digital Microfluidics (DMF)," "Digital Microfluidics Module (DMF Module)," or "Digital Microfluidics Device (DMF Device)" used interchangeably refer to modules or devices that utilize digital or droplet-based microfluidics techniques to provide manipulation of liquids in the form of dispersed, small-volume droplets. Digital microfluidics uses the principles of emulsion science to establish fluid-fluid dispersions (primarily water-in-oil emulsions) within channels. It allows for the generation of monodisperse droplets/bubbles or droplets with very low polydispersity. Digital microfluidics is based on the micromanipulation of discontinuous fluid droplets within a reconfigurable network. Complex instructions can be programmed by combining basic operations of droplet formation, transfer, splitting, and merging.

Digital microfluidics operate on discrete volumes of fluid that can be manipulated by binary electrical signals. By using discrete unit-volume droplets, microfluidic manipulation can be defined as a set of repetitive basic operations: moving one unit of fluid one unit distance. Droplets can be formed using the surface tension properties of the liquid. The actuation of the droplets is based on the presence of electrostatic forces generated by electrodes placed below the bottom surface where the droplets are located. Different types of electrostatic forces can be used to control the shape and movement of the droplets. One technique for establishing these electrostatic forces is based on dielectric electrophoresis, which relies on the permittivity difference between the droplets and the surrounding medium and can utilize a high-frequency AC electric field. Another technique for establishing these electrostatic forces is based on electrowetting, which relies on the dependence of the surface tension between the liquid droplets present on the surface and the surface on the electric field applied to the surface.

"Drag tag" refers to a migration modifier. A drag tag can be a genetically engineered, highly reproducible polypeptide ("protein polymer") designed to be large, water-soluble, and completely monodisperse. Positively charged arginines can be intentionally introduced into the amino acid sequence at regular intervals to increase hydrodynamic drag without increasing the drag tag length. A description of drag tags is given in U.S. Patent Publication No. 20120141997, which is incorporated herein by reference.

As used herein, "enzyme-cleavable sequence" refers to any nucleic acid sequence that can be cleaved by an enzyme. For example, the enzyme can be a protease or an endonuclease, such as a restriction endonuclease (also called a restriction enzyme). Restriction endonucleases are capable of recognizing and cleaving DNA molecules at specific DNA cleavage sites between predetermined nucleotides. Some endonucleases (e.g., Fokl) contain a cleavage domain that nonspecifically cleaves DNA at a specific site, regardless of the nucleotide present at that site. In some embodiments, the specific DNA cleavage site and the DNA recognition site of the restriction endonuclease are the same.

"Globular proteins" refers to water-soluble proteins that are approximately spherical in shape. Examples of globular proteins include, but are not limited to, ovalbumin, β-globulin, C-reactive protein, fibrin, hemoglobin, IgG, IgM, and thrombin.

The term “tag” or “detectable tag” may be used interchangeably in this document to refer to a tag attached to a specific binding member or analyte via a cuttable connector.

"Nanoparticles" and "nanoparticles" are used interchangeably herein and refer to nanobeads or nanoparticles having dimensions that transfer through or across a nanopore used to count the number of nanobeads/nanoparticles passing through it.

"Nucleoside base" or "base" refers to those naturally occurring and synthetic heterocyclic moieties commonly known in the fields of nucleic acid or polynucleotide technology or peptide nucleic acid technology for the production of polymers. Non-limiting examples of suitable nucleoside bases include: adenine, cytosine, guanine, thymine, uracil, 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine, N9-(7-deazo-guanine), N9-(7-deazo-8-aza-guanine), and N8-(7-deazo-8-aza-adenine). Nucleoside bases can be linked to other moieties to form nucleosides, nucleotides, and nucleoside/nucleotide analogs.

"Nucleoside" refers to a compound consisting of a purine, denitropurine, or pyrimidine nucleoside base (e.g., adenine, guanine, cytosine, uracil, thymine, 7-denitroadenine, 7-denitroguanosine) linked at the 1' position to an isomeric carbon of a pentose sugar (such as ribose, 2'-deoxyribose, or 2',3'-dideoxyribose).

As used in this article, “nucleotide” refers to the phosphate ester of a nucleoside, such as a mono-, di-, or tri-phosphate, the most common esterification site of which is the hydroxyl group attached to the C-5 position of the pentose sugar.

"Nucleoside base polymer" or "nucleoside base oligomer" refers to two or more nucleoside bases linked by bonds to form an oligomer. Nucleoside base polymers or oligomers include, but are not limited to: polynucleotides and oligonucleotides (e.g., DNA and RNA polymers and oligomers), polynucleotides and oligonucleotide analogs, and polynucleotides and oligonucleotide mimics, such as polyamides or peptide nucleic acids. Nucleoside base polymers or oligomers can vary in size from a few nucleoside bases to hundreds or even thousands of nucleoside bases. The nucleoside base polymer or oligomer may comprise about 2-100 nucleoside bases or about 8000-10000 nucleoside bases. For example, the nucleoside base polymer or oligomer may have at least about 2 nucleoside bases, at least about 5 nucleoside bases, at least about 10 nucleoside bases, at least about 20 nucleoside bases, at least about 30 nucleoside bases, at least about 40 nucleoside bases, at least about 50 nucleoside bases, at least about 60 nucleoside bases, at least about 70 nucleoside bases, at least about 80 nucleoside bases, at least about 90 nucleoside bases, at least about 100 nucleoside bases, at least about 200 nucleoside bases, at least about 300 nucleoside bases, at least about 400 nucleoside bases, or at least... Approximately 500 nucleoside bases, at least approximately 600 nucleoside bases, at least approximately 700 nucleoside bases, at least approximately 800 nucleoside bases, at least approximately 900 nucleoside bases, at least approximately 1000 nucleoside bases, at least approximately 2000 nucleoside bases, at least approximately 3000 nucleoside bases, at least approximately 4000 nucleoside bases, at least approximately 5000 nucleoside bases, at least approximately 6000 nucleoside bases, at least approximately 7000 nucleoside bases, at least approximately 8000 nucleoside bases, at least approximately 9000 nucleoside bases, or at least approximately 10000 nucleoside bases.

"One or more nanopores in a layer" means that in a single-film or multi-film structure, there is a single nanopore, or multiple nanopores (e.g., two or more) that are close to each other (e.g., in parallel). When one or more nanopores are present (e.g., 1, 2, 3, 4, 5, 6, or other numbers of nanopores, as long as technically feasible), they may optionally be in parallel (e.g., close to each other) or in series (e.g., a nanopore in one layer is present as follows: separate from another nanopore in another layer, or stacked on top of another nanopore in another layer (e.g., on top of or on top of), etc.), or in an alternating structure, as those skilled in the art will understand. Optionally, such nanopores are independently addressable, for example, by placing each in its own separate compartment (e.g., spaced apart from any other nanopores by a wall), or alternatively, by means of independent detection circuitry.

A "polymer brush" refers to a polymer layer attached to a surface at one end. The polymers are brought close together to form a layer or coating, which creates its own environment. The brush can be in a solvent state (when the suspended chain is immersed in a solvent) or a molten state (when the suspended chain completely fills the available space). Additionally, a separate category of polyelectrolyte brushes exists when the polymer chains themselves carry an electrostatic charge. The brush can be characterized by a high density of transplanted chains. The limited space then leads to strong chain elongation and rare system properties. Brushes can be used to stabilize colloids, reduce friction between surfaces, and provide lubrication in artificial joints.

"Polynucleotide" or "oligonucleotide" refers to a polymer or oligomer of nucleoside bases in which nucleoside bases are linked by sugar-phosphate bonds (a sugar-phosphate backbone). Exemplary polynucleotides and oligonucleotides include polymers of 2'-deoxyribonucleotides (DNA) and polymers of ribonucleotides (RNA). Polynucleotides may consist entirely of ribonucleotides, entirely of 2'-deoxyribonucleotides, or a combination thereof. "Nucleic acid" includes both "polynucleotides" and "oligonucleotides," and includes single-stranded and double-stranded polymers of nucleotide monomers.

"Polynucleotide analogues" or "oligonucleotide analogues" refer to nucleoside base polymers or oligomers in which nucleoside bases are linked by a glycophosphate backbone comprising one or more glycophosphate analogues. Typical glycophosphate analogues include, but are not limited to: glycoalkylphosphonates, glycoaminophosphites, glycoalkyl phosphate triesters or substituted alkyl phosphate triesters, glycothiophosphates, glycodithiophosphates, glycophosphates, and glycophosphate analogues, wherein the sugar is a sugar other than 2'-deoxyribose, and the nucleoside base polymer has positively charged sugar-guanidino linkages, such as those described in U.S. Patent Nos. 6,013,785 and 5,696,253.

As used herein, “pore” (or alternatively “nanopore”) or “channel” (or alternatively “nanopore” or “nanochannel”) refers to an opening, gap, conduit, or groove in a membrane/layer, wherein the pore or channel is of sufficient size to allow the passage or analysis of individual molecules (e.g., tags) at a time (e.g., one by one, such as in a series).

As used in this article, "receptor" refers to a protein-molecule that recognizes and responds to endogenous chemical signals. When such endogenous chemical signals bind to receptors, they cause some form of cellular/tissue response. Examples of receptors include, but are not limited to, neural receptors, hormone receptors, nutrient receptors, and cell surface receptors.

As used herein, "spacer" refers to a chemical portion that extends a cleavable group from a specific binding member, or provides a link between the binding member and a support, or extends a tag/label from a lightly cleavable portion. In some embodiments, one or more spacers may be included at the N-terminus or C-terminus of a peptide- or nucleotide-based tag or label to provide an optimal distance between the sequence and the specific binding member. Spacers may include, but are not limited to: 6-aminohexanoic acid; 1,3-diaminopropane; 1,3-diaminoethane; polyethylene glycol (PEG) polymer groups; short amino acid sequences of 1-5 amino acids; and sequences such as polyglycine. In some embodiments, the spacer is nitrobenzyl, dithioethylamino, a 6-carbon spacer, a 12-carbon spacer, or 3-(9-((3-carboxypropyl)(toluenesulfonyl)carbamoyl)acridin-10-onthium-10-yl)propane-1-sulfonate.

In this document, the terms "specific binding pair" or "specific binding member" used interchangeably refer to the specific recognition (of a much smaller size relative to the recognition of other molecules) of two or more different molecules. One of the two different molecules has a region, on its surface or within its lumen, that specifically binds to the other molecule in a particular spatial and polar configuration and is therefore defined as complementary to the latter. The molecule can be a member of a specific binding pair. For example, a specific binding member can include, but is not limited to, proteins such as receptors, enzymes, and antibodies.

As used herein, “tag” or “tag molecule” refers to a molecule (e.g., an aptamer cleaved from a second binding member or dissociated from a target analyte) that translocates through or across a nanopore and provides an indication of analyte levels in the sample. These terms refer to a single-tag molecule or multiple identical-tag molecules. Similarly, unless otherwise stated, “tag” means one label or one or more labels.

The term "threshold" used in this paper refers to an empirically determined and subjective cutoff level; data exceeding this threshold is considered a "signal," and data below it is considered "noise." Figure 29 illustrates the use of thresholds for digital signal counting. A computer program based on CUSUM (cumulative summation algorithm) processes the acquired data and detects events based on a threshold input from the user. Detection of any large number of events can only prevent variations among users, and the data is subsequently filtered for specific purposes. For example, as can be seen from the figure, events detected above a set threshold can affect the group of events counted as signals. For a "loose" threshold, fewer events will be counted as signals. For a "tight" threshold, a larger number of events will be counted as signals. Setting the threshold to loose or tight is a subjective choice based on the desired sensitivity or specificity of the measurement and whether there is a preference for false positives or false negatives in a given assessment. The current blocking marker from DNA transfer was calculated to be 1.2 nA, based on an empirical formula that correlates current change with the diameter of the DNA and the thickness of the nanopore membrane (H. Kwok, et al., PLoSONE, 9(3), 392880, 2014).

As used herein, the reference to “movement through or across” a nanopore (e.g., the movement of nanoparticles, tags, tag molecules or other objects) may alternatively mean moving through or across, in other words, from one side of the nanopore to the other, for example, from the front side to the back side, or vice versa.

As used herein, "tracer" refers to an analyte or analyte fragment conjugated to a tag or label, wherein the analyte conjugated to the tag or label can effectively compete with the analyte for a site on the antibody that is specific to the analyte. For example, the tracer can be an analyte or an analogue of an analyte, such as cyclosporine or its analogue ISA247, vitamin D and its analogues, sex hormones and their analogues, etc.

As used in this paper, “transfer event” refers to an event in which a tag is transferred across or across (e.g., from the front side to the back side, or vice versa) a layer or nanopore.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, this document (including the definitions) shall prevail. Preferred methods and materials are described below, although similar or equivalent methods and materials may be used to practice or test the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety to disclose and describe the cited publications and their associated methods and/or materials. The materials, methods, and embodiments disclosed herein are merely exemplary and are not intended to be limiting.

2. Methods used for analyte analysis

This document provides methods for analyte analysis. These methods may involve single-molecule counting. In some embodiments, the methods for analyte analysis may involve evaluating the presence of an analyte in a sample. In some embodiments, the evaluation may be used to determine the presence and/or concentration of an analyte in the sample. In some embodiments, the methods may also be used to determine the presence and/or concentration of multiple different analytes in a sample.

This document provides a method for measuring or detecting an analyte present in a biological sample. The method includes: contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds the analyte; contacting the analyte with a second binding member, wherein the second binding member specifically binds the analyte and wherein the second binding member includes a cleavable tag attached thereto; removing the second binding member from the analyte not bound to the first binding member; cutting the tag attached to the second binding member, the second binding member binding to the analyte bound to the first binding member; transferring the cut tag through or across one or more nanopores in a layer; detecting or measuring the tag transferred through the layer; and evaluating the tag transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tags transferred through the layer detects the analyte present in the sample. In some embodiments, the evaluation of the tags transferred through the layer is performed, wherein the number of tags transferred through the layer measures the amount of analyte present in the sample. In some implementations, the label that detects transfer across the layer is evaluated, wherein the label that detects transfer across the layer detects the analyte present in the sample.

This document provides a method for measuring or detecting an analyte present in a biological sample. The method includes: contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds the analyte; contacting the analyte with a second binding member, wherein the second binding member specifically binds the analyte and wherein the second binding member includes an aptamer; removing aptamers not bound to the analyte bound to the solid substrate; dissociating the aptamers bound to the analyte and transferring the dissociated aptamers through or across one or more nanopores in a layer; and evaluating the aptamers transferred through the layer, wherein measuring the number of aptamers transferred through the layer measures the amount of analyte present in the sample, or detecting the aptamers transferred through the layer detects the analyte present in the sample. In some embodiments, the evaluation of measuring the aptamers transferred through the layer is performed, wherein the number of aptamers transferred through the layer measures the amount of analyte present in the sample. In some embodiments, the evaluation of detecting the aptamers transferred through the layer is performed, wherein detecting the aptamers transferred through the layer detects the analyte present in the sample.

In some embodiments, each tag (such as an aptamer) transferred across the layer is a transfer event. Measuring the number of transfer events measures the amount of analyte present in the sample. In some embodiments, the amount of analyte present in the sample can be determined by counting the number of transfer events over a set time period and correlating the number of transfer events with a control. A standard curve can be determined by measuring the number of transfer events at a control concentration of the analyte over the set time period. In some embodiments, the amount of analyte present in the sample can be determined by measuring the time required for a set number of transfer events to occur and correlating it with a control. A standard curve can be determined by measuring the time required for a set number of transfer events to occur at a control concentration of the analyte. In some embodiments, the amount of analyte present in the sample can be determined by measuring the average time between transfer events and correlating it with a control. A standard curve can be determined by measuring the average time between transfer events at a control concentration of the analyte. In some embodiments, the control may be a reference standard comprising a calibration curve, a standard addition, or a digital polymerase chain reaction.

In an exemplary embodiment, the method may include: contacting the sample with a first binding member (“binding member” may alternatively be referred to as a “specific binding member,” and as described in section c below) wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds the analyte; contacting the analyte with a second binding member, the second binding member specifically binding the analyte and the second binding member including a cleavable tag (“tag” as defined herein and as described in section d below) attached thereto); removing the second binding member from the analyte not bound to the first binding member; cleaving the tag attached to the second binding member, the second binding member binding to the analyte bound to the first binding member; transferring the tag through nanopores in the layer; determining the number of tags transferred through the layer; and determining the concentration of the analyte in the sample based on the number of tags transferred through the layer. In some embodiments, the concentration of the analyte may be determined by counting the number of tags transferred through the layer per unit time. In other embodiments, the concentration of the analyte may be determined by determining the time when the number of tags transferred through the layer reaches a threshold.

The sample may be any test sample containing or suspected of containing the target analyte. The terms "analyte," "target analyte," and "target analyte" are used interchangeably herein and refer to the analyte measured in the methods and apparatus disclosed herein. The target analyte is further described below.

As used herein, the term "contact" and its grammatical equivalents refer to any type of combination action that brings a binding member close enough to a target analyte in a sample that a binding interaction will occur if a target analyte specific to the binding member is present in the sample. Contact can be achieved in a variety of different ways, including combining the sample with a binding member, exposing the target analyte to the binding member by introducing the binding member into close proximity to the analyte, etc.

In some cases, the first binding member can be immobilized on a solid support. As used herein, “immobilization” means a stable binding of the first binding member to the surface of a solid support. “Stable binding” refers to a physical binding between two entities where the average half-life of the binding is one day or more, for example, under physiological conditions. In some aspects, the physical binding between two entities has an average half-life of 2 days or more, 1 week or more, 1 month or more (including 6 months or more, for example, 1 year or more) in PBS at 4°C. According to some embodiments, the stable binding originates from a covalent bond between the two entities, a non-covalent bond (e.g., an ionic or metallic bond) between the two entities, or other forms of chemical attraction such as hydrogen bonding, van der Waals forces, etc.

The solid support having a surface on which the binding agent is immobilized can be any convenient surface with a planar or non-planar conformation, such as the surface of a microfluidic chip, the inner surface of a compartment, the outer surface of a bead (as defined herein), or the inner and/or outer surface of a porous bead. For example, the first binding member can be covalently or non-covalently attached to the bead, such as latex, agarose, sepharose, streptomycin, toluenesulfonyl-activated, epoxy resin, polystyrene, amino beads, amine beads, carboxyl beads, etc. In some embodiments, the beads can be particles, such as microparticles. In some embodiments, the microparticles can be about 0.1 nm to about 10 micrometers, about 50 nm to about 5 micrometers, about 100 nm to about 1 micrometer, about 0.1 nm to about 700 nm, about 500 nm to about 10 micrometers, about 500 nm to about 5 micrometers, about 500 nm to about 3 micrometers, about 100 nm to 700 nm, or about 500 nm to 700 nm. For example, the particles may be about 4-6 micrometers, about 2-3 micrometers, or about 0.5-1.5 micrometers. Particles smaller than about 500 nm are sometimes considered nanoparticles. Thus, the particles may optionally be nanoparticles of about 0.1 nm to about 500 nm, about 10 nm to about 500 nm, about 50 nm to about 500 nm, about 100 nm to about 500 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, or about 500 nm.

In some embodiments, the beads may be magnetic beads or magnetic particles. Magnetic beads/particles may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or ferrofluidic. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, _CrO₂ , MnAs, MnBi, EuO, _and NiO _/ Fe. Examples of ferrimagnetic materials include _NiFe₂O₄ , _CoFe₂O₄ , _{and Fe₃O₄} (or _{FeO.Fe₂O₃} ). The beads may have a solid core portion that is magnetic and surrounded by one or more _non -magnetic layers. Alternatively, the magnetic portion may be a layer surrounding the non-magnetic core. The solid support on which the first binding member is immobilized may be stored in a dry or liquid form. The magnetic beads may be placed in a magnetic field before or after contacting a sample with the magnetic beads on which the first binding member is immobilized.

Following the contact step, the sample and the first binding member can be incubated for a sufficient period to allow binding interactions between the binding member and the analyte to occur. Alternatively, this incubation can be performed in a binding buffer that promotes specific binding interactions. By changing the binding buffer, the binding affinity and/or specificity of the first and/or second binding members can be manipulated or altered during the assay. In some embodiments, changing the binding buffer can increase the binding affinity and/or specificity. In some embodiments, changing the binding buffer can decrease the binding affinity and/or specificity.

The binding affinity and/or specificity of the first binding member and/or the second binding member can be measured using the methods and apparatus disclosed below. In some embodiments, an aliquot of a sample is measured using a set of conditions and compared with another aliquot of a sample measured using a different set of conditions, thereby determining the effect of the conditions on binding affinity and/or specificity. For example, changing or altering the conditions can be one or more of the following: removing the target analyte from the sample, adding a molecule that competes with the target analyte or ligand for binding, and changing the pH, salt concentration, or temperature. Additionally or alternatively, the duration can be variable, and changing the conditions can include waiting for the duration before re-executing the detection method.

In some embodiments, after the tag or aptamer has passed through the pores of the nanopore device, the device can be reconfigured to reverse the movement of the tag or aptamer, so that the tag or aptamer can pass through the pores again and be remeasured or re-detected, for example, in confirmatory assays for infectious disease assays to confirm the results of the measurement.

The binding buffer may include molecular standards for antigen-antibody binding buffers, such as albumin (e.g., BSA), nonionic detergents (Tween-20, Triton X-100), and/or protease inhibitors (e.g., PMSF). In some cases, the binding buffer may be added to a microfluidic chip, compartment, etc., before or after sample addition. In some cases, the first binding member may be present in the binding buffer before contact with the sample. The duration for which the binding interaction between the binding member and the analyte occurs can be determined empirically and may depend on the binding affinity and binding affinity between the binding member and the analyte. In some embodiments, the contact or incubation may last for a period of 5 seconds to 1 hour, for example, 10 seconds to 30 minutes, or 1 minute to 15 minutes, or 5 minutes to 10 minutes, for example, 10 seconds, 15 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, or 2 hours. Other conditions for the binding interaction (e.g., temperature, salt concentration) may also be determined empirically or may be based on the manufacturer's instructions. For example, the contact can be performed at room temperature (21°C-28°C, e.g., 23°C-25°C), 37°C, or 4°C. In some embodiments, optional mixing of the sample with the first binding member can be performed during the contact step.

Once the immobilized first binding member and analyte complex has formed, any unbound analyte can be removed from the vicinity of the first binding member along with the sample, while the complex of the first binding member and analyte can be retained due to its binding to the solid support. Optionally, the solid support can be contacted with a washing buffer to remove any molecules that are nonspecifically bound to the solid support.

Following the first contact step and optional sample removal and/or optional washing steps, the complex of the first binding member and the analyte can be contacted with the second binding member, thereby resulting in the formation of a sandwich complex, wherein the two binding members bind the analyte. Optional mixing of the second member with the first binding member-analyte complex can be performed in a second contact step. In some embodiments, immobilization of the analyte molecules relative to the surface can facilitate the removal of any excess second binding member from the solution without concern for removing the analyte molecules from the surface. In some embodiments, the second binding member may include a tag attached thereto, such as a cuttable tag.

As noted above, the second contact step can be performed under conditions sufficient for the binding interaction between the analyte and the second binding member. Following the second contact step, any unbound second binding member can be removed, followed by an optional washing step. Any unbound second binding member can be separated from the first binding member-analyte-second binding member complex by suitable means, such as microdroplet actuation, electrophoresis, electrowetting, dielectrophoresis, electrostatic actuation, electric field mediation, electrode mediation, capillary force, chromatography, centrifugation, or aspiration. After removing any unbound second binding member from the vicinity of the first binding member-analyte-second binding member complex, the tag attached to the second binding member present in the first binding member-analyte-second binding member complex can be separated by suitable means. In some embodiments, the tag is cut or separated from the complex remaining after the removal of unbound reagents. For example, the tag can be attached to the second binding member via a cuttable connector (“cuttable connector” as described in section f below). The complex of the first binding member-analyte-second binding member can be exposed to a cutting agent that mediates the cutting of the cutable joint.

In some embodiments, the separation of the tag from the first binding member-analyte-second binding member complex is performed without causing damage to the complex, resulting in the release of only the tag from the complex. In other embodiments, the separation of the tag from the first binding member-analyte-second binding member complex is performed under conditions that could cause damage to the complex, resulting in the release of the tag, and one or more of the second binding member, the analyte, and the first binding member from the complex. In some embodiments, the size of the nanopores used to count the tag can prevent the second binding member, the analyte, and the first binding member from transferring through the nanopores. In other embodiments, where the complex of the second binding member, the analyte, and the first binding member is retained on a solid support, the nanopores may not be large enough to exclude the second binding member, the analyte, and the first binding member.

The separation step results in the generation of free tags, which, under the influence of an electric field, can cause the free tags to transfer through or across nanopores or nanopore layers (as described in section f below). In some cases, the cleavage step can result in the separation of substantially all tag molecules attached to each of the second binding members in the first binding member-analyte-second binding member complex. The number of tag molecules can be correlated with the number of analyte molecules in the complex, which is proportional to the concentration of the analyte in the sample. In some embodiments, the correlation between the number of tags and the analyte concentration can be direct (a higher number of tag molecules is associated with a higher analyte concentration). In embodiments where a tagged competitor or a tagged analyte (such as a tracer (as defined herein)) is combined with the sample, the tagged competitor or analyte competes with the analyte in the sample for binding to the first binding member, and the correlation between the number of tags and the analyte concentration can be inverse (a lower number of tag molecules is associated with a higher analyte concentration). The correlation between the number of tag molecules and the analyte concentration (whether direct or inverse) can be linear or logarithmic. Therefore, the number of tag molecules transferred through the nanopore can be used to determine the analyte concentration in the sample. In some embodiments, the concentration of the analyte can be determined by counting the number of tags transferred through the layer per unit time. In other embodiments, the concentration of the analyte can be determined by determining the time when the number of tags transferred through the layer reaches a threshold. In some embodiments, the number of tag molecules transferred through or across the nanopore can be determined by the current blocking frequency at the nanopore per unit time. Signal detection is further described in section g) below. As described in section d) below, the tag molecules can be nanoparticles or nanobeads (“nanoparticles” and “nanobeads” are as defined herein).

The number of tags incorporated into the second binding member (i.e., the number of tags in the tag/second binding member conjugate) provides a definite stoichiometry for the analyte. In some embodiments, tags can be attached to the second binding member using a procedure that produces a consistent number of tags attached to each second binding member. The number of tags can be optimized based on the counting rate. A faster readout rate can be obtained by including more tags on the binding member, since the counting rate depends on the concentration. The number of tags can be optimized based on the stoichiometry of tag incorporation, such as a 1:1 or 1:4 incorporation ratio. In some embodiments, a 1:5 incorporation ratio is present. For example, a second binding member may have 1, 2, 3, 4, or up to 10 tag molecules attached to it. In some embodiments, a second binding member may have 5 tag molecules attached to it. Many conjugation methods for tagging to second binding members (e.g., peptides, polypeptides, nucleic acids) are known, any of which can be used to prepare the tagged second binding member used in the methods and apparatus of the present invention. For example, using antibodies with C- or N-terminal hexahistine tags, antibodies with aldehyde tags, copper-free click reactions, etc., site-specific conjugation of tags to analyte-specific antibodies can be achieved using thiol-maleimide chemistry, amine-succinimide chemistry, THIOBRIDGE ^™ technology.

In some embodiments, the method measures the amount of analyte by determining the number of transfer events. In some embodiments, one or more transfer events may correspond to binding events between a binding member and the analyte, depending on the chemostoichiometry of the tag incorporated into the specific binding member. For example, if one tag is incorporated into each binding member, then one transfer event represents the binding of the binding member to the analyte; if two tags are incorporated into each binding member, then two transfer events represent the binding of the binding member to the analyte; if three tags are incorporated into each binding member, then three transfer events represent the binding of the binding member to the analyte, and so on.

In another embodiment, the second binding member may be an aptamer that specifically binds to the analyte. In this embodiment, the tag may not be attached to the aptamer. Instead, the aptamers are counted as they transfer through or across the nanopore; that is, the aptamer serves a dual function as both a second binding member and a tag. In these embodiments, the aptamer in the first binding member-analyte-aptamer complex can be dissociated from the complex by any suitable method. For example, the aptamer bound to the first binding member-analyte complex can be dissociated by a denaturing step before transfer through or across the nanopore. The denaturing step may involve exposure to a dissociating agent, a high-salt solution, an acidic reagent, a basic reagent, a solvent, or a heating step. The aptamer can then be transferred through or across the nanopore, and the number of aptamer molecules transferred through or across the nanopore can be used to determine the concentration of the analyte in the sample.

As noted herein, the tag or aptamer may comprise nucleic acids. In some embodiments, the counting step using nanopores does not include identifying the tag or aptamer by determining the identity of at least a portion of the nucleic acid sequence present in the tag/aptamer. For example, the counting step may not include determining the sequence of the tag/aptamer. In other embodiments, the tag/aptamer may not be sequenced; however, the identity of the tag/aptamer can be determined to the extent that one tag/aptamer can be distinguished from another, based on a distinguishable signal associated with the tag/aptamer caused by its size, conformation, charge, charge quantity, etc. Tag/aptamer identification can be useful in methods involving the simultaneous analysis of multiple different analytes in a sample (e.g., two, three, four, or more different analytes in a sample).

In some implementations, simultaneous analysis of multiple analytes in a single sample can be performed by using multiple different first binding members and second binding members (where a pair of first binding members and second binding members is specific to a single analyte in the sample). In these implementations, a tag bound to a second binding member of a first pair of first binding members and second binding members that is specific to a single analyte can be distinguished from a tag bound to a second binding member of a second pair of first binding members and second binding members that is specific to a different analyte. As noted above, a first tag can be distinguished from a second tag based on differences in size, charge, etc.

In some implementations, the concentration of the analyte in the fluid sample can be determined substantially accurately to be less than about 5000 fM ( ^10⁻¹⁵ mol), less than about 3000 fM, less than about 2000 fM, less than about 1000 fM, less than about 500 fM, less than about 300 fM, less than about 200 fM, less than about 100 fM, less than about 50 fM, less than about 25 fM, less than about 10 fM, less than about 5 fM, less than about 2 fM, less than about 1 fM, less than about 500 aM ( ^10⁻¹⁸ mol), less than about 100 aM, less than about 10 aM, less than about 5 aM, less than about 1 aM, less than about 0.1 aM, less than about 500 zM ( ^10⁻²¹ mol), less than about 100 zM, less than about 10 zM, less than about 5 zM, less than about 1 zM, less than about 0.1 zM, or less.

In some cases, the detection limit (e.g., the lowest concentration of an analyte that can be determined in solution) is about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, about 1 fM, about 500 aM ( ^10⁻¹⁸ mol), about 100 aM, about 50 aM, about 10 aM, about 5 aM, about 1 aM, about 0.1 aM, about 500 zM ( ^10⁻²¹ mol), about 100 zM, about 50 zM, about 10 zM, about 5 zM, about 1 zM, about 0.1 zM or less. In some embodiments, the concentration of the analyte in the fluid sample, which can be determined substantially accurately, is between about 5000 fM and about 0.1 fM, between about 3000 fM and about 0.1 fM, between about 1000 fM and about 0.1 fM, between about 1000 fM and about 0.1 ozM, between about 100 fM and about 1 ozM, between about 100 a M and about 0.1 ozM or less.

The detection limit (e.g., the higher concentration of the analyte that can be determined in solution) is at least about 100 fM, at least about 1000 fM, at least about 10 pM (picomolar), at least about 100 pM, at least about 100 pM, at least about 10 nM (nanomolar), at least about 100 nM, at least about 1000 nM, at least about 10 μM, at least about 100 μM, at least about 1000 μM, at least about 10 mM, at least about 100 mM, at least about 1000 mM or greater.

In some cases, the presence and/or concentration of an analyte in a sample can be detected rapidly, typically within less than about 1 hour, for example, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 1 minute, or 30 seconds.

In some embodiments, at least some steps of the methods described herein can be performed on a digital microfluidic device, such as the device described in Section 3 below. In some embodiments, the methods of the invention are performed using a digital microfluidic device combined with a nanopore device. For example, the digital microfluidic device and the nanopore device can be separate devices, and droplets containing cleaved tags or dissociated aptamers can be generated in the microfluidic device and transported to the nanopore device. In some embodiments, droplets containing cleaved tags or dissociated aptamers can be aspirated from the microfluidic device and transported to the nanopore device using a user- or robot-operated pipette.

In some embodiments, the method of the present invention is implemented using a device in which a digital microfluidic module and a nanopore module are integrated, such as the device described below. In some embodiments, the digital microfluidic module and the nanopore module can be reversibly integrated. For example, the two modules can be physically combined to form an integrated device, and the device can then be separated into individual modules. In some embodiments, the method of the present invention is implemented using a disposable cartridge comprising a microfluidic module having an embedded nanopore module. Exemplary embodiments of the apparatus for performing the methods provided herein are further described in the next section.

In some cases, a microfluidic device or microfluidic module integrated with a nanoporous module (reversibly or completely) may include a first substrate and a second substrate arranged at intervals, wherein the first substrate and the second substrate are separated by a gap/space, and wherein at least the steps of contacting the sample with a first binding member, contacting the analyte with a second binding member, removing the second binding member of the analyte that is not bound to the first binding member, and cutting the label attached to the second binding member (which retains the analyte bound to the first binding member) are performed in the space/gap between the first substrate and the second substrate.

An exemplary embodiment of the method of the present invention includes: generating microdroplets of a sample, and combining the microdroplets of the sample with microdroplets containing a first binding member to generate a single microdroplet. The first binding member may be immobilized on a solid substrate such as beads (e.g., magnetic beads). The single microdroplet may be incubated for a time sufficient to allow the first binding member to bind with the analyte present in the sample microdroplet. Optionally, the single microdroplet may be stirred to promote mixing of the sample with the first binding member. Mixing may be achieved by moving the single microdroplet back and forth, moving the single microdroplet around multiple electrodes, splitting and then merging the microdroplets, or using SAW, etc. The single microdroplet may then be subjected to magnetic force to retain the beads in one position in the device, while the microdroplet may move away and be replaced by a microdroplet containing a second binding member. An optional washing step may be performed before adding the second binding member by moving the microdroplet of washing buffer to the position where the beads are retained using magnetic force. After a sufficient period of time for the second binding member to bind to the analyte bound to the first binding member, the droplet containing the second binding member can be moved away, while the bead remains in the first position. The bead can be washed with a droplet of washing buffer, followed by contact with a droplet containing a cleaving agent to cleave the tag attached to the second binding member. In embodiments where the tag is attached to the second binding member via a photocuttable connector, the bead can be exposed to light of an appropriate wavelength to cleave the connector. In some cases, the bead can be exposed to a droplet of buffer before cleaving the photocuttable connector. Optionally, after a washing step to remove any unbound second binding members, the droplet containing buffer can be left covering the bead, the magnetic force holding the bead in the first position can be removed, and the buffer droplet containing the bead can be moved to a second position where photocutting is possible. The droplet containing the cleaved tag can then be moved to the nanoporous device or nanoporous module portion of the integrated device. In embodiments using aptamers as the second binding member, after a washing step to remove any unbound aptamers, droplets containing buffer solution can be left covering the beads, the magnetic force holding the beads in the first position can be removed, and the buffer droplets containing the beads can be moved to a second position where aptamer dissociation can occur. In other embodiments, after the washing step, the beads can be exposed to droplets of reagent for dissociating the aptamers bound to the analyte. Droplets containing dissociated aptamers can be moved to nanopores while the beads are held in place using magnets. Droplets containing dissociated aptamers can be moved to the nanopore device or nanopore module portion of an integrated device.

In an alternative embodiment, the first binding member may be immobilized on the surface of the first or second substrate at the location in the gap/space. The step of contacting the sample with the first binding member may include moving a droplet of the sample to the location in the gap/space where the first binding member is immobilized. Subsequent steps may be substantially similar to those described above regarding first binding members immobilized on magnetic beads.

Following the cutting/dissociation step, microdroplets containing the cut tag/dissociated aptamer can be moved to the nanoporous device or nanoporous module of the integrated device. As noted above, the microdroplets can be moved using a liquid transfer system, such as a pipette. In some cases, the microfluidic module can be fluidly connected to the nanoporous module. This fluid connection can be achieved by connecting the microfluidic module to the nanoporous module via a channel, or reversibly or during the fabrication of the integrated device by placing the nanoporous module within the microfluidic module. Such devices are further described in the following sections.

In the above embodiments, optionally, after assembly, the droplets can be manipulated (e.g., moved back and forth, moved in a circular direction, oscillated, split/merged, exposed to SAW, etc.) to promote mixing of the sample with the assay reagents (e.g., first binding member, second binding member, etc.).

The movement of microdroplets in integrated microfluidic nanopore devices can be achieved using electrodynamic forces (e.g., electrowetting, dielectrophoresis, electrode-mediated, photoinduced electrowetting, electric field-mediated, and electrostatically driven), pressure, surface acoustic waves, etc. The forces used to move the microdroplets can be determined based on the details of the device, which are described in sections a) through g) below, and for the specific device described in section 3.

a) Multiplexing

The method may include one or more (or alternatively two or more) specific binding members to detect one or more (or alternatively two or more) target analytes in a multiplexed assay. Each of the one or more (or alternatively two or more) specific binding members binds to a different target analyte, and each specific binding member is labeled with a different tag and/or aptamer. For example, a first specific binding member binds to a first target analyte, a second specific binding member binds to a second target analyte, a third specific binding member binds to a third target analyte, etc., and the first specific binding member is labeled with a first tag and/or aptamer, the second specific binding member is labeled with a second tag and/or aptamer, the third specific binding member is labeled with a third tag and/or aptamer, etc. In some embodiments, a first condition causes the cleavage or release of a first tag (if the first specific binding member is tagged) or the dissociation or release of a first aptamer (if the first specific binding member is aptamer-tagged), a second condition causes the cleavage or release of a second tag (if the second specific binding member is tagged) or the dissociation or release of a second aptamer (if the second specific binding member is aptamer-tagged), a third condition causes the cleavage or release of a third tag (if the third specific binding member is tagged) or the dissociation or release of a third aptamer (if the third specific binding member is aptamer-tagged), and so on. In some embodiments, sample conditions can be varied at different times during the assay, thereby allowing the detection of a first tag or aptamer, a second tag or aptamer, a third tag or aptamer, etc., thereby detecting one or more (or alternatively two or more) target analytes. In some embodiments, based on the residence time in the nanopore, the order of magnitude of the current impedance, or a combination thereof, one or more (or alternatively two or more) cleaved tags and/or dissociated aptamers are simultaneously detected through the pore.

b) Exemplary target analytes

Those skilled in the art will understand that, using the methods and apparatus of the present invention, any analyte that can be specifically bound by a first binding member and a second binding member can be detected and optionally quantified.

In some embodiments, the analyte may be a biomolecule. Non-limiting examples of biomolecules include macromolecules, such as proteins, lipids, and carbohydrates. In some cases, the analyte may be a hormone, antibody, growth factor, cytokine, enzyme, receptor (e.g., nerve, hormone, nutrient, and cell surface receptors) or their ligands, cancer markers (e.g., PSA, TNF-α), markers of myocardial infarction (e.g., troponin, creatine kinase, etc.), toxin, drug (e.g., addictive drugs), metabolizer (e.g., including vitamins), etc. Non-limiting embodiments of protein analytes include peptides, polypeptides, protein fragments, protein complexes, fusion proteins, recombinant proteins, phosphoproteins, glycoproteins, lipoproteins, etc.

In some embodiments, the analyte may be a post-translational modified protein (e.g., phosphorylated, methylated, or glycosylated protein), and the first or second binding member may be an antibody specific to the post-translational modification. The modified protein may bind to the first binding member immobilized on a solid support, wherein the first binding member binds to the modified protein but not to unmodified proteins. In other embodiments, the first binding member may bind to both unmodified and modified proteins, and the second binding member may be specific to post-translational modified proteins.

In some embodiments, the analyte may be cells, such as circulating tumor cells, pathogenic bacteria, viruses (including retroviruses, herpesviruses, adenoviruses, lentiviruses, filoviruses (Ebola virus), hepatitis viruses (e.g., hepatitis A, B, C, D, and E); HPV; spores, etc.

A non-limiting list of analytes that can be analyzed using the methods presented herein includes Aβ42 amyloid-β protein, fetoglobulin-A, tau, secretory granulin II, prions, α-synuclein, tau protein, neurofilament light chain, parkin, PTEN-induced putative kinase 1, DJ-1, leucine-rich repeat kinase 2, mutant ATP13A2, Apo H, ceruloplasmin, peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), transthyretin, vitamin D-binding protein, apoptosis-promoting kinase R (PKR) and its phosphorylated PKR (pP). KR), CXCL13, IL-12p40, CXCL13, IL-8, Dkk-3 (sperm), p14endocan fragment, serum, ACE2, autoantibody against CD25, hTERT, CAI25 (MUC 16), VEGF, sIL-2, osteopontin, human epididymal protein 4 (HE4), alpha-fetoprotein, albumin, albuminuria, microalbuminuria, neutrophil gelatinase-associated lipotransferase (NGAL), interleukin-18 (IL-18), kidney injury molecule-1 (KIM-1), liver fatty acid-binding protein (L-FABP), LMP1, BARF1 IL-8, Carcinoembryonic antigen (CEA), BRAF, CCNI, EGRF, FGF19, FRS2, GREB1 and LZTS1, α-amylase, CEA, CA 125, IL-8, thioredoxin, β-2 microglobulin levels - monitoring viral activity, tumor necrosis factor-α receptor - monitoring viral activity, CA15-3, follicle-stimulating hormone (FSH), luteinizing hormone (LH), T-cell lymphoma invasion and metastasis 1 (TIAM1), N-cadherin, EC39, bimodalin, deoxyuridine triphosphatase, secretory coagulant protein (pGSN), PSA (prostate-specific antigen), thyroxine, thoraxine, thoraxine, β-2-microglobulin levels - monitoring viral activity, tumor necrosis factor-α receptor - monitoring viral activity, CA15-3, follicle-stimulating hormone (FSH), luteinizing hormone (LH), T-cell lymphoma invasion and metastasis 1 (TIAM1), N-cadherin, EC39, bimodalin, deoxyuridine triphosphatase, secretory coagulant protein (pGSN), PSA (prostate-specific antigen), thyroxine, thora ... Adenosine β15, insulin, plasma C-peptide, glycosylated hemoglobin (HBA1c), C-reactive protein (CRP), interleukin-6 (IL-6), ARHGDIB (Rho GDP dissociation inhibitor 2), CFL1 (filoprotein-1), PFN1 (profilin-1), GSTP1 (glutathione S-transferase P), S100A11 (protein S100-A11), PRDX6 (peroxoreductin-6), HSPE1 (10kDa heat shock protein, mitochondrial), LYZ (lysozyme C precursor), GPI (glucose-6-phosphate isomerase), HIST2H2A A (histone H2A type 2-A), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), HSPG2 (basement membrane-specific heparan sulfate proteoglycan core protein precursor), LGALS3BP (galactoglobin-3-binding protein precursor), CTSD (cathepsin D precursor), APOE (apolipoprotein E precursor), IQGAP1 (Ras GTPase-activator-like protein IQGAP1), CP (ceruloplasmin precursor) and IGLC2 (IGLC1 protein), PCDGF/GP88, EGFR, HER2, MUC4, IGF-IR, p27 (kip1), Akt, HER3, HER4, PTEN, PIK3CA, SHIP, Grb2, Gab2, PDK-1 (phosphoinositol 3-phosphokinase-1), TSC1, TSC2, mTOR, MIG-6 (ERBB receptor feedback inhibitor 1), S6K, src, KRAS, MEK mitogen-activated protein kinase 1, cMYC, TOPO II topoisomerase (DNA) II α 170kDa, FRAP1, NRG1, ESR1, ESR2, PGR, CDKN1B, MAP2K1, NEDD4-1, FOXO3A, PPP1R1B, PXN, E LA2, CTNNB1, AR, EPHB2, KLF6, ANXA7, NKX3-1, PITX2, MKI67, PHLPP, adiponectin (ADIPOQ), fibrinogen α chain (FGA), leptin (LEP), advanced glycation end products-specific receptor (AGER aka RAGE), α-2-HS-glycoprotein (AHSG), angiopoietin (ANG), CD14 molecule (CD14), ferritin (FTH1), insulin-like growth factor binding protein 1 (IGFBP1), interleukin 2 receptor, α (IL2RA), vascular cell adhesion molecule 1 (VCAM1) And Von Willebrand factor (VWF), myeloperoxidase (MPO), IL1α, TNFα, perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA), lactoferrin, calprotectin, Wilmanoma-1 protein, aquaporin-1, MLL3, AMBP, VDAC1, E. coli enterotoxin (heat unstable exotoxin, heat stable enterotoxin), influenza HA antigen, tetanus toxin, diphtheria toxin, botulinum toxin, Shiga toxin, Shiga-like toxin I, Shiga-like toxin II, Clostridium difficile toxin A and B, etc.

Exemplary targets of nucleic acid aptamers that can be measured in samples (such as environmental samples, biological samples obtained from patients or subjects in need) using the subject-specific methods and apparatus include: abused drugs (e.g., cocaine), protein biomarkers (including, but not limited to, nucleolin, nuclear factor-κB essential regulator (NEMO), CD-30, protein tyrosine kinase 7 (PTK7), vascular endothelial growth factor (VEGF), MUC1 glycoform, immunoglobulin μ heavy chain (IGHM), immunoglobulin E, αvβ3 integrin, α-coagulation... Enzymes, HIV gp120, NF-κB, E2F transcription factor, HER3, plasminogen activator inhibitor, tendonin C, CXCL12/SDF-1, prostate-specific membrane antigen (PSMA), gastric cancer cells, HGC-27); cells (including but not limited to, non-small cell lung cancer (NSCLC), colorectal cancer cells, (DLD-1), H23 lung adenocarcinoma cells, Ramos cells, T-cell acute lymphoblastic leukemia (T-ALL) cells, CCRF-CEM, acute myeloid leukemia AML cells (HL60), small cell lung cancer (SCLC) cells, NCIH69, human glioblastoma cells, U118-MG, PC-3 cells, HER-2 overexpressing human breast cancer cells, SK-BR-3, pancreatic cancer cell line (Mia-PaCa-2)); and infectious pathogens (including but not limited to Mycobacterium tuberculosis, Staphylococcus aureus, Shigella dysenteriae, Escherichia coli O157:H7, Campylobacter jejuni, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella O8, and Salmonella enteritidis).

Exemplary targets of protein or peptide aptamers that can be measured in samples obtained from patients or subjects in need using the subject-specific methods and apparatus include, but are not limited to: HBV nucleocapsid protein, CDK2, E2F transcription factor, thymidine synthase, Ras, EB1, and receptor for advanced glycation end products (RAGE). Reviews of aptamers, their applications, and manufacturing methods are available in, for example, Shum et al., J Cancer Ther. 20134:872; Zhang et al., Curr Med Chem. 2011; 18:4185; Zhu et al., Chem Commun (Camb). 201248:10472; Crawford et al., Brief Function Genomic Proteomic. 20032:72; Reverdatto et al., PLoS One. 20138:e65180.

c) Sample

As used herein, “sample,” “test sample,” and “biological sample” refer to fluid samples containing or suspected of containing the target analyte. The sample may originate from any suitable source. In some cases, the sample may comprise a liquid, a flowing particulate solid, or a fluid suspension of solid particles. In some cases, the sample may be treated prior to the analysis described herein. For example, the sample may be isolated or purified from its source prior to analysis; however, in some embodiments, the untreated sample containing the analyte may be directly measured. The source of the analyte molecule may be synthetic (e.g., produced in a laboratory), environmental (e.g., air, soil, fluid samples, e.g., water supply), animal (e.g., mammal), plant, or any combination thereof. In one particular embodiment, the source of the analyte is human matter (e.g., bodily fluids, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, lymph, amniotic fluid, interstitial fluid, bronchoalveolar lavage fluid, cerebrospinal fluid, feces, tissue, organ, etc.). Tissues may include, but are not limited to, skeletal muscle tissue, liver tissue, lung tissue, kidney tissue, myocardial tissue, brain tissue, bone marrow, cervical tissue, skin, etc. The sample may be a liquid sample or a liquid extract of a solid sample. In some cases, the sample may be derived from an organ or tissue, such as a biopsy sample, which can be dissolved through tissue disintegration/cell lysis.

Fluid samples with a wide range of volumes can be analyzed. In several exemplary embodiments, the sample volume can be about 0.5 nL, about 1 nL, about 3 nL, about 0.01 μL, about 0.1 μL, about 1 μL, about 5 μL, about 10 μL, about 100 μL, about 1 mL, about 5 mL, about 10 mL, etc. In some cases, the volume of the fluid sample is between about 0.01 μL and about 10 mL, between about 0.01 μL and about 1 mL, between about 0.01 μL and about 100 μL, or between about 0.1 μL and about 10 μL.

In some cases, the fluid sample may be diluted before use in the assay. For example, in embodiments where the analyte molecules are derived from human fluids (e.g., blood, serum), the fluid may be diluted with a suitable solvent (e.g., a buffer such as PBS buffer). The fluid sample may be diluted by approximately 1, 2, 3, 4, 5, 6, 10, 100 times, or more before use.

In some cases, the sample may undergo pre-analytical processing. Pre-analytical processing can provide additional functionality such as non-specific protein removal and/or efficient, still inexpensive, mixed functionality. General methods for pre-analytical processing may include the use of electrokinetic trapping, AC electrokinetics, surface acoustic waves, isovelocity electrophoresis, dielectric electrophoresis, electrophoresis, or other pre-concentration techniques known in the art. In some cases, fluid samples may be concentrated prior to use in an assay. For example, in embodiments where the analyte molecules are derived from human fluids (e.g., blood, serum), the fluid may be concentrated by precipitation, evaporation, filtration, centrifugation, or a combination thereof. Fluid samples may be concentrated by about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold, or more prior to use.

In some implementations, the analyte is not amplified before measurement (i.e., the copy number of the analyte is not increased). For example, where the analyte is DNA or RNA, the analyte is not copied to increase its copy number. In some cases, the analyte is a protein or a small molecule.

d) Specific binding members

Those skilled in the art will understand that the binding members will depend on the analyte being analyzed. The binding members of many target molecules are known, or can be readily discovered or developed using known techniques. For example, when the target analyte is a protein, the binding member may include proteins, particularly antibodies or fragments thereof derived from camels (e.g., antigen-binding fragments (Fab), Fab' fragments, F(ab') ₂ fragments, recombinant antibodies, chimeric antibodies, single-chain Fv (“scFv”), single-chain antibodies, single-domain antibodies such as variable heavy chain domains (“VHH”; also referred to as “VHH fragments”) (VHHs and methods for their preparation are described in Gottlin et al., Journal of Biomolecular Screening, 14:77-85 (2009)), recombinant VHH single-domain antibodies and V _NAR fragments, disulfide-linked Fv (“sdFv”) and anti-idiotype (“anti-Id”) antibodies and functionally active epitope-binding fragments of any of the above, full-length polyclonal or monoclonal antibodies, antibody-like fragments, etc.), other proteins such as receptor proteins, protein A, protein C, etc. In cases where the analyte is a small molecule (e.g., steroids, ketones, retinoids, and lipids), the first binding member and/or the second binding member may be a scaffold protein (e.g., a lipid transporter) or a receptor. In some cases, the binding member for a protein analyte may be a peptide. For example, when the target analyte is an enzyme, suitable binding members may include an enzyme substrate and/or an enzyme inhibitor, which may be a peptide, a small molecule, etc. In some cases, when the target analyte is a phosphorylated substance, the binding member may comprise a phosphate binder. For example, the phosphate binder may comprise metal ion affinity mediators such as those described in U.S. Patent No. 7,070,921 and U.S. Patent Application No. 20060121544.

In some cases, at least one of the binding members may be an aptamer, such as those described in U.S. Patent Nos. 5,270,163, 5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867, and 5,705,337. Nucleic acid aptamers (e.g., single-stranded DNA or single-stranded RNA molecules) can be developed to capture virtually any target molecule. Aptamers bind to target molecules in a highly specific, conformation-dependent manner, typically with very high affinity, but aptamers with lower binding affinity can also be selected. Aptamers can be distinguished between target analyte molecules based on very small structural differences, such as the presence or absence of a methyl or hydroxyl group, and some aptamers can be distinguished between D- and L-enantiomers and diastereomers. Aptamers can bind to small molecule targets, including drugs, metal ions and organodyes, peptides, biotin, and proteins. Aptamers can retain their functional activity after biotinylation, fluorescein labeling, and when attached to glass surfaces and microspheres.

Nucleic acid aptamers are oligonucleotides that can be single-stranded oligodeoxynucleotides, oligoribonucleotides, or modified oligodeoxynucleotides or oligoribonucleotides. "Modification" includes nucleotides having covalently modified bases and/or sugars. For example, modified nucleotides include nucleotides having sugars covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3′ position and a phosphate ester group at the 5′ position. Thus, modified nucleotides may also include 2′-substituted sugars such as 2′-O-methyl-; 2-O-alkyl; 2-O-allyl; 2′-S-alkyl; 2′-S-allyl; 2′-fluoro-; 2′-halogenated or 2-azido-ribose, carbocyclic sugar analogs, α-terminal isomers; and epimeric sugars such as arabinose, xylose or lythose, pyranose, furanose, and sedoheptulose. In some embodiments, the binding member comprises a nucleic acid containing the nucleotide sequence shown in any one of SEQ ID NO: 1-11.

Peptide aptamers can be designed to interfere with protein-protein interactions. An aptamer can be a protein scaffold with a variable peptide ring attached thereto, thereby restricting the conformation of the aptamer. In some cases, the scaffold portion of the aptamer is derived from bacterial thioredoxin A (TrxA).

When the target molecule is a carbohydrate, potentially suitable capture components (as defined herein) include, for example, antibodies, lectins, and selectins. Those skilled in the art will appreciate that any molecule that can specifically bind to the target molecule can potentially be used as a binding member.

For certain implementation schemes, suitable target analytes/binding member complexes may include, but are not limited to, antibodies/antigens, antigens/antibodies, receptors/ligands, ligands/receptors, proteins/nucleic acids, enzymes/bases and/or inhibitors, carbohydrates (including glycoproteins and glycolipids)/lectins and/or selectins, proteins/proteins, proteins/small molecules, etc.

In one particular embodiment, the first binding member can be attached to a solid support via a link, the link potentially comprising any portion of the support and/or the binding member that facilitates attachment of the binding member to the support, functionalization, or modification. The link between the binding member and the support can comprise one or more chemical or physical (e.g., non-specific attachment via van der Waals forces, hydrogen bonding, electrostatic interactions, hydrophobic/hydrophilic interactions, etc.) bonds and/or chemical spacers providing such bonds.

In some embodiments, the solid support may also comprise a protective, blocking, or passivating layer that can eliminate or reduce nonspecific adhesion of non-capturing components (e.g., analyte molecules, binding members) to the binding surface during assaying, which may lead to false positives or signal loss during detection. Examples of materials that may be used to form the passivating layer in some embodiments include, but are not limited to: polymers, such as polyethylene glycol, which repel nonspecific binding of proteins; naturally occurring proteins with this property, such as serum albumin and casein; surfactants, such as zwitterionic surfactants, such as sulfobetaine; naturally occurring long-chain lipids; polymer brushes; and nucleic acids, such as salmon sperm DNA.

Some embodiments utilize binding members of proteins or peptides. As is known in the art, peptides can be attached to a variety of solid supports using any number of techniques. Various techniques for adding reactive portions to proteins are known, for example, the method described in U.S. Patent No. 5,620,850. Furthermore, methods for attaching proteins to surfaces are known, for example, see Heller, Acc. Chem. Res. 23:128 (1990).

As explained herein, the binding between the binding member and the analyte is specific (e.g., when the binding member and the analyte are complementary portions of a binding pair). In some embodiments, the binding member specifically binds the analyte. “Specifically binding” or “binding specificity” means that the binding member specifically binds to the analyte molecule in a manner sufficient to distinguish it from other components or contaminants in the test sample. For example, according to one embodiment, the binding member may be an antibody that specifically binds to an epitope on the analyte. According to one embodiment, the antibody may be any antibody capable of specifically binding to a target analyte. For example, suitable antibodies include, but are not limited to, monoclonal antibodies, bispecific antibodies, microbodies, domain antibodies (dAbs) (e.g., as described in Holt et al. (2014) Trends in Biotechnology 21:484-490), and include single-domain antibodies sdAbs that are naturally occurring, such as in cartilaginous fish and camelids, or that are synthetic, such as nanobodies, VHHs, or other domain structures), synthetic antibodies (sometimes referred to as antibody mimics), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as “antibody conjugates”), and fragments of each. As another embodiment, the analyte molecule may be an antibody, and the first binding member may be an antigen and the second binding member may be a second antibody that specifically binds to the target antibody, or the first binding member may be a second antibody that specifically binds to the target antibody and the second binding member may be an antigen.

In some embodiments, the binding member may be a chemically programmed antibody (cpAb) (described in Rader (2014) Trends in Biotechnology 32:186-197), a bispecific cpAb, an antibody recruitment molecule (ARM) (described in McEnaney et al. (2012) ACS Chem. Biol. 7:1139-1151), a branched trapping agent such as a triligand trapping agent (described in Millward et al. (2011) J. Am. Chem. Soc. 133:18280-18288), an engineered binding protein derived from a non-antibody scaffold such as a monoclonal antibody (derived from the tenth fibronectin type III domain of human fibronectin), an affinity protein (derived from immunoglobulin-binding protein A), DARPins (based on ankyrin repeat modules), and anticalins (derived from lipid transport proteins post-cholechrome-binding protein and human...). Lipid transporter 2) and cysteine knottin (described in Gilbreth and Koide, (2012) Current Opinion in Structural Biology 22:1-8; Banta et al. (2013) Annu. Rev. Biomed. Eng. 15:93-113), WW domain (described in Patel et al. (2013) Protein Engineering, Design & Selection 26(4):307-314), target-resetting receptor ligand, affitins (described in Béhar et al. (2013) 26:267-275) and/or adhirons (described in Tiede et al. (2014) Protein Engineering, Design & Selection 27:145-155).

According to an embodiment in which the analyte is a biological cell (e.g., mammal, bird, reptile, other vertebrate, insect, yeast, bacteria, cell, etc.), the binding member can be a ligand with specific affinity for cell surface antigens (e.g., cell surface receptors). In one embodiment, the binding member can be an adhesion molecule receptor or a portion thereof, which has binding specificity to cell adhesion molecules expressed on the surface of a target cell type. In use, the adhesion molecule receptor binds to adhesion molecules on the extracellular surface of a target cell, thereby immobilizing or capturing the cell, and the bound cell can then be detected using a second binding member, which may be the same as the first binding member or may bind to different molecules expressed on the cell surface.

In some embodiments, the binding affinity between the analyte molecule and its binding member should be sufficient to maintain binding under assay conditions, including a washing step to remove non-specifically bound molecules or particles. In some cases, such as in the detection of certain biomolecules, the binding constant of the analyte molecule to its complementary binding member may be between at least about ^10⁴ and about ^{10⁶ M⁻¹} , at least about ^10⁵ and about ^{10⁹ M⁻¹} , at least about ^10⁷ and about ^{10⁹ M⁻¹} , greater than about ^{10⁹ M⁻¹} , or greater.

e) Labels or markings

The methods described herein may include specific binding members that bind to tags (such as markers) to analyze analytes by impedance. The incorporated tags or markers do not materially interfere with the reaction protocol. For example, the incorporated tags or markers do not interfere with the binding constant or interaction between the analyte and its complementary binding member. The size and number of incorporated tags or markers may be related to capture rate and readout rate. Capture rate and readout rate can be increased by increasing the size and/or number of incorporated tags or markers. For example, the size and number of incorporated tags or markers can increase the charge and increase the capture area of the nanopore. The incorporated tags or markers do not alter the binding member kinetics (e.g., antibody kinetics) or reaction protocol. Exemplary tags include: polymers, such as anionic or cationic polymers (e.g., peptides with a net positive charge, such as polyhistidine or polylysine), wherein said polymers are about 5-1000 residues in length; proteins (e.g., globular proteins) that do not cross-react with binding members and/or interfere with the assay; dendritic polymers, such as DNA dendritic polymers; and charged nanoparticles, such as nanobeads. Polymer tags may include nucleic acids, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Polymer tags may also include nucleoside base polymers. In some cases, the tag may be a DNA or RNA aptamer, wherein the aptamer does not bind to the analyte. In the case where the tag is an aptamer, it may optionally be denatured before transfer through a nanopore. The polymer tag or nanoparticle (e.g., nanobeads) may be large enough to generate a reproducible signal as it transfers through or across a nanopore. The aptamer may be 20-220 bases in length, for example, 20-60 bases. The size of the nanoparticles (e.g., nanobeads or dendritic polymers) can range from about 1 nm to about 950 nm in diameter, for example, 10 nm-900 nm, 20 nm-800 nm, 30 nm-700 nm, 50 nm-600 nm, 80 nm-500 nm, 100 nm-500 nm, 200 nm-500 nm, 300 nm-500 nm, or 400 nm-500 nm in diameter, for example, 10 nm, 20 nm, 30 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, or 900 nm. When used as tags, the preferred size of the nanoparticles is one that can pass through or traverse nanopores (as further described herein). In some cases, the nanobeads/nanoparticles may be made of materials with a net negative or positive charge, or may be processed to have a net negative or positive charge. Exemplary nanobeads/nanoparticles include those made of organic or inorganic polymers. Organic polymers include polymers such as polystyrene, carbon, polyacrylamide, etc. Inorganic polymers include silicon or metal nanobeads/nanoparticles. In some cases, the nanobeads/nanoparticles may not be magnetic.

In some cases, the tag may be single-stranded DNA or RNA. The single-stranded DNA or RNA may hybridize with probe molecules before being transferred through or across the nanopore. In some cases, the method may include analyzing multiple analytes in a single sample. A second binding member binding to different analytes in the sample may include different single-stranded DNA or RNA to which it is attached as a tag, and the different single-stranded DNA or RNA may hybridize with different probes, which further distinguish the different single-stranded DNA or RNA from each other as they pass through the nanopore. In other embodiments, tags attached to different second binding members may have different hairpin structures (e.g., the length of the hairpin structure) that are distinguishable when the tag passes through or across the nanopore. In another embodiment, tags attached to different second binding members may have different lengths that are distinguishable when the tag passes through or across the nanopore—for example, the tag may be a double-stranded DNA of different lengths (e.g., 25 base pairs, 50 base pairs, 75 base pairs, 100 base pairs, 150 base pairs, 200 base pairs or more). In some cases, tags attached to different second binding members can have different polyethylene glycol (PEG) lengths, or can be DNA or RNA that has been differentially modified with PEG.

It should be noted that references to tags or tag molecules include single tags or single-tag molecules as well as multiple tags (all of which may be identical). It should also be noted that the nanopores include single nanopores present in a single layer (e.g., substrate, membrane, etc.) as well as multiple nanopores. Thus, counting the number of tags transferred through or across nanopores in a layer/sheet/film represents counting multiple tags transferred through or across one or more nanopores in a layer/sheet/film. Nanopores may be present in a single layer (such as a substrate or membrane), which may be made of any suitable material that is electrically insulating or has high resistance, such as lipid bilayers, dielectric materials, such as silicon nitride and silicon dioxide, atomic-level thin films such as graphene, silicon, silicene, molybdenum disulfide ( _MoS₂ ), etc., or combinations thereof.

The label can be of any size or shape. In some embodiments, the label can be nanoparticles or nanobeads with a diameter of approximately 10 to 950 nm, for example, with diameters of 20-900 nm, 30-800 nm, 40-700 nm, 50-600 nm, 60-500 nm, 70-400 nm, 80-300 nm, 90-200 nm, 100-150 nm, 200-600 nm, 400-500 nm, 2-10 nm, 2-4 nm, or 3-4 nm. The label can be substantially spherical, such as spherical beads or nanobeads, or hemispherical. The tag can be a protein of about 0.5 kDa to about 50 kDa, for example, about 0.5 kDa to about 400 kDa, about 0.8 kDa to about 400 kDa, about 1.0 kDa to about 400 kDa, about 1.5 kDa to about 400 kDa, about 2.0 kDa to about 400 kDa, about 5 kDa to about 400 kDa, about 10 kDa to about 400 kDa, about 50 kDa to about 400 kDa, about 100 kDa to about 400 kDa, about 150 kDa to about 400 kDa, about 200 kDa to about 400 kDa, about 250 kDa to about 400 kDa, about 300 kDa to about 400 kDa, about 0.5 kDa to about 300 kDa, about 0.8 kDa to about 300 kDa, about 1.0 kDa to about 300 kDa, about 1.5 kDa to about 400 kDa. Approximately 300kDa, approximately 2.0kDa to approximately 300kDa, approximately 5kDa to approximately 300kDa, approximately 10kDa to approximately 300kDa, approximately 50kDa to approximately 300kDa, approximately 100kDa to approximately 300kDa, approximately 150kDa to approximately 300kDa, approximately 200kDa to approximately 300kDa, approximately 250kDa to approximately 300kDa, approximately 0.5kDa to approximately 250kDa, approximately 0.8kDa to approximately 250kDa, approximately 1.0kDa to approximately 250kDa, approximately 1.5kDa to approximately 250kDa, approximately 2.0kDa to approximately 250kDa in size, approximately 5kDa to approximately 250kDa, approximately 10kDa to approximately 250kDa, approximately 50kDa to approximately 250kDa, approximately 100kDa to approximately 250kDa, approximately 150kDa to approximately 2 50kDa, approximately 200kDa to approximately 250kDa, approximately 0.5kDa to approximately 200kDa, approximately 0.8kDa to approximately 200kDa, approximately 1.0kDa to approximately 200kDa, approximately 1.5kDa to approximately 200kDa, approximately 2.0kDa to approximately 200kDa in size, approximately 5kDa to approximately 200kDa, approximately 10kDa to approximately 200kDa, approximately 50kDa to approximately 200kDa, approximately 100kDa to approximately 200kDa, approximately 150kDa to approximately 200kDa, approximately 0.5kDa to approximately 100kDa, approximately 0.8kDa to approximately 100kDa, approximately 1.0kDa to approximately 100kDa, approximately 1.5kDa to approximately 100kDa, approximately 2.0kDa to approximately 100kDa, approximately 5kDa to approximately 100kDa, approximately 10kDa to approximately 10 0 kDa, approximately 50 kDa to approximately 100 kDa, approximately 0.5 kDa to approximately 50 kDa, approximately 0.8 kDa to approximately 50 kDa, approximately 1.0 kDa to approximately 50 kDa, approximately 1.5 kDa to approximately 50 kDa, approximately 2.0 kDa to approximately 50 kDa, approximately 5 kDa to approximately 50 kDa, approximately 10 kDa to approximately 50 kDa, approximately 10 kDa to approximately 90 kDa, approximately 10 kDa to approximately 80 kDa, approximately 10 kDa to approximately 70 kDa, approximately 10 kDa to approximately 60 kDa, approximately 20 kDa to approximately 90 kDa, approximately 20 kDa to approximately 80 kDa, approximately 20 kDa to approximately 70 kDa, approximately 20 kDa to approximately 60 kDa, approximately 40 kDa to approximately 90 kDa, approximately 40 kDa to approximately 80 kDa, approximately 40 kDa to approximately 70 kDa, or approximately 40 kDa to approximately 60 kDa.

In some embodiments, the tag may be a nanoparticle. As noted herein, the nanoparticle may be reversibly (e.g., cleavably) attached to a second binding member. In some aspects, the nanoparticle may be a nanobead of a defined diameter, the defined diameter being a property of the nanobead measured through a nanoporous layer. In certain cases, the methods, systems, and apparatus of the present invention can be used to simultaneously analyze multiple different analytes in a sample. For such analyses, multiple second binding members, each specifically binding to a corresponding analyte, may be used. Each of the different second binding members may be attached to nanobeads of different sizes, which may be used to identify the second binding member. For example, the different nanobead tags can have different diameters, such as 1nm, 2nm, 4nm, 6nm, 8nm, 10nm, 12nm, 14nm or larger, such as up-20nm, 30nm, 50nm, 100nm, 200nm, 300nm, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, 950nm or 990nm.

In some embodiments, nanobeads of different diameters can all transfer through a nanopore layer having nanopores of a single diameter, wherein nanobeads of different sizes can be identified based on residence time in the nanopore, the order of magnitude of current impedance, or a combination thereof. In some cases, a stacked nanopore layer device containing multiple nanopore layers (where the first layer may have nanopores of a first diameter and the second layer may have nanopores of a second diameter) can be used to detect and count nanobeads transferring through or across the nanopores. The multiple nanopore layers can be arranged in a specific manner such that a layer with a larger diameter nanopore is upstream of a layer with a smaller diameter nanopore. An exemplary stacked nanopore layer is disclosed in US20120080361.

Exemplary nanoparticles that can be used as labels in the methods of the present invention include gold nanoparticles or polystyrene nanoparticles in the diameter range of 5 nm to 950 nm.

In some cases, the tag may be a polymer, such as a nucleic acid. The presence of the tag can be determined by detecting tag-specific signals, such as those related to the size or length of the polymer tag. The size or length of the polymer tag can be determined by measuring its residence time in a pore or channel, for example, by measuring the duration of a brief interruption of current.

Elements that can be part of, in combination with, or attached to a label or mark include: nanoparticles; gold particles; silver particles; coatings or deposits of silver, copper, zinc, or other metals; polymers; drag tags (as defined herein); magnetic particles; floatable particles; metal particles; charged portions; dielectric electrophoretic tags with and without impurities, such as silica (e.g., quartz, glass, etc.); polymethyl methacrylate (PMMA); polyimide; silicon nitride; gold; silver; quantum dots (including CdS quantum dots); carbon dots; fluorophores; quenchers; polymers; polystyrene; Janus particles; scattering particles; fluorescent particles; phosphorescent particles; spheres; cubes; insulators; conductors; Body; barcode-labeled or marked particles; porous particles; solid particles; nanoshells; nanorods; microspheres; analytes such as viruses, cells, parasites, and organisms; nucleic acids; proteins; molecular recognition elements; spacers; PEG; dendritic polymers; charge-changing agents; magnetic materials; enzymes; DNA, including aptamer sequences; amplifiable DNA; repetitive DNA sequences; fusions or conjugates of detectable elements with molecular recognition elements (e.g., engineered binding members); anti-antibody aptamers; aptamers against antibody-binding proteins; absorbable or adsorbed detectable compounds; heme; luciferin; phosphor; azide or alkyne (e.g., terminal or non-terminal alkynes) or other click chemical participants.

In some embodiments, the tag can be selected to provide a capture rate high enough to enable rapid analysis of the sample. In some embodiments, the capture rate of the tag can be about 1 event/10 sec, 1 event/5 sec, 1 event/sec, or higher. In some embodiments, linear polymer tags can be used, such as ribopolymers, deoxyribopolymers, oligonucleotides, DNA, or RNA. Typically, for a 1 nM DNA solution, using solid-state nanopores ( _Si₃N₄ ), a zero salt gradient, a voltage of 200–800 _mV , and a salt (KCl) concentration of 1 M, the capture rate is approximately 1 event/sec.

In some cases, linear polymer tags (e.g., ribopolymers, deoxyribopolymers, oligonucleotides, DNA, or RNA) cannot be used because their capture rates may be too low for certain applications. Tags with hemispherical, spherical, or substantially spherical shapes transfer rapidly through nanopores, thus shortening the assay duration usable in applications requiring faster tag counting. In some cases, the size of the spherical or hemispherical tag can be selected based on the capture rate required for the assay. For example, larger spherical or hemispherical tags can be selected for higher capture rates. In some cases, the tag can be a spherical tag, for example, nanoparticles/nanobeads having a capture rate approximately 10, 30, 50, 100, 300, 500, or 1000 times faster than linear tags (e.g., DNA tags) under the same measurement conditions.

In some embodiments, the tag may be conjugated to an antibody, such as a CPSP antibody conjugate. In some embodiments, the tag may be conjugated to an antibody having a spacer, such as a CPSP antibody conjugate with a spacer. In some embodiments, the tag may be conjugated to an oligonucleotide and an antibody, such as a CPSP oligonucleotide-antibody conjugate. In some embodiments, the tag may be conjugated to an oligonucleotide, such as a CPSP oligonucleotide conjugate. In some embodiments, the spacer comprises nitrobenzyl, dithioethylamino, a 6-carbon spacer, a 12-carbon spacer, or 3-(9-((3-carboxypropyl)(toluenesulfonyl)carbamoyl)acridin-10-onthiol-10-yl)propane-1-sulfonate. In some embodiments, the spacer comprises nitrobenzyl, and the tag is a DNA molecule. In some embodiments, the spacer is a dithioethylamino group, and the tag is a carboxylated nanoparticle. In some embodiments, the spacer is 3-(9-((3-carboxypropyl)(toluenesulfonyl)carbamoyl)acridin-10-onthiol-10-yl)propane-1-sulfonate, and the tag is an oligonucleotide. In some embodiments, the spacer comprises a 6-carbon spacer or a 12-carbon spacer, and the tag is biotin.

f) Cuttable connector

The tag used in the methods described herein can be attached to a specific binding member via a universal adapter. A cleavable adapter ensures that the tag can be removed. The universal adapter can be a cleavable adapter. For example, the tag can be attached to a second binding member via a cleavable adapter. The complex of the first binding member-analyte-second binding member can be exposed to a cleaving agent mediating the cleavage of the cleavable adapter. The adapter can be cleaved by any suitable method, including exposure to acids, bases, nucleophiles, electrophiles, residues, metals, reducing or oxidizing agents, light, temperature, enzymes, etc. Suitable adapters can be modified from standard chemical blocking groups, as disclosed in Greene & Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons. Other suitable cleavable adapters for use in solid-phase synthesis are disclosed in Guillier et al. (Chem. Rev. 100:2092-2157, 2000). The adapter can be acid-cleavable, base-cleavable, or photo-cleavable. Redox reactions can be part of the cleavage scheme. The cuttable connector can be a charged polymer.

The connector can be optically cleavable, chemically cleavable, or thermally cleavable. In embodiments, the connector can be a heat-sensitive cleavable connector. When the connector is an optically cleavable group, the cleaving agent can be light of an appropriate wavelength that destroys or cleaves the optically cleavable group. In many embodiments, the wavelength range of the light used to cleave the optically cleavable linker is about 180 nm to 400 nm, for example, about 250 nm to 400 nm, or about 300 nm to 400 nm. Preferably, the light required to activate the cleavage does not affect other components of the analyte. Suitable connectors include those based on O-nitrobenzyl compounds and nitroveratrol compounds. Benzoin-based connectors can also be used (Lee et al., J. Org. Chem. 64:3454-3460, 1999). In some embodiments, the optically cleavable connector can be derived from the following:

Alternatively, when the linker is a chemically cleavable group, the cleaving agent can be a chemical reagent capable of cleaving said group. Chemically cleavable links can be cleaved by oxidation/reduction-based cleavage, acid-catalyzed cleavage, base-catalyzed cleavage, or nucleophilic substitution. For example, when the linker is a disulfide bond, the tag can be released using thiol-mediated cleavage with dithiothreitol or β-mercaptoethanol. In further embodiments where the linker is a restriction site, the reagent is a catalyst, such as an enzyme, which can be a hydrolase, a restriction enzyme, or another enzyme that cleaves the linker. For example, the restriction enzyme can be a type I, type II, type II, type III, or type IV restriction enzyme.

In some embodiments, the cleavage adapter is an enzyme-cleavable sequence. In one aspect of any embodiment herein, the enzyme-cleavable sequence is a nucleic acid sequence of 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In one embodiment, the enzyme-cleavable sequence comprises a sequence of at least 10 nucleotides. In one embodiment, the enzyme-cleavable sequence comprises a sequence between 2 and 20 nucleotides. In one embodiment, the enzyme-cleavable sequence comprises a sequence between 2 and 15 nucleotides. In one embodiment, the enzyme-cleavable sequence comprises a sequence between 4 and 10 nucleotides. In one embodiment, the enzyme-cleavable sequence comprises a sequence between 4 and 15 nucleotides.

For example, the cleavable linker may be an acridine, an ether such as a substituted benzyl ether or its derivative (e.g., benzyl hydroxyl ether, indane ether, etc.) (which can be cleaved under acidic or mild reducing conditions (e.g., hydrogen peroxide to produce acridine and sulfonamide)), a charged polymer produced by P-elimination (where a weak base can be used to release the product), an acetal, including its thio analogues (where separation is achieved by a weak acid, particularly in the presence of a carbonyl-capturing compound), a photolabile bond (e.g., O-nitrobenzoyl, 7-nitroindane, 2-nitrodiphenylmethyl ether or ester, etc.), or a peptide linker to which it is enzymatically hydrolyzed (e.g., an enzyme-cleavable linker), particularly where the enzyme recognizes a specific sequence, such as a peptide or enterokinase targeting factor Xa. Examples of joints include, but are not limited to, disulfide joints, acid-labile joints (including dialkoxybenzyl joints), Sieber joints, indole joints, tert-butyl Sieber joints, electrophilically cleavable joints, nucleophilically cleavable joints, photo-cleavable joints, cleavage under reducing conditions, cleavage under oxidizing conditions, cleavage by using a safety trap joint, and cleavage by an elimination mechanism.

Electrophilically cleaved linkers are typically proton-cleaved and include acid-sensitive cleavage. Suitable linkers include modified benzyl systems such as triphenylmethyl, p-alkoxybenzyl esters, and p-alkoxybenzylamides. Other suitable linkers include tert-butoxycarbonyl (Boc) groups and acetal systems. To prepare suitable linker molecules, the use of chalcophilic metals (such as nickel, silver, or mercury) in the cleavage of thioacetals or other sulfur-containing protecting groups can also be considered.

For nucleophilic cleavage, groups such as esters that are unstable in water (i.e., can be easily cleaved at alkaline pH) and groups that are unstable to non-aqueous nucleophiles can be used. Fluoride ions can be used to cleave silicon-oxygen bonds in groups such as triisopropylsilane (TIPS) or tert-butyldimethylsilane (TBDMS).

Reduction-sensitive junctions can be used in conjunction with processes such as disulfide bond reduction. Catalytic hydrogenation using palladium-based catalysts has been used to cleave benzyl and benzyloxycarbonyl groups.

Oxidation-based approaches are well known in the art. These include the oxidation of alkoxybenzyl groups and the oxidation of sulfur and selenium joints. Iodine solutions used for cleaving disulfide bonds and other sulfur- or selenium-based joints can also be used.

Safe capture connectors are those that cleave in two steps. In a preferred system, the first step is the generation of a reactive nucleophilic center, followed by a second step that involves intramolecular cyclization leading to cleavage. For example, the levulinate bond can be treated with hydrazine or photochemically to release an active amine, which can then be cyclized to cleave the ester elsewhere in the molecule (Burgess et al., J.Org.Chem.62:5165-5168, 1997).

Elimination reactions can also be used. For example, base-based elimination with groups such as Fmoc and cyanoethyl can be used, as well as palladium-catalyzed reductive elimination in allyl systems.

In cases where the connector is a thermally cleavable or heat-sensitive connector, the cleaving agent may be a localized temperature increase above a threshold to destroy or cleave the thermally cleavable group. In some embodiments, microwave radiation may be used to locally increase the temperature to induce particle overheating. Particle overheating methods, such as those reviewed in Dutz and Hergt (Nanotechnology, 25:452001 (2014)) and described in U.S. Patent No. 7,718,445, U.S. Patent Publication No. 20030082633, International Patent Publication No. WO 2002029076, and U.S. Patent Publication No. 20020197645, each of which is incorporated herein by reference. In some embodiments, the temperature increase may be photothermally achieved by transferring energy from light to an absorbing target (such as a dye, pigment, or water). In one aspect, the source of light is a laser. In some embodiments, the increased temperature may cause thermal separation of double-stranded DNA.

g) Nanoporous layer

In this invention, the detection and/or counting of the tags (e.g., polymers, aptamers, nanoparticles) can be accomplished by transferring the tags through or across nanopores or nanochannels. In some embodiments, the detection and/or counting of the tags (e.g., polymers, aptamers, nanoparticles) can be accomplished by transferring the tags through or across at least one or more nanopores or nanochannels. In some embodiments, at least one or more nanopores or nanochannels exist in parallel or in series. In some embodiments, the size of the nanopores or nanochannels is suitable for transferring no more than one tag at a time. Thus, in some embodiments, the size of the nanopores typically depends on the size of the tag to be examined. Tags with double-stranded regions may require larger nanopore sizes than those for transferring tags that are sufficiently single-stranded. Additionally, nanoparticle tags such as nanobead tags may require larger pores or channels than oligomeric tags. Typically, a pore with a diameter of about 1 nm can allow the passage of single-stranded polymers, while a pore size of 2 nm or larger will allow the passage of double-stranded nucleic acid molecules. In some embodiments, the nanopores or nanochannels are selectable for single-stranded tags (e.g., about 1 nm to less than 2 nm in diameter), while in other embodiments, the nanopores or nanochannels have a sufficient diameter to allow the passage of double-stranded polynucleotides (e.g., 2 nm or larger). The selected pore size provides the optimal signal-to-noise ratio for the target analyte.

In some embodiments, the aperture may be about 0.1 nm to about 1000 nm in diameter, about 50 nm to about 1000 nm, about 100 nm to 1000 nm, about 0.1 nm to about 700 nm, about 50 nm to about 700 nm, about 100 nm to 700 nm, about 0.1 nm to about 500 nm, about 50 nm to about 500 nm, or about 100 nm to 500 nm. For example, the aperture can be approximately 0.1 nm, approximately 0.2 nm, approximately 0.3 nm, approximately 0.4 nm, approximately 0.5 nm, approximately 0.6 nm, approximately 0.7 nm, approximately 0.8 nm, approximately 0.9 nm, approximately 1.0 nm, approximately 1.5 nm, approximately 2.0 nm, approximately 2.5 nm, approximately 3.0 nm, approximately 3.5 nm, approximately 4.0 nm, approximately 4.5 nm, approximately 5.0 nm, approximately 7.5 nm, approximately 10 nm, approximately 15 nm, approximately 20 nm, approximately 25 nm, approximately 30 nm, approximately 35 nm, approximately 40 nm, approximately 45 nm, approximately 50 nm. nm, approximately 55nm, approximately 60nm, approximately 65nm, approximately 70nm, approximately 75nm, approximately 80nm, approximately 85nm, approximately 90nm, approximately 95nm, approximately 100nm, approximately 150nm, approximately 200nm, approximately 250nm, approximately 300nm, approximately 3500nm, approximately 400nm, approximately 450nm, approximately 500nm, approximately 550nm, approximately 600nm, approximately 650nm, approximately 700nm, approximately 750nm, approximately 800nm, approximately 850nm, approximately 900nm, approximately 950nm, or approximately 1000nm diameter.

Generally, nanopores are shorter than nanochannels. Nanochannels are much longer than nanopores and are therefore useful in applications where it is desirable to increase the time required for molecules to transfer through them (relative to the time required to transfer through or across a nanopore of the same diameter). The length of nanopores can range from about 0.1 nm to less than about 200 nm. The length of nanochannels can range from about 500 nm to about 100 μm or longer. The diameters of nanopores and nanochannels can be similar.

Various types of nanopores can be used to analyze the tags/aptamers. These include, in particular, bio-nanopores employing biopores or channels embedded in a membrane. Another type of nanopore layer is the solid-state nanopore, wherein the channels or pores are entirely or partially made of a fabricated or sculpted solid component (such as silicon). In some embodiments, the nanopores are solid-state nanopores produced using controlled dielectric breakdown. In some embodiments, the nanopores are solid-state nanopores produced by methods other than controlled dielectric breakdown.

In some embodiments, the length of the nanopores can be up to about 200 nm, for example, about 0.1 nm to about 30 nm, about 10 to about 80 nm, about 1 to about 50 nm, about 0.1 nm to about 0.5 nm, about 0.3 nm to about 1 nm, about 1 nm to about 2 nm, about 0.3 nm to about 10 nm, or about 10 to about 30 nm. The number of nanopores in the nanopore layer can be about 1, 2, 3, 4, 5, 10, 30, 100, 300, 1000, 3000, 10000, 30000, 100000, 300000, or more. The center-to-center distance between the nanopores in the layer can be about 100 nm to about 300 nm, about 300 nm to about 500 nm, or about 500 nm to about 1000 nm, for example, 100 nm, 150 nm, 200 nm, or 300 nm.

In some embodiments, multiple nanopore layers (each containing one or more nanopores) may be arranged in series for detecting and/or counting the tags (e.g., polymers, aptamers, nanoparticles). In this case, detecting and/or counting the tags can be achieved by transferring the tags through or across each nanopore layer. Thus, counting the number of tags transferred through or across the nanopores in a layer/sheet/film represents counting multiple tags transferred through or across one or more nanopores in one or more layers/sheets/films. In some embodiments, when more than one nanopore layer is present (e.g., 1, 2, 3, 4, 5, 6, or other numbers of nanopore layers, as long as technically feasible), they are optionally present in series, wherein at least one nanopore in one layer is separated from or stacked on top of another nanopore in another layer (e.g., on top or on top), etc. In the case of the nanopore layers in series, at least two electrodes can be used to establish an electric field to drive the tags through the pores, and optionally, additional electrodes located between the nanopore layers can further provide a driving current.

i) Biopores

To detect and optionally count tags/aptamers, any biopore with a channel size that allows tag transfer can be used. Two main classes of biochannels are suitable for the methods disclosed herein. Non-voltage-gated channels allow molecules to pass through the pore without requiring a change in membrane potential to activate or open the channel. Voltage-gated channels, on the other hand, require a specific range of membrane potentials to activate the channel opening. Most studies using bionanopores have utilized α-hemolysin, a mushroom-shaped homooligomeric heptamer channel of approximately 10 nm in length found in Staphylococcus aureus. Each subunit contributes two β chains to form a 14-chain antiparallel β-tube. The pore formed by the β-tube structure has an entrance of approximately 2.6 nm in diameter, containing a lysine residue loop and opening into an inner lumen of approximately 3.6 nm in diameter. The backbone of the hemolysin pore (which penetrates the lipid bilayer) has an average inner diameter of approximately 2.0 nm and a 1.5 nm contraction between the lumen and the backbone. The size of the backbone is sufficient for single-stranded nucleic acids to pass through, but not for double-stranded nucleic acids. Therefore, α-hemolysin pores can be used as nanopores selective for single-chain polynucleotides and other polymers of similar size.

In other embodiments, the bio-nanopores have a size large enough for polymers to pass through, exceeding the size of single-stranded nucleic acids. An exemplary pore is mitochondrial porin, a voltage-dependent anion channel (VDAC) located in the outer mitochondrial membrane. Porins can be obtained in purified form and, when reconstituted into an artificial lipid bilayer, produce functional channels that allow double-stranded nucleic acids to pass through (Szabo et al., 1998, FASEB J.12:495-502). Structural studies suggest that porins also possess a β-tube structure containing 13 or 16 chains (Rauch et al., 1994, Biochem Biophys ResComm200:908-915). Porins exhibit greater conductivity compared to pores formed by α-hemolysin, maltoseporin (LamB), and bacitracin. The larger conductivity of porins supports the conclusion that porin channels have a size large enough for double-stranded nucleic acids to pass through. The estimated pore diameter of a porin molecule is 4 nm. It is estimated that the diameter of the unwound double-stranded nucleic acid is about 2 nm.

Another biological channel potentially suitable for scanning double-stranded polynucleotides is the channel found in Bacillus subtilis (Szabo et al., 1997, J. Biol. Chem. 272:25275-25282). Plasma membrane vesicles made from Bacillus subtilis and incorporated into artificial membranes allow the transmembrane passage of double-stranded DNA. The conductivity of channels formed from Bacillus subtilis membrane fabrications is similar to that of mitochondrial membrane porins. Although incomplete characterization of these channels exists (e.g., in purified forms), a purified form is not necessary for the purposes of this paper. Individual channels can be separated in a detection device by diluting the plasma membrane fabrication (by dissolving it in a suitable detergent or incorporating it into an artificial lipid membrane with sufficient surface area). Limiting the duration of contact between the membrane fabrication (or protein fabrication) and the artificial membrane through appropriately timed washing provides another method for incorporating individual channels into an artificial lipid bilayer. The channels incorporated into the bilayer can be characterized using conductivity properties.

In some cases, the nanopores can be hybrid nanopores, in which biological pores are introduced into solid nanopores, such as nanopores fabricated in non-biological materials. For example, α-hemolysin pores can be inserted into solid nanopores. In some cases, the nanopores can be hybrid nanopores as described in Hall et al., Nature Nanotechnology, November 28, 2010, Vol. 5, pp. 874-877.

ii) Solid-state holes

In other embodiments, tag analysis is performed by transferring the tag through or across nanopores or nanochannels made of non-biological materials. Many different techniques can be used to fabricate nanopores or nanochannels from a variety of solid materials, including in particular chemical deposition, electrochemical deposition, electroplating, electron beam engraving, ion beam engraving, nanolithography, chemical etching, laser ablation, focused ion beam, atomic layer deposition, and other methods well known in the art (see, for example, Li et al., 2001, Nature 412:166-169; and WO 2004/085609).

In a particular embodiment, the nanopore may be the nanopore described in WO13167952A1 or WO13167955A1. As described in WO13167952A1 or WO13167955A1, nanopores with accurate and uniform pore sizes can be formed by precisely enlarging the nanopores formed in the film. The method may include: enlarging the nanopore by applying a high potential across the nanopore; measuring the current flowing through the nanopore; determining the size of the nanopore in part based on the measured current; and removing the potential applied to the nanopore when the size of the nanopore corresponds to a desired size. In some cases, the applied potential may have a pulsed waveform oscillating between high and low values, allowing the current flowing through the nanopore to be measured while the potential is applied to the nanopore at a low value.

By way of example and not limitation, solid materials include any known semiconductor materials, insulating materials, and metals coated with insulating materials. Therefore, at least a portion of the nanopores may comprise, but is not limited to, silicon, silicon dioxide, silylene, silicon oxide, graphene, silicon nitride, germanium, gallium arsenide, or metals, metal oxides, and metal colloids coated with insulating materials.

To fabricate nanoscale pores, various feedback procedures can be employed during the manufacturing process. In embodiments where ions permeate the pore, detecting the ion flow through the solid material provides a way to measure the pore size created during manufacturing (see, for example, U.S. Publication No. 2005/0126905). In other embodiments where the pore size is defined by electrodes, the electron tunneling current between the electrodes provides information about the gap between them. An increase in the tunneling current indicates a decrease in the gap space between the electrodes. Other feedback techniques will be apparent to those skilled in the art.

In some embodiments, ion beam engraving is used to fabricate nanopores, as described in Li et al., 2003, Nature Materials s2:611-615. In some embodiments, high current is used to fabricate nanopores, as described in WO13167952A1 or WO13167955A1. In other embodiments, nanopores are fabricated by a combination of electron beam lithography and high-energy electron beam engraving (see, for example, Storm et al., 2003, Nature Materials s2:537-540). A similar scheme for producing suitable nanopores by ion beam sputtering is described in Heng et al., 2004, Biophy J 87:2905-2911. Nanopores are formed using lithography on metal-oxide-semiconductor (CMOS) with a focused high-energy electron beam in combination with general techniques for producing ultrathin films. In other embodiments, nanopores are constructed by sculpting silicon nitride, as provided in U.S. Patent Nos. 6,627,067, 6,464,842, 6,783,643 and U.S. Publication No. 2005/0006224.

In some implementations, nanochannels can be constructed as gold or silver nanotubes. These nanochannels are formed using templates of porous materials (such as polycarbonate filters prepared using orbital etching methods), and gold or other suitable metals are deposited on the surface of the porous material. Orbital-etched polycarbonate films are typically formed as follows: a solid film material is exposed to high-energy core particles, which establish orbitals in the film material. Chemical etching is then used to convert the etched orbitals into pores. The resulting pores have diameters of approximately 10 nm and larger. Adjusting the intensity of the core particles controls the density of pores formed in the film. Nanotubes are formed on the etched film by depositing metal (typically gold or silver) into the orbital-etched pores via an electroless electroplating method (Menon et al., 1995, Anal Chem 67:1920-1928). This metal deposition method uses a catalyst deposited on the surface of the porous material, which is then immersed in a solution containing Au(I) and a reducing agent. The reduction of Au(I) to metallic Au occurs on the surface containing the catalyst. The amount of gold deposited depends on the incubation time, such that increasing the incubation time reduces the inner diameter of the pores in the filter material. Therefore, the pore size can be controlled by adjusting the amount of metal deposited on the pores. The pore size is measured using various techniques, such as gas transport properties using simple diffusion or by measuring the ion flow through the pores using a membrane clamping system. The support material is either kept intact or removed to leave the gold nanotubes. Electroless plating techniques can be used to form pores with diameters from about 1 nm to about 5 nm or larger, depending on the requirements. Gold nanotubes with pore diameters of about 0.6 nm appear to distinguish between Ru(bpy)²⁺ and methyl viologen, exhibiting selectivity for gold nanopores (Jirage et al., 1997, Science 278:655-658). The surface of gold nanotubes can be easily modified by attaching thiol-containing compounds to the gold surface or by derivatizing the gold surface with other functional groups. This feature allows for the attachment of pore-modifying compounds as discussed herein, as well as probe markers. Devices (such as cis/trans devices for the biopores described herein) can be used with gold nanopores to analyze individually encoded molecules.

In cases where the detection mode of the tag involves a current passing through the tag (e.g., electron tunneling current), the solid film can be metallized using various techniques. A conductive layer can be deposited on both sides of the film to create electrodes suitable for querying the tag along the length of the chain, e.g., longitudinal electron tunneling current. In other embodiments, a conductive layer can be deposited on one surface of the film to form electrodes suitable for querying tags that pass through an aperture (e.g., lateral tunneling current). Various methods for depositing conductive materials are known, including sputtering deposition (i.e., physical vapor deposition), non-electrolyte deposition (e.g., colloidal suspensions), and electrolyte deposition. Other metal deposition techniques are filament evaporation, metal layer evaporation, electron beam evaporation, flash evaporation, and induced evaporation, and are as will be understood by those skilled in the art.

In some embodiments, the detection electrode is formed by sputter deposition, in which an ion beam bombards a metal block and evaporates metal atoms, which are then deposited as a thin film on the wafer material. Depending on the photolithography method used, the metal film is then etched using reactive ion etching or polished using chemical-mechanical polishing. The metal film can be deposited on pre-formed nanopores or prior to the fabrication of said pores.

In some embodiments, the detection electrode is fabricated by electrodeposition (see, for example, Xiang et al., 2005, Angew. Chem. Int. Ed. 44: 1265-1268; Li et al., Applied Physics Lett. 77(24): 3995-3997; and U.S. Publication No. 2003/0141189). This fabrication process is suitable for creating nanopores and corresponding detection electrodes on one side of a solid film, such as for detecting lateral electron tunneling. Initially, a pair of facing electrodes is formed on a silicon dioxide layer (supported on a silicon wafer) using conventional photolithography methods. An electrolyte solution is applied to the electrodes, and metal ions are deposited on one of the electrodes by passing a current through the electrode pair. The deposition of metal on the electrodes over time reduces the gap distance between the electrodes, establishing not only the detection electrode but also a nanoscale gap for transferring encoded molecules. The gap distance between the electrodes can be controlled by a number of feedback methods.

In the case of detection based on charge-induced field effects imaging, semiconductors can be fabricated as described in U.S. Patent No. 6,413,792 and U.S. Publication Application No. 2003/0211502. The methods for fabricating these nanopore devices can use techniques similar to those used for fabricating other solid-state nanopores.

As further described below, the detection of the tag (such as a polynucleotide) is performed. For tag analysis, nanopores can be constructed in various forms. In some embodiments, the device comprises a biological membrane or solid membrane containing nanopores held between two reservoirs (also referred to as cis and anti-cells) (see, for example, U.S. Patent No. 6,627,067). A conduit for electron migration between the two cells allows electrical contact between the two cells, and a voltage bias between the two cells drives the transfer of the tag through the nanopore. A variant of this configuration has been used to analyze the current flowing through the nanopore, as described in U.S. Patent Nos. 6,015,714 and 6,428,959 and Kasianowiscz et al., 1996, Proc Natl AcadSci USA 93:13770-13773, the disclosures of which are incorporated herein by reference.

A variation of the above device is disclosed in U.S. Application Publication No. 2003/0141189. A pair of nanoelectrodes, fabricated by electrodeposition, are located on a substrate surface. The electrodes face each other and have a gap distance sufficient for a single nucleic acid to pass through. An insulating material protects the nanoelectrodes, exposing only the tips of the nanoelectrodes for nucleic acid detection. The insulating material and nanoelectrodes separate a compartment that serves as a sample reservoir and a compartment for delivering a polymer via transfer. Cathode and anode electrodes provide an electrophoretic electric field for driving a tag from the sample compartment to the delivery compartment.

By applying an electric field oriented through the nanopore, a current bias can be generated to drive the tag through the nanopore. In some embodiments, the electric field is a constant voltage or a constant current bias. In other embodiments, the movement of the tag is controlled by pulsed manipulation of the electrophoretic electric field parameters (see, for example, U.S. Patent Application No. 2003/141189 and U.S. Patent No. 6,627,067). Pulsing of the current can provide a method for precisely transferring one or only a few bases of the oligonucleotide tag through the pore within a defined time period and briefly retaining the tag within the pore, thereby providing greater resolution of the tag's electrical properties.

The nanopore device may also include an electric or electromagnetic field for restricting the orientation of the oligonucleotide tag as it passes through the nanopore. This holding field can be used to reduce the movement of the oligonucleotide tag within the pore. In some embodiments, an electric field perpendicular to the direction of transfer is provided to restrict the movement of the tag molecule within the nanopore. This is explained in U.S. Application Publication No. 2003/0141189 using two parallel conductive plates above and below a sample plate. These electrodes generate an electric field perpendicular to the direction of transfer of the tag molecule and thus retain the tag molecule to one of the sample plates. The negatively charged backbone of DNA or nucleic acids modified to have a negative charge on one strand will be oriented on the anode plate, thereby restricting the movement of the tag molecule.

In other embodiments, the tag's position is controlled by a method described in U.S. Application Publication No. 2004/0149580, which employs an electromagnetic field established within the nanopore by a series of electrodes located near or on the surface of the nanopore. In these embodiments, one set of electrodes applies a DC voltage and a radio frequency potential, while a second set of electrodes applies an opposite DC voltage and a radio frequency potential, which is 180 degrees phase-shifted relative to the radio frequency potential generated by the first set of electrodes. This radio frequency quadrupole holds the charged particle (e.g., nucleic acid) at the center of the field (i.e., the center of the pore).

In an exemplary embodiment, the nanoporous membrane may be a multilayer stack of conductive and dielectric layers, wherein embedded conductive layers or conductive layer gates provide a well-controlled and measurable electric field in and around the nanopores through which the tag is transferred. In one aspect, the conductive layer may be graphene. Examples of stacked nanoporous membranes are found, for example, in US20080187915 and US20140174927.

It should be understood that the nanopores may be located in membranes, layers or other substrates, and the term has been used interchangeably to describe two-dimensional substrates containing nanopores.

In some embodiments, nanopores can be formed as part of a determination process using said nanopores to detect and/or determine analyte concentrations. Specifically, an apparatus for detecting and/or determining analyte concentrations using nanopores can be provided first, without nanopores formed in a membrane or layer. The apparatus may include a membrane separating two compartments (cis and anti-cis) on opposite sides of the membrane. The cis and anti-cis compartments may include a salt solution and may be connected to a power source. When a nanopore is to be formed in the membrane, a voltage is applied to the salt solution in the cis and anti-cis compartments, and the conductivity across the membrane is measured. Before the nanopore is formed, there is no current or only a very small current measured across the membrane. After the nanopore is formed, the current measured across the membrane increases. A voltage sufficient to form a nanopore of a desired diameter can be applied for a period of time. After the nanopore is formed, an analyte or tag can be transferred through the nanopore, and the transfer event can be detected. In some embodiments, the same salt solution can be used for both nanopore formation and for detecting the transfer of an analyte or tag through the nanopore. Any suitable salt solution can be used for nanopore formation and/or the transfer of an analyte or tag through the nanopore. Any salt solution that will not damage the counting markers can be used. Exemplary salt solutions include lithium chloride, potassium chloride, sodium chloride, calcium chloride, magnesium chloride, etc. The concentration of the salt solution can be selected based on the desired conductivity of the salt solution. In some embodiments, the salt solution may have a concentration in the range of 1 mM to 10 M, for example, 10 mM-10 M, 30 mM-10 M, 100 mM-10 M, 1 M-10 M, 10 mM-5 M, 10 mM-3 M, 10 mM-1 M, 30 mM-5 M, 30 mM-3 M, 30 mM-1 M, 100 mM-5 M, 100 mM-3 M, 100 mM-1 M, 500 mM-5 M, 500 mM-3 M, or 500 mM-1 M, for example, 10 mM, 30 mM, 100 mM, 500 mM, 1 M, 3 M, 5 M, or 10 M.

In some embodiments, the nanopores may become clogged, and the clogged nanopores can be cleaned by adjusting the pattern of the voltage applied across the nanopore layer or membrane by the electrodes. In some cases, the clogged nanopores are cleaned by reversing the polarity of the voltage applied across the nanopore layer or membrane. In other cases, the clogged nanopores are cleaned by increasing the magnitude of the voltage applied across the nanopore layer or membrane. The voltage increase can be a brief increase lasting 10 seconds (s) or less, for example, 8 s or less, 6 s or less, 5 s or less, 4 s or less, 3 s or less, 2 s or less, 1 s or less, 0.5 s or less, 0.4 s or less, 0.3 s or less, 0.2 s or less, including 0.1 s or less.

h) Signal detection

Transferring the query tag/aptamer through or across nanopores and detecting detectable properties generates a signal that can be used for counting (i.e., determining amount or concentration) and/or identifying (i.e., determining presence) the tag/aptamer. The type of detection method employed may correspond to the properties being detected for the tag.

In some embodiments, the detectable property is the effect of the tag's transfer through the nanopore on the electrical properties of the nanopore. The electrical properties of the nanopore include, in particular, current amplitude, impedance, duration, and frequency. In some cases, the tag can be identified using nanopore force spectroscopy (see, for example, Tropini C. and Marziali A., Biophysical Journal, 2007, Vol. 92, 1632-1637). Apparatus for detecting the electrical properties of the pore can include nanopores in a doped layer (e.g., a thin film or membrane) separating cis and anti-cis compartments connected by conductive bridges. The tag to be analyzed can be present on the cis side of the nanopore in an aqueous solution typically containing one or more dissolved salts (such as potassium chloride). Applying an electric field across the nanopore using electrodes located on both the cis and anti-cis sides causes the tag to transfer through the nanopore, which affects ion migration through the pore, thereby altering the electrical properties of the pore. The current can be measured at appropriate time frequencies to obtain sufficient data points to detect current signal patterns. The generated signal pattern can then be compared with a set of reference patterns, each derived from an examination of a single population of known tags bound to the analyte in a sample with a known analyte concentration. As previously noted, the number of tags of the same type transferred through the nanopore per unit time can be counted, for example, every 15 min, 13 min, 10 min, 8 min, 6 min, 4 min, 2 min, 1 min, 30 s, every 20 s, every 15 s, every 10 s, every 5 s, every 1 s, every 100 ms, every 10 ms, or every 1 ms. In some cases, the number of tags of the same type transferred through the nanopore can be counted for a specific time period to determine the amount of time required to reach a threshold count. Conversions in current amplitude, current duration, current frequency, and current magnitude can define the signal pattern of the tag and can be used to distinguish different tags from one another. Measurements of the current properties of nanopores (such as via patch clamping techniques) are described in the publications discussed above and in numerous references, e.g., Hille, B, 2001, Ion Channels of Excitable Membranes, 3rd Edition, Sinauer Associates, Inc., Sunderland, Mass. The number of counts measured over a time period (counts/time) is proportional to the concentration of molecules (e.g., tags) transferred through or across the nanopore. The tag concentration can be determined by generating a standard curve. For example, a series of standard molecules at different concentrations can be transferred through the nanopore and the counts/times measured to calculate the count rate for each concentration. The count rate of the tag being measured is compared to the standard curve to calculate the tag concentration.

In some embodiments, the detectable property of the tag can be quantum tunneling of electrons. Quantum tunneling is a quantum mechanical effect in which a particle's quantum wave properties allow it to transition through a classically forbidden energy state. Electron tunneling occurs in the presence of a potential barrier due to the movement of electrons between the donor and acceptor. To detect electron tunneling, a microelectrode tip can be positioned approximately 2 nanometers from the sample. At an appropriate separation distance, electrons tunnel through the region between the tip and the sample, and if a voltage is applied between the tip and the sample, a net electron current (i.e., tunneling current) flows through the gap in the direction of voltage bias. In cases where a detection electrode is used in the nanodevice to measure the tunneling current, the electrode is located near the transfer tag such that electron tunneling exists between the detection electrode and the tag. As discussed further below, the arrangement of the electrode relative to the transfer tag may determine the type of electron transport that occurs through the tag.

In some embodiments, analysis of the tag may involve detecting a current occurring across the nucleic acid chain (i.e., longitudinally along the nucleic acid chain) (Murphy et al., 1994, Proc Natl Acad Sci USA 91(12):5315-9). The exact mechanism of electron transfer is unknown, although electron tunneling is given as an explanation for the transport properties of DNA. However, the fundamental physics of electron transport across double-stranded nucleic acids is not limiting for the purposes of this paper, and the detection of the current flowing through the nucleic acid is used to distinguish one polymer tag from another. To detect the electron flow occurring longitudinally across the tag molecular chain, detection electrodes can be positioned longitudinally in the direction of tag molecule transfer such that there is a gap between electrodes parallel to the extended chain of the tag molecule. In different embodiments, the detection electrodes may be placed on opposite sides of the layers (e.g., membranes) separating the nanopores, while in other embodiments, the detection electrodes may be positioned within the layers separating the nanopores.

Another mode of electron flow in nucleic acids occurs across the nucleic acid, for example, in the transverse direction of the extended nucleic acid chain (e.g., across the diameter of a double-stranded nucleic acid). In double-stranded nucleic acids, electron transport occurs through paired bases, while in single-stranded nucleic acids, electron transport occurs through a single unpaired base. Furthermore, chemical composition, hydration structure, interaction with charged ions, spatial orientation of each base, and differences in different base pairing combinations can alter the transverse electron transport characteristics and thus provide a basis for distinguishing tag molecules that differ in sequence and/or polymer backbone. To detect electron flow across the tag molecule (i.e., in the transverse direction of the extended nucleic acid chain), a detection electrode is positioned on one side of a nanopore to query for tag molecules that cross the nanopore rather than pass through it.

In embodiments involving longitudinal or lateral detection, the thickness of the electrode can determine the total number of bases queried by the electrode. For lateral detection, the tip of the detection electrode can be configured to query a single nucleoside base (as defined herein), thereby achieving single-base resolution. In other embodiments, the size of the detection electrode is arranged to query more than one nucleoside base. Thus, in some embodiments, the number of nucleoside bases queried at any given time can be about 2 or more, about 5 or more, about 10 or more, or about 20 or more, depending on the resolution required to detect differences in the different polymer sequences of the tag molecule.

In other implementations, differences in tag structure can be detected as differences in capacitance. This type of measurement is explained in US2003/0141189. Capacitance causes a phase shift in an applied alternating voltage at a defined applied frequency and impedance. The phase shift characteristics of each nucleoside base are determined for nucleic acids with known sequences and structures and used as a reference standard for identifying individual base characteristics. Nearest neighbor analysis can allow capacitance measurements to be extended beyond a single nucleoside base.

In other embodiments, the detection technique may be based on imaging charge-induced fields, as described in U.S. Patent No. 6,413,792 and U.S. Publication No. 2003/0211502, the disclosures of which are incorporated herein by reference. For tag detection based on charge-induced fields, the semiconductor device described above is used. If a current channel is formed in the semiconductor, the application of a voltage between the source and drain regions will generate a current flow from the source to the drain region. Because each nucleoside base has an associated charge, the passage of the tag molecule through the semiconductor aperture induces a change in the conductivity of the semiconductor material lining the aperture, thereby inducing a current of a specific magnitude and waveform. Different bases produce currents of different magnitudes and waveforms due to differences in the charge, charge distribution, and size of the bases. In the embodiment disclosed in U.S. Patent No. 6,413,792, the polymer passes through an aperture formed from a p-type silicon layer. As described above, the transfer of the tag molecule is achieved by a method similar to those used for moving polymers through other types of channels. The expected current is on the order of microamperes, which is much higher than the expected picoampere current detected by electron tunneling. Because the polymer segment regions in the tag molecule contain more than a single nucleoside base, these block copolymer regions will generate characteristic signals reflecting the charge and charge distribution of the block copolymer regions.

It should be understood that although the above description involves various detection techniques, in some implementations, multiple different techniques may be used to examine single-tag molecules (see, for example, Kassies et al., 2005, J Microsc 217:109-16). Examples of multiple detection modes include, in particular, current blocking combined with electron tunneling current and current blocking combined with imaging charge-induced field. Parallel detection using different detection modes can be used to identify tagged molecules by correlating the detection times of the signals obtained between the different detection modes.

In some embodiments, measuring the number of tags transferred through the layer or detecting tags transferred through the layer includes observing the current-blocking effect of the tags on the nanopores. In some embodiments, the analyte is present in the sample when the current-blocking effect is above a threshold level.

3. Apparatus for the analysis of analytes

This invention describes microfluidic devices used in conjunction with nanoporous devices and integrated microfluidic nanoporous devices. The disclosed microfluidic devices used in conjunction with nanoporous devices and integrated microfluidic nanoporous devices can be used in the analyte analysis methods described above. However, in some cases, the devices described herein can be used for other applications. Similarly, in some cases, the methods described herein can be used in conjunction with other devices.

Figures 1A and 1B depict a microfluidic device used in conjunction with a nanoporous device. The microfluidic device 10 is depicted together with fluid droplets 11 to be analyzed in the nanoporous device 15. The fluid droplets may include tags (e.g., cut tags or aptamers) to be counted using the nanoporous device. The nanoporous device 15 includes a first compartment 16, a layer 17 having nanopores 18, and a second compartment 19. Figures 1A and 1B depict a liquid transfer step 1 in which fluid droplets 11 are removed from the microfluidic device 10 and placed in the nanoporous device 15. As depicted in Figure 1A, the fluid droplets 11 are placed on the layer 17 in a manner that causes the droplets to separate across the layer 17 and be positioned at the nanopores 18. The fluid droplets can be introduced into the nanoporous device 15 via an inlet port (not shown). The inlet port may be located on a portion of the layer 17. For example, the inlet port may be located in an opening within the wall of the compartment containing the nanopore layer. In Figure 1B, liquid droplets 11 are placed in the first compartment 16. Buffer addition step 2 introduces buffer into the second compartment 19. In other embodiments, buffer may be added to the second compartment 19 before liquid droplets 11 are introduced into the first compartment 16. In other embodiments, liquid droplets 11 may be placed in the second compartment 19 before or after buffer is added to the first compartment 16. In Figure 1A, the step of adding buffer to either compartment is not required.

In another embodiment, the device may be an integrated device. The integrated device may include microfluidic modules and nanopore modules, which may be constructed separately and then combined to form an integrated device, or the microfluidic modules and the nanopore modules may be embedded together in a single device.

Figures 2A and 2B depict schematic diagrams of an integrated device having microfluidic modules combined with nanopore modules, the two modules being integrated by connecting them using channels. Although Figures 2A and 2B depict devices comprising individual modules (which are combined to produce an integrated device), it should be understood that the devices of Figures 2A and 2B can also be fabricated as unit devices in which two modules are connected.

The top images of Figures 2A and 2B depict a microfluidic module 20 having fluid droplets 25 to be analyzed in a nanoporous device 30. The nanoporous module 30 includes a first compartment 31, a layer 32 having nanopores 33, and a second compartment 34. The microfluidic module 20 is integrated with the nanoporous module 30 via a channel 40. The channel fluidly connects the two modules and facilitates the movement of the droplets 25 from the microfluidic module 20 to the nanoporous module 30. The middle image illustrates the movement of the droplets 25 from the microfluidic module 20 to the nanoporous module 30 via the channel 40. As shown in Figure 2A, the channel can connect the microfluidic module 20 to an inlet port in the nanoporous module 30. The inlet port (not shown) can be positioned such that the fluid droplets 25 are placed on the layer 32 in a specific manner, causing the droplets to separate across the layer 32 and be positioned at the nanopores 33. At the end of the transfer process, the fluid droplets are positioned across the nanopores 33 (Figure 2A, bottom view). In other embodiments, channel 40 can connect the microfluidic module 20 to an inlet port in the first or second compartment of the nanopore module 30. One such embodiment is shown in FIG. 2B, where channel 40 connects the microfluidic module 20 to an inlet port in the first compartment 31 of the nanopore module 30. A buffer solution can be added to the second compartment before or after the transfer of the liquid droplet 25 into the first compartment 31. In step 2 of FIG. 2B, after the droplet 25 is transferred to the first compartment 31, a buffer solution is added to the second compartment 34. Optionally, after the transfer is complete, channel 40 can be removed and the two modules separated. The microfluidic and nanopore devices and modules shown in FIG. 1A, 1B, 2A, and 2B are each individually functional.

Figures 2C-2H depict one embodiment of an integrated device including a digital microfluidic module 50 and a nanopore module 60. The digital microfluidic module is depicted together with an electrode array 49 operatively connected to a plurality of reagent reservoirs 51 for generating microdroplets to be transported to the nanopore module. One or more of the reservoirs 51 may contain reagents or samples. Different reagents may be present in different reservoirs. A contact pad 53 connecting the electrode array 49 to a power source (not shown) is also depicted in the microfluidic module 50. No trace lines connecting the electrode array 49 to the contact pad are depicted. The electrode array 49 transports one or more microdroplets (such as buffer droplets or droplets containing buffer and/or tags (e.g., cleaved tags or dissociated aptamers)) to one or both of transfer electrodes 71 and 72 located at an interface 100 between the digital microfluidic module 50 and the nanopore module 60. The digital microfluidic module 50 and the nanopore module 60 are operatively connected at the interface 100. The nanopore module 60 includes at least two microfluidic capillary channels 61 and 62 that intersect each other at the location where the nanopore layer 70 is disposed. The two microfluidic capillary channels 61 and 62 are located in two different substrates within the nanopore module (depicted in FIG. 2D). Thus, the nanopore module includes a first substrate 63 (e.g., a bottom substrate) including the microfluidic capillary channel 61 in the top surface of the first substrate 63, and also includes a second substrate 64 (e.g., a top substrate) having the microfluidic capillary channel 62 in a first surface of the second substrate. The second substrate 64 covers the microfluidic capillary channel 61, and the first substrate 63 is deposited beneath the microfluidic capillary channel 62. At the intersection of the two channels in the nanopore layer 70, the capillary channel 62 covers the capillary channel 61 (see also FIG. 2D, bottom view). The two capillary channels are physically separated at the intersection by the nanopore layer 70 at the intersection. The nanopore layer 70 includes at least one nanopore (not shown) located at the intersection of capillary channels and allows molecules to be transported from one capillary channel through the nanopore to other capillary channels. Capillary channels 61 and 62 open at an interface 100 at a first end of the capillary channels and open at a second end of the capillary channels to a reservoir/discharge outlet (84 and 85, as seen in FIG. 2C). A cover substrate 101 positioned above the electrode array 49 is also depicted in FIG. 2C. The cover substrate 101 defines a gap in the microfluidic module in which microdroplets are manipulated. The cover substrate 101 may optionally include electrodes 55 (e.g., reference electrodes) disposed on a bottom surface of the cover substrate 101, thereby providing a biplanar electrode configuration for manipulating microdroplets in the microfluidic module 50. Without the biplanar electrode configuration, microdroplets can be manipulated in the microfluidic module 50 by using coplanar electrode actuation, such as using the electrode array 49 or another coplanar electrode configuration. For example, the coplanar electrode described in US6911132 can be used to manipulate microdroplets in microfluidic module 50.

Figure 2D top view shows a schematic front view of a cross-section of interface 100, at which the digital microfluidic module 50 and the nanopore module 60 are operatively connected. The bottom view of Figure 2D depicts a schematic side view of a cross-section of the device at transfer electrode 72. Figure 2D top view shows two microdroplets (65a and 65b) positioned on two transfer electrodes 71 and 72 at interface 100 between the microfluidic module 50 and the nanopore module 60. As shown in the top view of Figure 2D, microdroplet 65a positioned at electrode 71 is aligned with an opening in capillary channel 61, while microdroplet 65b positioned at electrode 72 is aligned with an opening in capillary channel 62. The bottom view of Figure 2D explains the side view of a cross-section of the integrated device, showing the arrangement of microdroplet 65b on transfer electrode 72. Microdroplet 65b is positioned to move into capillary channel 62. Capillary channel 61 is also shown; however, the capillary channel is a short distance from and aligned with transfer electrode 72 (not shown). A covering substrate 101 is also depicted having electrodes 55 disposed on its bottom surface. In an embodiment of the integrated device depicted in Figures 2D-2H, the electrode arrays of the nanopore module and the microfluidic module are disposed on the same substrate.

The vertical distance between the top surface of the transfer electrode and the inlet to the capillary channel can depend on the thickness of the substrate forming the lower portion of the microfluidic module and the nanopore module. The vertical distance can be set based on the volume of the droplet to be transferred to the nanopore module. The vertical distance can be adjusted by changing the thickness of the substrate. For example, the substrate of the nanopore module (e.g., substrate 63) can remain relatively thin, or the thickness of the substrate on which the transfer electrode is disposed can be increased (e.g., by using a thicker substrate) to ensure that the droplet is aligned with the inlet to the capillary channel. An exemplary device is depicted in Figure 2E, in which the droplet is aligned with the inlet to the capillary channel by using a microfluidic module with a thicker bottom substrate. The device shown in Figure 2E has the same configuration as described with respect to Figures 2C-2D. However, the thickness of the substrate 59a on which the electrode array is positioned is increased relative to the thickness of the substrate portion on which the nanopore module is disposed. The top view of Figure 2E depicts a front view of a cross-section at the interface 100 between the microfluidic module and the nanopore module. Figure 2E, in its base diagram, depicts a side view of a cross-section at the location of transfer electrode 72 and capillary channel 62. As shown in Figure 2E, the substrate 59a on which the electrode array 49 and transfer electrodes 71 and 72 are disposed is thicker than the substrate 59b on which the nanopore module is disposed. As shown in the base diagram of Figure 2E, substrate 59a has a first height H1, while substrate 59b has a second height H2, where H1 is greater than H2. The height difference between substrates 59a and 59b results in the alignment of capillary channels 61 and 62 in the nanopore module with the microdroplets positioned on electrodes 71 and 72, respectively. Channel 61 is also depicted in the base diagram of Figure 2E. As can be seen from Figure 2C, capillary channel 62 is perpendicular to capillary channel 61 at the location of nanopore layer 70. Channel 61 is aligned with transfer electrode 71 and configured to receive microdroplets 65a positioned on transfer electrode 71. Although the two capillary channels are depicted as perpendicular to each other at the point of intersection, other configurations are also anticipated, in which the two channels intersect at an angle of more than 90 degrees.

Upon contact with a capillary channel, the droplets move into the capillary channel via any suitable means (e.g., capillary action). Other methods/materials can facilitate the movement of droplets into the capillary channel. For example, the droplets can move into the capillary channel via diffusion, Brownian motion, convection, pumping, applied pressure, gravity-driven flow, density gradient, temperature gradient, chemical gradient, pressure gradient (positive or negative), aerodynamic pressure, chemical reactions that generate gas, wicking, capillary pressure, wicking, electric field-mediated flow, electrode-mediated flow, electrophoresis, dielectroelectrophoresis, magnetomigration, magnetic field, magnetically driven flow, optical force, chemotaxis, phototaxis, surface tension gradient-driven flow, Marangoni stress, thermo-capillary convection, surface energy gradient, acoustic migration, surface acoustic waves, electroosmosis, thermal migration, electrowetting, photoinduced electrowetting, or combinations thereof. Alternatively or alternatively, the movement of droplets into the capillary channel can be facilitated by, for example, using driving forces, such as those disclosed herein; using a hydrophilic coating in the capillary; or changing the size of the capillary channel (e.g., width and/or height and/or diameter and/or length)).

In the embodiments depicted in Figures 2C-2H, the flow of fluid across capillary channels 61 and 62 is controlled at least in part by altering the cross-section of the capillary—the fluid initially moves relatively rapidly until it enters a narrower portion of the capillary. One or two droplets may be droplets containing an analyte to be detected or counted (or a cleaved tag or dissociated aptamer) or a conductive solution (e.g., a buffer solution without analyte) for analysis through the nanopore. In some cases, one droplet 65a may be a droplet containing an analyte/tag/aptamer, while other droplets 65b may be buffer droplets. Although a single droplet is depicted for each channel, in practice, multiple droplets may be transported to the nanopore module. For example, multiple droplets may be transported to the nanopore module sequentially. In some cases, multiple droplets may be collected at one or two transfer electrodes to generate a larger droplet transported to the nanopore module.

Figure 2F illustrates exemplary configurations of various electrodes used in the integrated device. As noted above, a single, continuous electrode 55 (not shown in Figure 2F) is positioned in the microfluidic module 60 at intervals from the electrode array 49. The electrode array comprises a series of individually controllable electrodes. Electrode 55 is disposed on the lower surface of the covering substrate 101. Electrode 55 and the electrode array move droplets toward the transfer electrode. Although electrode 55 is depicted without covering transfer electrodes 71 and 72, in some exemplary devices, the covering substrate 101 and electrode 55 may extend over the transfer electrode. In embodiments where electrode 55 does not cover the transfer electrode, coplanar electrodes may be used to move droplets toward the transfer electrode (e.g., coplanar drive as described in US6911132). As described herein, a single electrode 55 may serve as a reference or ground electrode, while the electrode array 49 may be individually controllable (e.g., the electrode array may be independently drivable drive electrodes). Electrode pairs: pairs 80a and 80b and pairs 90a and 90b are positioned in the nanopore module. Electrode pairs 80a, 80b and 90a, 90b are used to establish opposite polarities across the nanopore layer 70 to drive charged molecules through the nanopores in the nanopore layer 70. In some embodiments, electrode pairs 80a and 80b can be positive electrodes, and electrode pairs 90a and 90b can be negative electrodes. Figure 2G illustrates alternative electrode configurations for the nanopore module where two electrodes 80 and 90 (instead of four) are used to establish the polarity difference across the nanopore layer 70. These embodiments demonstrate the application of symmetrical (four electrodes) or asymmetrical (two electrodes) electrode configurations that generate a potential gradient across the nanopore layer for transferring charged molecules through the nanopores.

Figure 2H illustrates an alternative configuration of the capillary channel, where only one channel 61 is connected to the microfluidic module at interface 100. The other channels 62 are connected to two reservoirs that can be filled with a conductive liquid to facilitate the transfer of charged molecules across the nanopores.

In some cases, the integrated device provided herein can be fabricated by forming a reservoir and electrode array of a digital microfluidic module portion on a first region of the top surface of a first substrate. A second substrate can be fabricated by placing a single electrode (e.g., electrode 55) on the bottom surface of a second substrate and positioning it at intervals over an array of individually controllable electrodes to provide a face-to-face orientation between the single electrode and the electrode array for biplane droplet actuation. As used herein, “droplet actuation” refers to the operation of a droplet using the microfluidic device disclosed herein or using a droplet actuator disclosed in US6,911,132, US6,773,566, or US6,565,727 (the disclosures of which are incorporated herein by reference). Thus, the configuration of the biplane electrodes or electrode arrays of the device disclosed herein can be similar to those disclosed in US6,911,132, US6,773,566, or US6,565,727. Electrode 55 on the second substrate can also be referred to as a reference electrode. Electrodes in the microfluidic module can optionally be coated with a dielectric material. Hydrophobic coatings can also be applied to dielectrics.

In some embodiments, microchannels can be formed on a third substrate, which may be disposed on a second region of the first substrate where the electrode array 49 is located. For example, the third substrate may be bonded to a second region of the first substrate, where the microfluidic electrode array is disposed in the first region. The substrate may have pre-formed microchannels, or the microchannels may be formed after the bonding step. A fourth substrate having second microchannels may be disposed on top of the substrate containing the microchannels to provide the integrated device depicted in Figures 2C-2H. The nanopore layer may be disposed on either microchannel at the intersection of the two microchannels. Thus, the substrate forming the nanopore module may include microchannels open at either end and on one side. The arrangement of the fourth substrate on the third substrate closes the microchannels, thereby forming capillary channels (e.g., 61 and 62).

In some embodiments, microchannels can be formed on a separate substrate, which may be disposed on a first substrate on which the microfluidic electrode array is disposed. For example, another substrate may be bonded to a second region of the first substrate, wherein the microfluidic electrode array is disposed in the first region. The substrate may have pre-formed microchannels, or the microchannels may be formed after the bonding step. Another substrate having a second microchannel may be disposed on top of the substrate containing the microchannel to provide the integrated device depicted in Figures 2C-2H. The nanoporous layer may be disposed on either microchannel at the intersection of the two microchannels 61 and 62.

In some embodiments, microchannels can be introduced into a second region adjacent to a first region on a first substrate where the microfluidic electrode array is disposed. For example, the microchannels can be etched onto the top surface of the second region. A nanopore layer can be placed at a location on the microchannels. The nanopore layer may include pre-formed nanopores. In an alternative embodiment, the nanopores can be formed after the layer is positioned at a location on the microchannels. A third substrate can be fabricated by introducing microchannels onto the bottom surface of a third substrate. The third substrate can be positioned over the second region on the first substrate such that the top surface of the second region of the first substrate contacts the bottom surface of the third substrate across its top surface, thereby establishing closed capillary channels 61 and 62.

Figures 2I-2K depict an apparatus in which a digital microfluidic module 250 and a nanopore module 260 share a common bottom (first) substrate 210 on which an electrode array 249 (a series of individually controllable electrodes) of the microfluidic module is disposed on a first region, and microfluidic channels 261 are formed in a second region. The microfluidic channels 261 in the first substrate are aligned with transfer electrodes 271. A second substrate 220 having a single continuous electrode 255 (e.g., a reference electrode) is disposed spaced apart from the electrode array 249 in the digital microfluidic module 250. A third substrate 230 containing microfluidic channels 262 formed in the lower surface of a third substrate is placed over the second region of the first surface 210, thereby covering the top surface of the first substrate in which the microfluidic channels 261 are formed. The first and third substrates in the nanopore module enclose the microfluidic channels 261 and 262, thereby providing capillary channels 261 and 262. It should be understood that “microfluidic channel” and “microchannel” are used interchangeably herein to refer to channels or cutouts in the substrate surface. After the substrate is placed on the channel, the channel is closed to form a capillary channel. Similar to FIG2C, the capillary channel may be fluidly connected to the microfluidic module at one end at the interface 100 between the microfluidic module 250 and the nanopore module 260, and have a reservoir or outlet at the other end. In other embodiments, the second capillary channel 262 may be constructed similarly to the capillary channel 62 in FIG2H, i.e., the second capillary channel 262 may not be connected to the microfluidic module at either end, and may be connected to a reservoir/outlet at both ends. A top view of the device is depicted in FIG2I, and a front view of the device at the interface between the modules is depicted in FIG2I(continued). As can be seen from the front view, the microdroplet 265a is on a plane above the inlet leading to the capillary channel 261. To allow the microdroplet 265a to flow into the capillary channel 261, a groove 280 is formed in the side edge of the third substrate 230 to provide space for the microdroplet to move downward into the microchannel 261. Therefore, a fluid connection between the microfluidic module and the nanopore module is provided through a vertical port formed by the groove 280, thereby providing an opening in the top portion of the first capillary channel 261 at one end of the interface 100. It should be understood that the groove 280 in FIG. 2I is not drawn to size and can have any suitable size, allowing fluid communication between the transfer electrode 271 and the first capillary channel 261 at the interface 100. Furthermore, the groove can vary in size. For example, the groove can be a cutout extending along the length of the side edge of the third substrate 230 at the interface 100, and can be proportional to match the width of the transfer electrode 271 or the width of the capillary channel 261, or a length in between. The cutout can nominally extend along the width of the third substrate 230 such that a relatively small area of the capillary channel 261 is not covered. In other embodiments, the cutout can extend over a large length of the capillary channel 261. A nanoporous layer 270 is positioned across a first capillary channel 261 at the intersection of two capillary channels. Layer 270 is positioned within a supporting substrate 275. In some cases, the first substrate 210 may be a glass substrate, and the supporting substrate 275 may be a PDMS gasket.

Figure 2J depicts a side view of a cross-section of the device shown in Figure 2I. This cross-section is located in the region of the device where the first capillary channel 261 is aligned with the first transfer electrode 271. A portion of the microfluidic module 250 with an electrode array 249 is also depicted, with a second substrate 220 having a single electrode 255 (e.g., a reference electrode) positioned spaced apart from the electrode array 249. As shown in Figure 2J, the single electrode 255 does not cover the transfer electrode. Although not illustrated in these figures, the second substrate 220 and the single electrode 255 (which may be a reference electrode) may cover the transfer electrodes 271 and 272, thereby providing a biplane electrode configuration. In this embodiment, the biplane electrode configuration allows the droplet to be moved toward the transfer electrodes 271 and 272. The first capillary 261 is located in the first substrate 210 and lies in a plane below the plane where the droplet 265a is located. A third substrate 230, including a second microchannel (which is closed by the top surface of the first substrate 210 to provide a capillary channel 262), is disposed on the first substrate. The third substrate 230 includes a groove 280 (or cutout) at the interface 100 adjacent to the side edge of the microfluidic module. The groove 280 opens at the top portion of the capillary channel 261 at its end, providing a vertical port for entry into the capillary channel 261. As indicated by the arrows, the droplet moves downwards into the capillary channel 261 and then forwards to flow towards the intersection of the first and second capillary channels. The second capillary channel 262 intersects the first capillary 261 at the location of the nanoporous layer 270. A support substrate 275 is depicted positioned above the first capillary channel 261 (and below the second capillary channel 262). The support substrate 275 includes the nanoporous layer 270. As shown in the top view of the nanoporous layer in the illustration, the support substrate 275 surrounds the nanoporous layer. In some embodiments, the support substrate may be a first layer with a central cutout and a second layer with a central cutout. The nanoporous layer can be disposed at the cutout between the first and second layers. The nanoporous layer in the supporting substrate can be used in devices where the bottom substrate 210 is made of glass.

Figure 2K shows another side view of the cross-section of the device shown in Figure 2I. In Figure 2J, the cross-section is at the location of the first transfer electrode 271. In Figure 2K, the cross-section is at the location of the second transfer electrode 272. As shown in Figure 2K, the inlet to the second capillary channel 262 is aligned with the location of the microdroplet 265b present on the second transfer electrode 272. The first capillary channel 261 is also depicted in Figure 2K, intersecting the second capillary channel 262 at the location of the nanopore layer 270.

In another embodiment, as shown in FIG2L, the first substrate 210 may include: a first portion 210a on which the electrode array 249 and transfer electrodes 271 and 272 are disposed; and a second portion 210b on which a substrate 290 containing a capillary channel 261a is disposed. Similar to the device shown in FIG2I-2K, the capillary channel 261a is below the plane of the transfer electrodes. The capillary channel 262 is located in the substrate 230, wherein the inlet to the capillary channel 262 is in the same plane as the transfer electrodes in the microfluidic module 250. Furthermore, similar to FIG2I-2K, the inlet to the capillary channel 262 is aligned with the transfer electrodes 272. Thus, microdroplets positioned on the electrodes 272 can move substantially horizontally to the capillary channel 262. Similar to the device shown in Figures 2I-2K, substrate 230 includes grooves 280 in its side edges to provide space for microdroplets positioned on transfer electrode 271 to move downwards to capillary 261a located in substrate 290. A nanoporous layer 270 is also depicted in Figure 2L. In this embodiment, the nanoporous layer is disposed directly on substrate 290 without the presence of support layer 275. For example, in an embodiment where both substrates containing the channels are formed of PDMS, the nanoporous layer can be disposed directly between the substrates without the presence of a support substrate. The top view of Figure 2L depicts a side view of a cross-section of the device at the location of transfer electrode 271 and capillary channel 261a. The bottom view of Figure 2L depicts a side view of a cross-section of the device at the location of transfer electrode 272 and capillary channel 262. From the top, the device appears identical to the device shown in Figure 2I. Thus, transfer electrodes 271 and 272 are spaced as they are in the device shown in Figure 2I.

Once the nanoporous layer is positioned within the device, electrodes can be fabricated within the nanoporous module for transporting molecules across the nanoporous layer via the nanopores. For example, the electrodes can be positioned in openings introduced into the substrate and located within capillary channels, exposing them to the capillary channels and allowing them to contact the fluid present within them. The distance between the electrodes and the nanopores can be empirically determined based on the resistance, width, diameter, and/or length of the capillary channels.

The nanoporous layer can be disposed on any of the channels. The nanoporous layer can be adhered to the surface of a substrate containing microchannels via plasma bonding or via a compressible element (such as a gasket). In some cases, the substrate containing the first channel can be a glass substrate. In this embodiment, a support substrate (e.g., a PDMS layer) can be used to position the nanoporous layer. For example, a PDMS gasket can be provided to the nanoporous layer.

Channels can be formed on the substrate using any suitable method. In some cases, photolithography or relief etching can be used to create channels for the nanopore module. In other embodiments, channels can be etched into the substrate. In some embodiments, a combination of suitable methods can be used to form channels in the substrate. For example, an etching method can be used to form one channel in a glass substrate, and another channel can be formed in a PDMS substrate using appropriate methods such as soft photolithography, nanoimprint lithography, laser ablation, or relief etching (e.g., soft relief etching). The height/width/diameter of the microchannels can be determined empirically. The height/width/diameter of the microchannel can be in the range of approximately 0.5 μm to 50 μm, for example, 0.5 μm-40 μm, 1 μm-30 μm, 2 μm-20 μm, 3 μm-10 μm, 5 μm-10 μm, for example, 0.5 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 15 μm, 20 μm, 30 μm, 40 μm, or 50 μm. As noted herein, the height/width/diameter of the channel can vary along the length of the channel.

In some embodiments, the nanoporous layer (e.g., 70 or 270) may include an insulating material coating on one or both sides of the nanoporous layer. The insulating material can reduce contact capacitance and decrease noise associated with the detection of molecular transfer through the nanopores in the nanoporous layer. In another embodiment, the surface area of the nanoporous layer exposed to a fluid in capillary channels (e.g., capillary channels 61 and 62, or 261 and 262) in contact with the nanoporous layer can be reduced. Reducing the surface area of the nanoporous layer in contact with the fluid containing molecules to be detected or counted through the nanopores can minimize contact capacitance and reduce background noise. The surface area of the nanoporous layer in contact with the fluid in the capillary channels can be reduced by reducing the size of the capillary channels at the location of the nanoporous layer. For example, the height or width, or both (e.g., diameter), of the capillary channels at the location of the nanoporous layer can be reduced. In another embodiment, the surface area of the nanoporous layer can be reduced. In some devices, a combination of these embodiments for reducing contact capacitance may be included. For example, in some embodiments, the integrated device as included herein may include capillary channels having reduced dimensions at the location of the nanoporous layer, and/or may include a nanoporous layer covered with an insulating material (e.g., PDMS) on one or both sides of the nanoporous layer, and/or may include a nanoporous layer having a minimum surface area.

Figure 3 illustrates another exemplary integrated device including a microfluidic module 300 and a nanopore module 325. Unlike the nanopore modules in Figures 1A, 1B, 2A, and 2B, the nanopore module 325 does not function as a standalone device but rather functions as a nanopore after integration with the microfluidic module 300. The microfluidic module 300 includes an opening 302 sized to allow insertion of the nanopore module 325. As depicted in Figure 3, the microfluidic module includes fluid droplets 301 to be analyzed using the nanopore module 325, which contains a layer 311 having nanopores 305. After the nanopore module 325 is inserted into the microfluidic module 300, a first compartment 306 and a second compartment 307 are established, spaced apart by the layer 311. The layer 311 also divides the fluid droplets 301 across the nanopores 305.

Figure 4 provides an integrated device 400 in which a digital microfluidic module includes an embedded nanopore module. In Figure 4, the nanopore module is positioned downstream of the region in the microfluidic module that generates fluid droplets 401. The microfluidic module moves the droplets 401 toward the nanopore module, causing the droplets 401 to be segmented across layer 402 and positioned at nanopores 403. Figure 4 shows a top view of the device. For clarity, the top substrate is not shown. Nanopores 403 in the nanopore layer 402 have been depicted, although they are not visible from the top view. The nanopore layer 402 may be attached to either a bottom substrate or a top substrate.

In Figures 1A, 1B, 2A, 2B, 3, and 4, although a single nanopore is shown, it should be understood that the layer may comprise one or more nanopores. Additionally, more than one droplet may be positioned within the nanopore module or device. The droplets can be analyzed by applying a voltage across the nanopore. Applying a voltage causes charged molecules to move across the nanopore. A decrease in the current across the nanopore provides an indication of transfer as the tag is transferred. In some embodiments, the compartments of the nanopore module may not be filled with a conductive solution (e.g., a buffer solution)—this conductive solution can be provided immediately after the fluid droplet is positioned across the nanopore layer. In some cases, the walls and nanopore layer of the nanopore device may define a first and a second compartment across which a voltage is applied to measure the transfer of a tag/aptamer present in the fluid droplet (e.g., see Figures 1B and 2B). The first and second compartments may be empty or may contain a conductive fluid before the fluid droplet is introduced. In other cases, the first and second compartments may be defined by the walls and nanopore layer of the microfluidic module (e.g., see Figure 3). In other cases, the first and second compartments may be defined by fluid droplets traversing the nanopore layer (e.g., see Figures 1A, 2A, and 4). In some cases, a voltage (used to guide charged molecules across the nanopores) may be applied to the fluid droplets, for example, in embodiments where a conductive solution is not present in the compartments. The voltage may be applied to the fluid droplets via electrodes in direct or indirect contact with them. It should be understood that the size of the nanopore layer is larger than the size of the droplets, such that the droplets traverse the layer divisions and are connected only via the nanopores.

Figures 5A, 5B, 6, and 7 illustrate droplet movement in a device with a digital microfluidic module and a nanopore layer. In Figure 5A, components of an integrated digital microfluidic/nanopore device 450 are depicted. The top view shows that a droplet 401, to be analyzed using nanopores 403 in the nanopore layer 402, is positioned across the nanopore layer 402. For illustrative purposes, nanopores 403 are shown here, although they are not visible from the top view. The device 450 includes a substrate 411 on which an electrode array 405 is disposed. The electrode array positions the droplet 401 by segmenting the droplet across the nanopore layer 402. Arrows 451 and 452 depict the directions in which the droplet 401 can move across the electrode array toward the nanopore layer 402. After the microdroplet 401 is positioned across the nanoporous layer 402, electrodes 404 and 406 positioned below the microdroplet 401 can be activated to provide a differential voltage across the nanoporous layer 402, thereby facilitating the movement of molecules (e.g., cleaved tags or aptamers) in the microdroplet 401 across the nanopore 403. Electrodes 404 and 406 are bifunctional electrodes used to move the microdroplet towards the nanoporous layer and drive the tag/aptamer across the nanopore 403.

Figure 5B depicts a side view of device 450, illustrating the top substrate 412 omitted in the top view shown in Figure 5A. The shown top substrate 412 includes an electrode 414. The electrode 414 can be a single electrode or an array of electrodes. A nanoporous layer extends from the top substrate to the bottom substrate. A microdroplet 401 bisects the nanoporous layer 402. Although a biplanar electrode is depicted in Figure 5B, the device may not include electrodes in both substrates; instead, the top or bottom substrate may include coplanar electrodes. Electrodes 404 and 406 near the microdroplet 401 have opposite polarities and drive the tag/aptamer across the nanopore 403.

Figure 6 illustrates the segmentation of a droplet 401 across a nanoporous layer 402 having nanopores 403. 1a depicts an electrode 405 causing the droplet to move towards the nanoporous layer 402 in the direction indicated by the arrow. In 2a, the droplet 401 has been segmented and positioned by the nanoporous layer 402 such that the droplet is connected via the nanopores 403. In 3a, an electrode positioned below the droplet 401 across the nanoporous layer 402 is activated to provide an anode (-) and a cathode (+). The activated electrode drives negatively charged molecules present in the droplet 401 (including the counted tags/aptamers) through the nanopores 403. As the tags/aptamers transfer through the nanopores 403, the number of tags/aptamers can be counted as explained herein. Step 3a is used to collect all tags/aptamers segmented on one side of the droplet as it is segmented across the nanoporous layer.

Once substantially all the tags/aptamers have been transferred to one side of the nanoporous membrane, the polarity of the electrode can be reversed, as shown in 4a, and the tags/aptamers can be transferred to the other side of the nanoporous layer 402 and counted. The number of tags counted in step 3a should be approximately half the count obtained in step 4a. The steps of reversing the electrode polarity and counting the tags/aptamers can be repeated any number of times to obtain multiple readouts of the number of tags/aptamers in the droplets.

Figures 7, 1b, and 2b show two microdroplets 600a and 600b moving to the nanoporous layer 604 in the direction indicated by the arrows. Once at the nanoporous layer 604, the microdroplets wet the nanoporous layer and are fluidly connected via nanopores 605 (3b). In step 4b, the electrode positioned below microdroplet 600a is activated to act as a cathode, and the electrode positioned below microdroplet 600b is activated to act as an anode, driving negatively charged cleaved tag/dissociated aptamers to microdroplet 600a and counting them. In step 5b, the polarity of the electrodes is reversed, driving negatively charged cleaved tag/dissociated aptamers present in microdroplet 600a to microdroplet 600b and counting them. The steps of reversing the polarity of the electrodes and counting the cleaved tag/dissociated aptamers can be repeated any number of times to obtain multiple readouts of the number of cleaved tag/dissociated aptamers in the microdroplet. The two droplets 600a and 600b can both be sample droplets (e.g., droplets containing the molecules to be counted) or buffer droplets (e.g., droplets used to wet the nanolayer before positioning the sample droplets at the nanopore). In some embodiments, one of the droplets can be a buffer droplet, while the other droplet can be a sample droplet. The tag/aptamer can be counted once or multiple times.

Figure 8 illustrates the integrated digital microfluidic and nanopore device from a side view. Substrates 91 and 92 are positioned at intervals. Substrate 92 includes electrodes 97, and substrate 91 includes an electrode array 95. A support structure 98 attaches a nanopore layer 94 to substrate 92. In other embodiments, the support structure 98 may be attached to substrate 91. Electrode array 95 is used to move droplets 99 to nanopore layer 94, where the nanopore layer cleaves the droplets and fluidly connects the two sides of the droplets via nanopores 93. Electrodes 96 and 97 are used to drive a tag/aptamer in the droplet 99 through nanopores 93. As noted above, the polarity of electrodes 96 and 97 can be reversed to transfer the tag/aptamer through the nanopores multiple times.

Although the figures depict a single nanopore, it should be understood that more than one nanopore can exist within the nanopore layer. Electrodes side-mounted to the nanopore layer and used to provide a voltage difference across it may or may not be in direct contact with the microdroplets positioned at the nanopore layer.

The movement of fluid droplets in microfluidic and nanoporous devices, modules, and integrated devices can be achieved in any suitable manner. Where applicable, the methods used to move fluid droplets in different devices/modules and channels can be the same or different. For example, fluid manipulation forces, such as electrowetting, dielectrophoresis, photoinduced electrowetting, electrode-mediated, electric field-mediated, electrostatic actuation, etc., or combinations thereof, can be used to move fluid droplets in microfluidic devices or modules. The movement of fluid droplets from a microfluidic module to a nanopore module via fluid connections (e.g., channels) can be via diffusion, Brownian motion, convection, pumping, applied pressure, gravity-driven flow, density gradient, temperature gradient, chemical gradient, pressure gradient (positive or negative), aerodynamic pressure, chemical reactions that generate gas, wicking, capillary pressure, wicking, electric field-mediated, electrode-mediated, electrophoresis, dielectric electrophoresis, magnetomigration, magnetic field, magnetically driven flow, optical force, chemotaxis, phototaxis, surface tension gradient-driven flow, Marangoni stress, thermo-capillary convection, surface energy gradient, acoustic migration, surface acoustic waves, electroosmosis, thermal migration, electrowetting, photoinduced electrowetting, or combinations thereof. Fluid droplets can move and be positioned across the nanopore layer within the nanopore module via fluid manipulation forces (e.g., electrowetting, dielectric electrophoresis, photoinduced electrowetting, electrode-mediated, electric field-mediated, electrostatically driven, etc., or combinations thereof). Tags/aptamers in microdroplets can be transferred through nanopores using electric potential, electrostatic potential, electrodynamic flow, electroosmotic flow, pressure-induced flow, electrophoresis, electrophoretic transport, electroosmotic transport, diffusion transport, electric field-mediated flow, dielectric electrophoretic-mediated tag/aptamer transport, and other methods or combinations thereof known to those skilled in the art.

An exemplary embodiment of the present invention includes counting the number of tags present in droplets positioned across a nanoporous layer as follows: first, substantially all tags are transferred to the same side of the nanoporous layer to collect all tags in cis or anti-cisional compartments, and then the tags are transferred to the other side of the nanoporous layer and the number of tags transferred through the nanopores in the nanoporous layer is counted. As used herein, “cis” and “anti-cisional” refer to opposite sides of the nanoporous layer in the context of the nanoporous layer. These terms are used in the context of both one side of the nanoporous layer and a compartment on one side of the nanoporous layer. From the description of the device, it will be understood that the cis and anti-cisional compartments can be defined by physical structures defined by walls, substrates, etc. In some cases, the cis and anti-cisional compartments can be defined by droplets placed across the nanoporous layer. The droplets can contact a wall or substrate on one or more sides of the droplets. In some cases, the cis and anti-cisional compartments can be defined by droplets, with cis compartments extending from the cis side of the nanoporous layer to the periphery of the droplet portion on the cis side, and anti-cisional compartments extending from the anti-cisional side of the nanoporous layer to the periphery of the droplet portion on the anti-cisional side. The droplet portion on each cis and anti-cis side can contact the substrate. Thus, the cis and anti-cis compartments can be defined by a combination of the periphery of the droplet, a portion of the substrate, and a nanoporous layer.

In some cases, microfluidic devices and/or microfluidic modules may include inert fluids that are immiscible with sample and reagent droplets. For example, the inert fluid may be a heavy fluid denser than water, such as an oil immiscible with the fluid droplets generated and processed in the microfluidic module. The inert fluid may promote the formation of fluid droplets and increase the stability of their shape, and may further be used to keep different droplets spatially separated from each other. Exemplary inert fluids include polar liquids, silicone oils, fluorosilicone oils, hydrocarbons, alkanes, mineral oils, and paraffin oils. In some cases, the microfluidic device or module and the inert fluid may be as disclosed in US20070242105, which is incorporated herein by reference in its entirety. In other embodiments, no immiscible fluid is included in the device. In these embodiments, ambient air fills the space within the device.

As used herein, the terms “microdroplet” and “fluid microdroplet” are used interchangeably to refer to discrete liquid volumes that are generally spherical in shape and constrained on at least two sides by the walls or substrate of a microfluidic device, nanoporous device, microfluidic module, or nanoporous module. “Generally spherical” in the context of a microdroplet indicates a shape such as a sphere, particularly an oblate spheroid, for example, a disk, a rod, a truncated sphere, an ellipsoid, a hemisphere, or an oval. The volumes of microdroplets in the microfluidic and nanoporous modules and devices disclosed herein can range from about 10 μL to about 5 pL, for example, 10 μL–1 pL, 7.5 μL–10 pL, 5 μL–1 nL, 2.5 μL–10 nL, or 1 μL–100 nL, for example, 10 μL, 1 μL, 800 nL, 400 nL, 100 nL, 10 nL, or smaller.

In some embodiments, the integrated device may include a microfluidic module with an embedded nanoporous module. The integrated device may include a first substrate and a second substrate, and a gap separating the first and second substrates, the gap (which may be filled with air or an immiscible liquid) providing space in which sample droplets contact a first binding member (immobilized on a magnetic bead or one of the two substrates); optionally, a washing step is performed; subsequently, the analyte bound to the first binding member is contacted with the second binding member; optional mixing and washing steps may be performed; and a tag attached to the second binding member is cleaved to produce droplets containing the cleaved tag. The droplets containing the cleaved tag can then be positioned across a nanoporous layer located in the gap between the first and second substrates.

As noted herein, microdroplets can be moved in integrated devices in numerous ways, such as using programmable fluid manipulation forces (e.g., electrowetting, dielectrophoresis, electrostatic actuation, electric field-mediated, electrode-mediated forces, SAW, etc.). In some cases, microfluidic devices and modules can move microdroplets of samples and reagents for analyte analysis by using electrodes. The electrodes may be coplanar, i.e., present on the same substrate, or face-oriented (biplanar), i.e., present in a first substrate and a second substrate. In some cases, microfluidic devices or modules may have electrode configurations as described in U.S. Patent No. 6,911,132 (which is incorporated herein by reference in its entirety). In some cases, the device may include a first substrate spaced from a second substrate by a gap; the first substrate may include a series of electrodes positioned on a superior surface; a dielectric layer may be disposed on the superior surface of the first substrate and cover the series of electrodes to provide a substantially planar surface for the movement of the microdroplets. Optionally, a hydrophobic material layer may be placed on the superior surface of the dielectric layer to provide a substantially planar surface. In some cases, the first substrate may include coplanar electrodes—for example, driving/control electrodes and reference electrodes present on a single substrate. In other cases, a second substrate positioned above the first substrate may include electrodes on a lower surface of the second substrate, wherein the lower surface of the second substrate faces the upper surface of the first substrate. The electrodes on the second substrate may be covered with an insulating material. The series of electrodes may be arranged longitudinally along the length of the microfluidic module, or laterally along the width of the microfluidic module, or both (e.g., a two-dimensional array or grid). In some cases, the electrode array may be activated (e.g., turned on and off) by a processor of a computer operatively coupled to a device for moving microdroplets in a programmable manner. Devices and methods for driving microdroplets in a microfluidic device are known. In exemplary cases, the microfluidic module may resemble a microdroplet driver known in the art. For example, the first (bottom) substrate may contain a patterned array of individually controllable electrodes, and the second (top) substrate may include a continuous ground electrode. A dielectric insulator coated with a hydrophobic material may be coated on the electrodes to reduce surface wettability and increase capacitance between the microdroplet and the control electrode (the patterned array of electrodes). To move a microdroplet, a control voltage can be applied to the electrodes adjacent to the microdroplet (in an electrode array), while simultaneously deactivating the electrode directly below the microdroplet. By changing the potential along the linear electrode array, electrowetting can be used to move the microdroplet along that row of electrodes.

The first and second substrates can be made of any suitable material. Suitable materials include, but are not limited to, paper, thin-film polymers, silica, silicon, treated silicon, glass (rigid or flexible), polymers (rigid, flexible, opaque or transparent) (e.g., polymethyl methacrylate (PMMA) and cyclic olefin copolymers (COC), polystyrene (PS), polycarbonate (PC), printed circuit boards, and polydimethylsiloxane (PDMS). In some cases, at least the first or second substrate can be substantially transparent. A substantially transparent substrate can be used in devices in which optical cutting of a tag attached to a second bonding member is performed. In embodiments in which coplanar electrodes are present in one of the substrates, the electrodes may or may not be transparent. In other embodiments, for example, where the electrodes are in a facing orientation (existing...) The electrode, located on at least one of two substrates, can be substantially transparent; for example, it can be made of indium tin oxide. The electrode can be made of any suitable material. It can be made of any conductive material, such as a pure metal or alloy, or other conductive material. Examples include aluminum, carbon (such as graphite), chromium, cobalt, copper, gallium, gold, indium, iridium, iron, lead, magnesium, mercury (as an amalgam), nickel, niobium, osmium, palladium, platinum, rhenium, rhodium, selenium, silicon (such as highly doped polycrystalline silicon), silver, tantalum, tin, titanium, tungsten, vanadium, zinc, zirconium, mixtures thereof, and alloys or metallic compounds of these elements. In some embodiments, the conductive material includes carbon, gold, platinum, palladium, iridium, or alloys of these metals, because such noble metals and their alloys are inert in aqueous environments.

In some cases, the first or second substrate may have a first binding member immobilized thereon within the gap. For example, the surface of the first substrate facing the surface of the second substrate may include a region on which the first binding member is disposed. As noted herein, the first binding member (e.g., a peptide, such as a receptor, antibody, or functional fragment thereof) can be immobilized on the surface of a solid substrate using any conventional method. In some cases, the first location on the surface of the first or second substrate within the gap may include only one type of binding member (e.g., a single type of antibody). In other embodiments, the first location on the surface of the first or second substrate within the gap may include only a plurality of different binding members for analyzing a variety of analytes. Alternatively, the device may include multiple locations on the surface of the first or second substrate, wherein each location may include a different first binding member immobilized thereon.

In one embodiment, the surface of a first or second substrate within the gap has multiple locations for immobilizing different first binding members, said locations being arranged linearly along the length of the device. Sample droplets can be moved linearly to sequentially contact each of said multiple locations. In another embodiment, the sample can be divided into multiple droplets, and each droplet can independently contact each of said multiple locations. As noted herein, the first binding member may not be attached to the first or second substrate and may be attached to beads, which can be introduced into the microfluidic device, for example, as droplets.

As noted herein, a sample and any reagents used to determine the sample can be manipulated into discrete volumes of fluid, which can be moved between a first and a second substrate using programmable fluid manipulation forces (e.g., electrowetting, dielectrophoresis, electrostatic actuation, electric field-mediated, electrode-mediated forces, etc.). For example, at least one of the first and second substrates may include an array of electrodes for manipulating discrete volumes of fluid, such as moving droplets from one location to another between the first and second substrates, mixing, fusing, splitting, diluting, etc. In another embodiment, surface acoustic waves can be used to move droplets for analyte analysis methods.

In another embodiment, the microfluidic module can be used for analyte analysis by moving microdroplets of samples and reagents using surface acoustic waves. In these embodiments, the first substrate can be a thin planar material that facilitates the propagation of surface acoustic waves. The first substrate can be a piezoelectric crystal layer, such as lithium niobate ( _LiNbO3 ), quartz, or _LiTaO3 wafers. In some cases, the piezoelectric wafer can be removably coupled to an upper substrate, wherein the surface acoustic waves (SAW) generated from the transducer are transported to the upper substrate via a coupling medium disposed between the piezoelectric crystal layer and the upper substrate. The upper surface of the upper substrate can be covered by a second substrate, and the microdroplets can move in the space between the second substrate and the upper surface of the upper substrate via SAW generated by mutually intersecting transducers connected to the piezoelectric crystal layer. In some cases, the microfluidic module can be a SAW microfluidic device as described in WO2011/023949 (which is incorporated herein by reference).

In an alternative embodiment, the microfluidic module may include a first surface spaced from a second surface, with a space between the first and second surfaces, wherein sample and reagent droplets are manipulated to perform the sample analyses disclosed herein. The microfluidic device may also include: a surface acoustic wave (SAW) generating material layer coupled to the first surface; and a transducer electrode structure arranged at the SAW generating material layer to provide surface acoustic waves (SAW) at the first surface for propagation to droplets on the first surface, wherein the first surface has at least one SAW scattering element for influencing the propagation, distribution, and/or behavior of the SAW at the first surface, and wherein the SAW generating material is selected from: polycrystalline materials, textured polycrystalline materials, biaxially textured polycrystalline materials, microcrystalline materials, nanocrystalline materials, amorphous substances, and composite materials. In some cases, the SAW generating material may be a ferroelectric, thermoelectric, piezoelectric, or magnetostrictive material. The arrangement of the SAW scattering element may substantially provide a phononic crystal structure that interacts with or influences the acoustic field at the first surface to affect the motion of droplets on the first surface. In some cases, the microfluidic module may be a SAW microfluidic device as described in US20130330247 (which is incorporated herein by reference). SAW microfluidic devices may be used in conjunction with nanoporous devices, or may have nanoporous modules integrated therewith.

The device described herein can be used in conjunction with one or more other devices (e.g., a power supply, a sound wave generator, etc.).

The apparatus that can be used to implement the method steps described herein may also include tools for supplying reagents and collecting waste. Such tools may include compartments, absorbent pads, reservoirs, etc. These tools may be fluidly connected to the apparatus.

Microfluidic modules can be fluidly connected to reservoirs for supplying sample analysis reagents (e.g., first binding members, second binding members, washing buffers, cleavage inducers, etc.). Nanopore modules can be fluidly connected to reservoirs for collecting waste, reservoirs for supplying conductive solutions to cis and reciprocating compartments, etc.

The integrated device can be automatic or semi-automatic and can be removably coupled to a housing containing a power supply for supplying voltage to the electrodes and a random access memory for storing instructions regarding: contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds the analyte; contacting the analyte with a second binding member, wherein the second binding member specifically binds the analyte and wherein the second binding member includes a severable tag attached thereto; removing the second binding member from the analyte not bound to the first binding member; cutting the tag attached to the second binding member, wherein the second binding member binds to the analyte bound to the first binding member; transferring the tag through or across nanopores in the layer; determining the number of tags transferred through the layer; and measuring the analyte in the sample based on the number of tags transferred through the layer in a fixed time interval or the time required to transfer a known number of tags. As noted herein, the analyte analysis method can be executed using a processor of a control device. For example, the device can be programmed to perform analyte analyses disclosed herein, including any optional mixing, incubation, and washing steps disclosed herein. The housing may also include a processor for executing instructions stored in the memory. The device described herein may include a data acquisition module (DAQ) for processing electrical signals from the nanopore device or module. In some cases, a sheet-clamped amplifier may also be included for processing the electrical signals and achieving an optimal signal-to-noise ratio.

In some cases, the apparatus described herein can be combined with a system for automating at least some steps of an analyte analysis method. An embodiment of such a system is shown in Figure 9. The exemplary system includes a processing unit 60 comprising a data processing unit 63 having a processor and memory, the data processing unit 63 being operatively coupled to a display 61 and a transmitter/receiver unit 62, the transmitter/receiver unit 62 communicating 64 with a receiver/transmitter unit 69 of the apparatus of the present invention 68. The apparatus 68 is controlled by the processing unit 60, which executes instructions (steps of a program) to perform at least some steps of the analyte analysis method disclosed herein. In some cases, the processing unit 60 may be a computer, a volumetric instrument having an opening for insertion into the integrated apparatus (the opening may be a slot, the size and shape of which are configured to adapt to and operatively connect to the apparatus), or a combination thereof. The communication 64 between the processing unit 60 and the apparatus 68 may be wired or wireless. The apparatus 68 may be any of the devices described herein with the functionality of microfluidics 66 and nanopores 67. In some cases, the motion of droplets in the devices disclosed herein can be programmed, as disclosed in U.S. Patent No. 6,294,063 (which is incorporated herein by reference in its entirety).

The various exemplary methods described herein can be implemented or performed using a general-purpose processor, digital signal processor (DSP), application-specific integrated circuit (ASIC), field-programmable gate array (FPGA) or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. The general-purpose processor may be a microprocessor, but alternatively, it may be any conventional processor, controller, microcontroller, or state machine. The processor may be part of a computing system that also has a user interface port for communicating with a user interface and receiving commands input by the user, and has at least one memory (e.g., a hard drive or other comparable memory and random access memory) for storing electronic information (including programs that run under the control of the processor and communicate via the user interface port) and a video output (which produces its output via any kind of video output format, such as VGA, DVI, HDMI, DisplayPort, or any other format).

The processor can also be implemented as a combination of computing devices, such as a combination of a DSP and a microprocessor, multiple microprocessors, one or more microprocessors combined with a DSP core, or any other such configuration. These devices can also be used to select the values of the devices described herein. The camera can be a camera based on a phototube, photodiode, automatic pixel sensor (CMOS), CCD, photoresistor, photovoltaic cell, or other digital image capture technology.

The steps of the methods or algorithms described in association with the embodiments disclosed herein can be embodied directly in hardware, in a software module executed by a processor, or in a combination of both. The software module can be housed in random access memory (RAM), flash memory, read-only memory (ROM), electrically programmable ROM (EPROM), electrically erasable programmable ROM (EEPROM), registers, hard disks, removable disks, CD-ROMs, clouds, or any other form of storage medium known in the art. An exemplary storage medium is coupled to a processor such that the processor can read information from and write information to the storage medium. Alternatively, the storage medium can be integrated with the processor. The processor and storage medium can be housed in an ASIC. The ASIC can be housed in a user terminal. Alternatively, the processor and storage medium can be housed as discrete components in the user terminal.

In one or more embodiment implementations, the functionality may be implemented in hardware, software, firmware, or any combination thereof. If implemented in software, the functionality may be stored on, transmitted on, or generated on a computer-readable medium as analytical/computational data output as one or more instructions, codes, or other information. Computer-readable media include computer storage media and communication media, including any medium that facilitates the transfer of a computer program from one place to another. Storage media may be any available non-transitory medium accessible to a computer. As examples, such computer-readable media may include RAM, ROM, EEPROM, CD-ROM or other optical disk storage, disk storage or other magnetic storage devices, or any other medium that can be used to carry or store required program code in the form of instructions or data structures and is accessible to a computer. The memory storage may also be a rotating magnetic hard disk drive, an optical disk drive, or a flash memory-based storage drive or other such solid-state, magnetic, or optical storage devices. The disks and discs used herein include compact disks (CDs), laser discs, optical discs, digital versatile optical discs (DVDs), floppy disks, and Blu-ray discs, wherein disks often magnetically reproduce data, while optical discs optically reproduce data using lasers. All of the above combinations should also be included within the scope of computer-readable media.

Within the scope of embodiments disclosed herein, including memory, storage, and/or computer-readable media or those operating therewith, such memory, storage, and/or computer-readable media is non-transitory. Therefore, within the scope of memory, storage, and/or computer-readable media covered by one or more claims, such memory, storage, and/or computer-readable media is merely non-transitory.

In some cases, the device may be a microfluidic device, such as a lab-on-chip device, a continuous flow microfluidic device, or a droplet-based microfluidic device, wherein analyte analysis can be performed in sample droplets containing or suspected of containing the analyte. Exemplary microfluidic devices that can be used in the methods of the present invention include those described in WO2007136386, US8287808, WO2009111431, WO2010040227, WO2011137533, WO2013066441, WO2014062551, or WO2014066704. In some cases, the device may be a digital microfluidic device (DMF), a surface acoustic wave-based microfluidic device (SAW), a fully integrated DMF and nanopore device, or a fully integrated SAW and nanopore device. In some embodiments, DMF elements and nanoporous elements are operatively coupled in a fully integrated DMF and nanoporous device, or SAW elements and nanoporous elements are operatively coupled in a fully integrated SAW and nanoporous device. In some embodiments, the DMF device or SAW device is fabricated using a roll-to-roll printed electronics method. In some embodiments, the DMF element or SAW element is fabricated using a roll-to-roll printed electronics method. In some embodiments, the fully integrated DMF and nanoporous device or the fully integrated SAW and nanoporous device includes a microfluidic conduit. In some embodiments, the microfluidic conduit couples the DMF element to the nanoporous element, and the microfluidic conduit includes a fluid flow induced by a powered or active force.

An exemplary electrowetting technique can be found in US8637242. Micrometer-scale electrophoresis, such as that described in WO2011057197, can also be utilized. An exemplary dielectric electrophoresis technique is described in US6294063.

The devices of the present invention typically do not require external pumps and valves, and are therefore economical in terms of preparation and application. The devices and related systems disclosed herein, as well as all methods disclosed herein, can be used for applications in the art, such as analyzing samples at the source of the sample, for example, at the point of care (e.g., in clinics, hospitals, physicians' offices, core laboratory equipment, at home, etc.). In some cases, the devices or systems of the present invention (e.g., also used as in the methods disclosed herein) include a heat source or light source configured to induce cutting of a thermally cuttable or optically cuttable connector that attaches a tag to the analyte when said heat source or light source is activated, as described herein.

This invention also describes microfluidic devices used in conjunction with nanopore realization devices and integrated microfluidic nanopore realization devices. A nanopore realization device refers to a device comprising a layer or membrane in which nanopores can be formed. The nanopore realization device of this invention comprises two compartments spaced by a layer or membrane, wherein the two compartments comprise an ionic liquid (e.g., a salt solution, containing or not containing a target analyte) for guiding an electric current. Nanopores can be formed in the layer of the nanopore realization device as follows: a voltage is applied across the layer using an ionic liquid (e.g., a salt solution, containing or not containing a target analyte) in the compartment. As will be understood, any nanopore device described herein (used in conjunction with a microfluidic device or integrated with a microfluidic module) can initially be provided as such a nanopore realization device: comprising a layer in which nanopores can be formed, but lacking nanopores. During use, for example, prior to the transfer of a nanopore detection tag, nanopores can be formed in the nanopore realization device. In some embodiments, an ionic liquid (e.g., a salt solution) containing a tag to be detected through the nanopore can be used to form the nanopore and for transferring the tag across the formed nanopore.

In some implementations, the properties of the nanopores established by applying voltage across layers, as described above, are evaluated by measuring the noise level in the current when a baseline voltage is applied across the nanopore layer or membrane.

In some cases, nanopores established by applying voltage across layers as described above can be modulated to physically alter the nanopores and obtain desired electroosmotic properties, for example, increasing the pore size and/or reducing noise in the current measured across the nanopore when a voltage is applied across the nanopore layer or membrane. Thus, in some embodiments, methods for generating nanopores in an integrated digital microfluidic nanopore realization device may include modulating the nanopores. Modulation may include: alternately applying a first voltage having a first polarity and a second voltage having a second polarity opposite to the first polarity across the nanopore layer or membrane, wherein the first voltage and the second voltage are each applied at least once; and measuring the electroosmotic properties in relation to the size of the nanopore. In some cases, the electroosmotic properties in relation to the size of the nanopore are measured prior to the modulation to obtain a preliminary estimate of the nanopore size.

The electroosmotic property can be any suitable property that provides an estimate of the size of the nanopores. In some cases, the electroosmotic property is represented by a current-voltage curve obtained over a voltage range (-1V to 1V, e.g., -500mV to 500mV, -250mV to 250mV, -200mV to 200mV, 10mV to 500mV, 10mV to 250mV, 10mV to 200mV, including 15mV to 200mV). In other cases, the electroosmotic property is the conductivity or resistance measured across the nanopore layer or membrane.

To modify the nanopores and obtain the desired electroosmotic properties, the first and second voltages can have any suitable order of magnitude. In some cases, the first and second voltages have an order of magnitude of 100 mV or more, such as 200 mV or more, 500 mV or more, 750 mV or more, 1.0 V or more, 2.0 V or more, 3.0 V or more, including 4.0 V or more, and in other cases, they have an order of magnitude of 10 V or less, such as 9.0 V or less, 8.0 V or less, 6.0 V or less, including 4.0 V or less. In some embodiments, the first and second voltages have an order of magnitude in the range of 100 mV to 10 V, such as 200 mV to 9.0 V, 250 mV to 9.0 V, 500 mV to 9.0 V, 1.0 V to 8.0 V, including 2.0 V to 6.0 V.

To modify the nanopores and obtain the desired electroosmotic properties, the first and second voltages can each be applied for any suitable duration. In some cases, the first and second voltages are each applied for 10 milliseconds (ms) or more, such as 100 ms or more, 200 ms or more, 500 ms or more, 1 second (s) or more, 2 s or more, including 3 s or more, and in other cases, they are applied for 10 s or less, such as 5 s or less, 4 s or less, 3 s or less, 2 s or less, 1 s or less, 500 ms or less, 200 ms or less, including 100 ms or less. In some cases, the first and second voltages are each applied for a duration ranging from 10 ms to 100 ms, 100 ms to 200 ms, 200 ms to 500 ms, 500 ms to 1 s, 1 s to 2 s, 2 s to 3 s, 3 s to 4 s, 3 s to 5 s, or 3 s to 10 s.

To modify the nanopores and obtain the desired electroosmotic properties, the first voltage and the second voltage can each be applied any suitable number of times. In some cases, the first voltage and the second voltage are each applied 2 or more, 3 or more, 4 or more, 5 or more, 7 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 200 or more, including 500 or more, and in some embodiments, applied 10,000 times or less, for example, 5,000 times or less, 1,000 times or less, 500 times or less, 400 times or less, 200 times or less, 100 times or less, including 50 times or less. In some implementations, the first voltage and the second voltage are each applied 2-50 times, 10-50 times, 30-50 times, 50-100 times, 100-200 times, 100-500 times, 500-1,000 times, 500-1,000 times, or 500-10,000 times.

4. Integration of nanopore modules on one side of a DMF module

One aspect of the invention includes an integrated device comprising a digital microfluidic (DMF) module and a nanopore layer positioned on an outer surface of the DMF module (FIG. 40). Microdroplets within the internal space of the DMF module can access the nanopores of the nanopore layer through pores (also referred to as “openings”) present in a first (e.g., top) or second (e.g., bottom) substrate of the DMF module or through a side of the DMF module between the first and second substrates. As described above, the nanopore layer may comprise a nanopore membrane or substrate, which in some cases may be a commercially available silicon nitride (SiN_{_x} ) film visible in a transmission electron microscope (TEM) window. The nanopore layer forms a seal over the pores such that, in the absence of nanopores (i.e., prior to the fabrication of nanopores, as described herein), the liquid volume within the DMF module is physically separated from any liquid volume on or around the outer surface of the nanopore layer. In some cases, the nanopore layer is part of the nanopore module, wherein the nanopore layer spaces the compartments within the nanopore module from the liquid volumes (e.g., liquid droplets in the substrate pores, as described above) within the DMF module. By sealing nanoporous layers or modules to the outer surface of a substrate, liquid volumes (e.g., liquid droplets in substrate pores) are physically separated from the external environment.

The size of the pores in the substrate (through which liquid droplets in the DMF approach the nanoporous layer) can be configured to accommodate the movement of liquid droplets through the pores via capillary action. Thus, the pores in the substrate can be capillary channels. The pores can have any suitable cross-sectional shape and size to support the passive (e.g., capillary action) movement of liquid droplets through the pores. In some cases, the diameter of the pores on the side surfaces of the DMF is wider than the diameter of the pores on the outer side surfaces (i.e., the side facing the nanoporous layer). In some cases, the angle between the bottom surface of the substrate and the wall of the pore is a right angle or an obtuse angle (e.g., 90° or greater, such as 95° or greater, including 100° or greater).

The integrated DMF-nanopore module device may include a pair of electrodes that can be used to fabricate nanopores in the nanopore layer and/or to detect target analytes that have been processed by the DMF module, as described elsewhere herein. The electrode pair may be made of any suitable material, including, but not limited to, indium tin oxide (ITO). The electrode pair may be constructed in any suitable manner. In some embodiments, one electrode is positioned within a compartment within the nanopore module, and a second electrode is positioned within the DMF module by physically penetrating the substrate to access a liquid volume on the other side of the nanopore layer (Figure 40).

In some embodiments, the first electrode may be the same electrode as a single continuous electrode (e.g., a reference electrode) used in the DMF module, and the second electrode may be disposed on the top surface (i.e., the outer surface) of the substrate, opposite to the bottom surface on which the first electrode is positioned (Figure 43). In such cases, the top surface may be treated in a similar manner to the bottom surface (e.g., coated with an electrode material such as indium tin oxide and a polymer such as polytetrafluoroethylene). Thus, in some cases, when the second electrode is an electrode on the top surface of the substrate (to which the nanopore layer/module is attached), the liquid volume relative to the outer surface of the nanopore layer of the DMF module makes electrical contact with the second electrode. The electrical channel for nanopore fabrication can be represented as: second electrode -> liquid (outside) -> nanopore membrane (without nanopores) -> liquid (inside the DMF module) -> first electrode (same as a single continuous electrode of the DMF). The second electrode may also lack the region to which the nanopore layer/module is attached, thereby forcing current into the liquid on the outside of the nanopore membrane, which in some cases may be contained within the nanopore module.

In some embodiments, as shown in FIG. 44, the first electrode is the same as the single continuous electrode (e.g., reference electrode) used in the first DMF module (e.g., the “bottom DMF chip” in FIG. 44), and the second electrode may be provided by a second DMF module (e.g., the “top DMF chip” in FIG. 44), which has pores in a corresponding top substrate associated with the single continuous electrode of the second DMF module, and a nanopore layer is inserted between the two DMF modules between the pores in the respective substrates. Thus, the first and second DMF modules may be oriented inverted relative to each other, such that the top substrate associated with the single continuous electrode of the first DMF module faces the top substrate associated with the single continuous electrode of the second DMF module and is proximal to the latter. The two DMF modules may be positioned relative to each other such that, when nanopores are present in the nanopore layer, the two DMF modules are fluidically and electrically coupled together via a nanopore membrane. Prior to nanopore formation, the two fluid volumes in the two DMF modules may be separated from each other. In some cases, a structural layer is inserted between the two DMF modules to provide structural support and reduce bending.

This document also provides a method for fabricating nanopores in an integrated DMF-nanopore module device within a nanopore realization layer, as described above. Implementation of the method may include: positioning an ionic liquid, such as a salt solution (e.g., LiCl, KCl, etc.), into a pore in the DMF module using any suitable method, as described herein, and allowing capillary action to move the liquid through the pore (see, for example, Figure 40). The ionic liquid (e.g., salt solution) may be positioned on the other side of the nanopore realization layer (i.e., the nanopore membrane prior to nanopore fabrication). Using any suitable method, such as, but not limited to, PDMS, pressure, wax, adhesives, etc., the nanopore module may be sealed from the DMF module such that the liquid volume within the pore is separated from the liquid volume on the other side of the nanopore membrane. The application of an electric field (such as voltage) across the nanopore realization layer leads to the eventual formation of the nanopore, which can be readily detected, for example, as dielectric breakdown in current traces.

After establishing nanopores in the nanoporous layer, in some cases, conditioning processes may be performed to physically alter the nanopores and cleaning signals. In some cases, this conditioning involves varying the voltage applied across the nanopores over time.

After the nanopores are fabricated, the DMF module can be reactivated to complete any liquid pretreatment steps required for transfer (e.g., replacing the solution in the DMF, such as replacing KCl with LiCl). Following pretreatment, a volume of DMF liquid (e.g., a liquid sample containing the target analyte) can be positioned within the pores. The DMF system can then be deactivated, and the nanopore module can be activated to allow and detect transfer events.

5. Variations of the method and variations of the apparatus used

The disclosed methods for determining the presence or amount of a target analyte in a sample and the applications of the microfluidic device can be as described above. The methods and applications of the disclosed microfluidic device can also be modified in consideration of other methods used to analyze the analyte. Examples of well-known variations include, but are not limited to, immunoassays, such as sandwich immunoassays (e.g., monoclonal-polyclonal sandwich immunoassays), including enzyme assays (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA), competitive inhibition immunoassays (e.g., forward and reverse), enzyme multiple immunoassay techniques (EMIT), competitive binding assays, bioluminescent resonance energy transfer (BRET), one-step antibody detection assays, homogeneous assays, heterogeneous assays, capture-on-the-fly assays, etc. In some cases, the following description may overlap with the methods described above; in other cases, the following description may provide an alternative.

a) Immunoassay

Immunoassays can be used to analyze target analytes and/or peptides or fragments thereof. The presence or amount of a target analyte can be determined by using the antibodies described herein and detecting specific binding to the target analyte. Any immunoassay can be used. The immunoassay can be, for example, an enzyme-linked immunosorbent assay (ELISA), a competitive inhibition assay such as a forward or reverse competitive inhibition assay, or a competitive binding assay. In some embodiments, a tag is attached to both a capture antibody and a detection antibody. Alternatively, microparticles or nanoparticles used for capture can also function for detection (e.g., where they are attached or bound in some way to a cleavable adapter).

Heterogeneous forms can be used. For example, after obtaining a test sample from a subject, a first mixture is prepared. The mixture contains the test sample to be evaluated against a target analyte and a first specific binding coupler, wherein the first specific binding coupler and any target analyte contained in the test sample form a first specific binding coupler-target analyte complex. Preferably, the first specific binding coupler is an anti-target analyte antibody or a fragment thereof. The order in which the test sample and the first specific binding coupler are added to form the mixture is not critical. Preferably, the first specific binding coupler is immobilized on a solid phase. The solid phase used in the immunoassay (for the first specific binding coupler, and optionally, the second specific binding coupler) can be any solid phase known in the art, such as, but not limited to, magnetic particles, beads, nanobeads, microbeads, nanoparticles, microparticles, membranes, scaffold molecules, thin films, filter paper, disks, or chips (e.g., microfluidic chips).

After forming a mixture containing a first specific binding partner-target analyte complex, any unbound target analyte is removed from the complex using any technique known in the art. For example, unbound target analytes can be removed by washing. However, ideally, the first specific binding partner is present in excess of any target analyte present in the test sample, such that all target analytes present in the test sample are bound by the first specific binding partner.

After removing any unbound target analyte, a second specific binding conjugate is added to the mixture to form a first specific binding conjugate-target analyte-second specific binding conjugate complex. The second specific binding conjugate is preferably an anti-target analyte antibody that binds to an epitope on the target analyte, the epitope being different from the epitope on the target analyte bound by the first specific binding conjugate. Furthermore, it is also preferred that the second specific binding conjugate contains or is labeled with a detectable marker (e.g., a cleavable adapter-attached tag, as described above).

The application of immobilized antibodies or fragments thereof can be integrated into immunoassays. The antibodies can be immobilized on a variety of supports, such as magnetic or chromatographic matrix particles, latex particles or modified surface latex particles, polymers or polymer films, plastics or plastic films, planar substrates, microfluidic surfaces, fragments of solid substrate materials, etc.

b) Sandwich immunoassay

Sandwich immunoassays measure the amount of antigen between two antibody layers (i.e., a capture antibody (i.e., at least one capture antibody) and a detection antibody (i.e., at least one detection antibody)). The capture antibody and the detection antibody bind to different epitopes on the antigen (e.g., the target analyte). Ideally, the binding of the capture antibody to the epitope should not interfere with the binding of the detection antibody to the epitope. Monoclonal or polyclonal antibodies can be used as both the capture and detection antibodies in sandwich immunoassays.

Typically, at least two antibodies are used to separate and quantify a target analyte in a sample. More specifically, the at least two antibodies bind to form an immune complex (referred to as a "sandwich") containing certain epitopes or fragments of the target analyte. One or more antibodies may be used to capture the target analyte in the test sample (these antibodies are often referred to as "capture" antibodies), and one or more antibodies that also bind to the target analyte and have a detectable label (e.g., a tag attached to a cleavable linker) may be used to complete the sandwich (these antibodies are often referred to as "detect" antibodies or "detection" antibodies). In some embodiments, an aptamer may be used as a second binding member and may act as a detectable tag. In a sandwich assay, the binding of any other antibody to its respective epitope in the assay ideally does not reduce the binding of an antibody to its epitope. In other words, antibodies are selected such that one or more first antibodies that come into contact with a test sample suspected of containing the target analyte do not bind to all or part of the epitopes recognized by a second or subsequent antibody, thereby interfering with the ability of one or more second detection antibodies to bind the target analyte.

In a preferred embodiment, a test sample suspected of containing a target analyte may be contacted simultaneously or sequentially with at least one capture antibody and at least one detection antibody. In a sandwich assay, the test sample suspected of containing a target analyte (membrane-associated target analyte, soluble target analyte, fragment of a membrane-associated target analyte, fragment of a soluble target analyte, variant of a target analyte (membrane-associated or soluble target analyte), or any combination thereof) is first contacted with at least one capture antibody that specifically binds to a specific epitope under conditions allowing the formation of an antibody-analyte complex. If more than one capture antibody is used, multiple capture antibody-analyte complexes are formed. In a sandwich assay, an antibody, preferably at least one capture antibody, is used in a maximum molar excess relative to the target analyte or target analyte fragment anticipated in the test sample.

Optionally, before contacting the test sample with at least one first capture antibody, the capture antibody may be bound to a solid support that facilitates the separation of the antibody-target analyte complex from the test sample. Any solid support known in the art may be used, including, but not limited to, solid supports made of polymeric materials in the form of planar substrates or beads. The antibody may be bound to the solid support by adsorption or by other means known in the art, using chemical coupling agents or through other means, provided that such binding does not interfere with the antibody's ability to bind the target analyte or target analyte fragments. Furthermore, if necessary, the solid support may be derivatized to allow reactivity with various functional groups on the antibody. Such derivatization requires the use of certain coupling agents, such as, but not limited to, maleic anhydride, N-hydroxysuccinimide, azide, alkynyl, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

After contacting a test sample suspected of containing the target analyte with at least one capture antibody, the test sample is incubated to allow the formation of a capture antibody-target analyte complex. The incubation may be performed at a pH of about 4.5 to about 10.0, at a temperature of about 2°C to about 45°C, and for a duration of at least about 1 minute to about 18 hours, about 2-6 minutes, or about 3-4 minutes.

After the capture antibody-target analyte complex is formed, the complex is then contacted with at least one detection antibody (under conditions that allow for the formation of the capture antibody-target analyte-detection antibody complex). If the capture antibody-target analyte complex is contacted with more than one detection antibody, then a capture antibody-target analyte-detection antibody detection complex is formed. Similar to the capture antibody, when at least one detection (and subsequently) antibody is contacted with the capture antibody-target analyte complex, the formation of the capture antibody-target analyte-detection antibody complex requires an incubation period under conditions similar to those described above. Preferably, at least one detection antibody contains a detectable label (e.g., a tag attached to a cleavable adapter). The detectable label may bind to at least one detection antibody before, during, or after the formation of the capture antibody-target analyte-detection antibody complex. Any detectable label known in the art can be used, such as cleavable adapters as discussed herein, and others known in the art.

The order in which the test sample and the specific binding partner are added to form the assay mixture is not critical. If the first specific binding partner is detectably labeled (e.g., a tag attached to a cuttable connector), then a detectably labeled first specific binding partner-target analyte complex is formed. Alternatively, if a second specific binding partner is used and the second specific binding partner is detectably labeled (e.g., a tag attached to a cuttable connector), then a detectably labeled first specific binding partner-target analyte-second specific binding partner complex is formed. Any unbound specific binding partners, whether labeled or unlabeled, can be removed from the mixture using any technique known in the art, such as washing.

Next, a signal is generated indicating the presence of the target analyte or a fragment thereof. Based on the parameters of the generated signal, the amount of the target analyte in the sample can be quantified. Optionally, a standard curve can be generated using a series of dilutions or solutions of the target analyte at known concentrations, by mass spectrometry, specific gravity determination methods, and other techniques known in the art.

c) Positive competitive inhibition

In the positive competition format, aliquots of a labeled target analyte (e.g., analytes with tags attached to a cleavable connector) at known concentrations compete with the target analyte in the test sample for binding to the target analyte antibody.

In a forward competitive assay, an immobilized specific binding partner (such as an antibody) can be contacted sequentially or simultaneously with the test sample and a labeled target analyte, its target analyte fragment, or target analyte variant. The target analyte peptide, target analyte fragment, or target analyte variant can be labeled with any detectable tag, including detectable tags containing tags attached to cleavable linkers. In this assay, the antibody can be immobilized on a solid support. Alternatively, the antibody can be coupled to an antibody already immobilized on a solid support (such as microparticles or a planar substrate), such as an anti-species antibody.

This document provides a method for measuring or detecting an analyte present in a biological sample. The method includes: contacting the sample with a binding member, wherein the binding member is immobilized on a solid support and wherein the binding member specifically binds the analyte; contacting the sample with a labeled analyte, the sample possibly containing the analyte bound to the binding member, wherein the labeled analyte is labeled with a severable tag; removing the labeled analyte not bound to the binding member; cutting the tag, the tag being attached to the labeled analyte bound to the binding member; transferring the cut tag through or across one or more nanopores in a layer; and evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or detecting the tags transferred through the layer detects the analyte present in the sample. In some embodiments, the evaluation of the tags transferred through the layer measures the amount of analyte present in the sample. In some implementations, the label that detects transfer across the layer is evaluated, wherein the label that detects transfer across the layer detects the analyte present in the sample.

This document provides a method for measuring or detecting an analyte present in a biological sample. The method includes: contacting the sample with a binding member, wherein the binding member is immobilized on a solid support, and wherein the binding member specifically binds the analyte; contacting the sample with a labeled analyte, the sample possibly containing the analyte bound to the binding member, wherein the labeled analyte comprises an aptamer; removing the labeled analyte not bound to the binding member; dissociating the aptamer bound to the labeled analyte bound to the binding member, and transferring the dissociated aptamer through or across one or more nanopores in a layer; and evaluating the aptamers transferred through the layer, wherein measuring the number of aptamers transferred through the layer measures the amount of analyte present in the sample, or detecting the aptamers transferred through the layer detects the analyte present in the sample. In some embodiments, the evaluation of the aptamers transferred through the layer is performed, wherein the number of aptamers transferred through the layer measures the amount of analyte present in the sample. In some implementations, the aptamer that has migrated across the layer is evaluated, wherein the tag that has migrated across the layer detects the analyte present in the sample.

Under conditions similar to those described above for sandwich assays, the labeled target analyte, test sample, and antibody are incubated. Two different antibody-analyte complexes can then be generated. Specifically, one of the generated antibody-analyte complexes contains a detectable label (e.g., a tag), while the other does not. The antibody-analyte complex may, but does not necessarily, separate from the remainder of the test sample before quantifying the detectable label. Regardless of whether the antibody-analyte complex is separated from the remainder of the test sample, the amount of the detectable label in the antibody-analyte complex is then quantified. The concentration of the target analyte (such as a membrane-associated target analyte, a soluble target analyte, a fragment of a soluble target analyte, a variant of the target analyte (membrane-associated or soluble), or any combination thereof) in the test sample can then be determined, for example, as described above. If advantageous, this can be determined by comparing the amount of the detectable label in the antibody-analyte complex with a standard curve. A standard curve can be generated using a series of dilutions of a target analyte of known concentration (such as a membrane-associated target analyte, a soluble target analyte, a fragment of a soluble target analyte, a variant of a target analyte (membrane-associated or soluble target analyte), or any combination thereof), wherein the concentration is determined by mass spectrometry, specific gravity determination, and other techniques known in the art.

Optionally, the antibody-target analyte complex can be separated from the test sample by binding the antibody to a solid support, such as the solid support discussed above with respect to sandwich assays, and then removing the remaining portion of the test sample from contact with the solid support.

d) Reverse competition measurement

In reverse competitive assays, immobilized target analytes can be sequentially or simultaneously exposed to the test sample and at least one labeled antibody.

This document provides a method for measuring or detecting an analyte present in a biological sample. The method includes: contacting the sample with a binding member, wherein the binding member specifically binds the analyte, and the binding member is labeled with a severable tag; contacting the sample with an immobilized analyte, the sample possibly containing the analyte bound to the binding member, wherein the immobilized analyte is immobilized on a solid support; removing binding members not bound to the immobilized analyte; slicing the tag, the tag being attached to a binding member bound to the immobilized analyte; transferring the sliced tag through or across one or more nanopores in a layer; and evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or detecting the tags transferred through the layer detects the analyte present in the sample. In some embodiments, evaluating the tags transferred through the layer measures the amount of analyte present in the sample. In some embodiments, evaluating the tags transferred through the layer detects the analyte present in the sample.

This document provides a method for measuring or detecting an analyte present in a biological sample. The method includes: contacting the sample with a binding member, wherein the binding member specifically binds the analyte and the binding member comprises an aptamer; contacting the sample with an immobilized analyte, the sample possibly containing the analyte bound to the binding member, wherein the immobilized analyte is immobilized on a solid support; removing binding members not bound to the immobilized analyte; dissociating the aptamer bound to the binding member bound to the immobilized analyte and transferring the dissociated aptamer through or across one or more nanopores in a layer; and evaluating the aptamers transferred through the layer, wherein measuring the number of aptamers transferred through the layer measures the amount of analyte present in the sample, or detecting the aptamers transferred through the layer detects the analyte present in the sample. In some embodiments, the evaluation of the aptamers transferred through the layer is performed, wherein the number of aptamers transferred through the layer measures the amount of analyte present in the sample. In some implementations, the aptamer that has migrated across the layer is evaluated, wherein the tag that has migrated across the layer detects the analyte present in the sample.

The target analyte can be bound to a solid support, such as the solid support discussed above regarding sandwich assays.

The target analyte, test sample, and at least one labeled antibody are incubated under conditions similar to those described above for sandwich assays. Two distinct target analyte-antibody complexes are then generated. Specifically, one of the generated target analyte-antibody complexes is immobilized and contains a detectable label (e.g., a tag attached to a cleavable adapter), while the other target analyte-antibody complex is not immobilized and also contains a detectable label. The unimmobilized target analyte-antibody complex and the remainder of the test sample are removed from the presence of the immobilized target analyte-antibody complex using techniques known in the art, such as washing. Once the unimmobilized target analyte-antibody complex is removed, the amount of detectable label in the immobilized target analyte-antibody complex is quantified after the label is cleaved. The concentration of the target analyte in the test sample can then be determined by comparing the amount of detectable label as described above. This can be accomplished using a standard curve, if advantageous. A standard curve can be generated using a series of dilutions of the target analyte or a fragment of the target analyte at known concentrations, wherein the concentration is determined by mass spectrometry, hydrometry, and other techniques known in the art.

e) One-step immunoassay or in-run capture assay

In a one-step immunoassay or in-run capture assay, a solid substrate is pre-coated with an immobilization reagent. The capture agent, analyte, and detector are added together to the solid substrate, followed by a washing step prior to detection. The capture agent may bind to the analyte and contain a ligand of the immobilization reagent. The capture agent and detector may be antibodies or any other portion described herein or known in the art capable of capturing or detecting. The ligand may contain a peptide tag, and the immobilization reagent may contain an anti-peptide-tagged antibody. Alternatively, the ligand and the immobilization reagent may be any reagent pair capable of binding together for use in an in-run capture assay (e.g., specific binding pairs and others, such as those known in the art). More than one analyte can be measured. In some embodiments, the solid substrate may be coated with an antigen, and the analyte to be analyzed is an antibody.

In some embodiments, a solid support (such as microparticles) pre-coated with an immobilization reagent (such as biotin, streptomycin, etc.) and at least a first specific binding member and a second specific binding member (which function as a capture reagent and a detection reagent, respectively) are used. The first specific binding member contains a ligand of the immobilization reagent (e.g., if the immobilization reagent on the solid support is streptomycin, the ligand on the first specific binding member may be biotin) and also binds the target analyte. The second specific binding member contains a detectable label and binds the target analyte. The solid support and the first and second specific binding members can be added to the test sample (sequentially or simultaneously). The ligand on the first specific binding member binds to the immobilization reagent on the solid support to form a solid support/first specific binding member complex. Any target analyte present in the sample binds to the solid support/first specific binding member complex to form a solid support/first specific binding member/analyte complex. The second specific binding member binds to the solid support/first specific binding member/analyte complex and detects the detectable label. An optional washing step may be performed prior to detection. In some embodiments, more than one analyte can be measured in a one-step assay. In some other embodiments, more than two specific binding members may be employed. In some other embodiments, multiple detectable markers may be incorporated. In some other embodiments, multiple target analytes may be detected.

The application of a one-step immunoassay or in-run capture assay can be performed in a variety of forms described herein and known in the art. For example, the forms described may be sandwich assays as described above, but alternatively, competitive assays may be used, employing a single specific binding member, or using other variants such as those known.

f) Combined determination (using Ag/Ab co-coated particles)

In combinatorial assays, a solid substrate (such as microparticles) is co-coated with antigens and antibodies to capture antibodies and antigens from the sample, respectively. A solid support can be co-coated with two or more different antigens to capture two or more different antibodies from the sample. A solid support can be co-coated with two or more different antibodies to capture two or more different antigens from the sample.

Additionally, the methods described herein can use blocking agents to prevent specific or non-specific binding reactions between the assay compounds (e.g., HAMA involved). Once the reagent (and optionally any control) is immobilized on a support, any remaining binding sites of the reagent can be blocked on the support. Any suitable blocking agent known to those skilled in the art can be used. For example, bovine serum albumin (“BSA”), casein in phosphate-buffered saline (“PBS”) solution, Tween 20™ (Sigma Chemical Company, St. Louis, Mo.), or other suitable surfactants, as well as other blocking agents, can be used.

As will be understood from this invention, the methods and apparatus disclosed herein (including variations) can be used to diagnose diseases, disorders, or conditions in subjects suspected of having them. For example, the sample analysis can be used to detect disease biomarkers, such as cancer biomarkers, cardiac biomarkers, toxins, pathogens, such as viruses, bacteria, or parts thereof. The methods and apparatus can also be used to measure analytes present in biological samples. The methods and apparatus can also be used in blood screening assays to detect target analytes. The blood screening assays can be used to screen blood supply.

6. Counting and Data Analysis

The number of transfer events can be determined qualitatively or quantitatively using any conventional techniques known in the art. In some embodiments, the number of transfer events can be determined by first calculating the expected current changes observed in double-stranded DNA transfer events under experimental test conditions using the following equation:

As cited in Kwok et al., “Nanopore Fabrication by controlled Dielectric Breakdown” Supplementary Information, Part 8, and Kwok, H.; Briggs, K.; and Tabard-Cossa, V.; “Nanopore Fabrication by Controlled Dielectric Breakdown” - PLoS ONE 9(3):e92880 (2014). Using the expected current blocking value, the binary data of the experimental nanopore output can be scanned visually or manually for acceptable expected current blocking events. Using these events, the threshold and hysteresis parameters required by the CUSUM nanopore software can be applied and executed. The output from the software can be further analyzed using the cusumtoolsreadevents.py software and filtering blocking events greater than 1000 pA (as determined from the first calculation). Flux events, the time between events, and other calculations can be determined from the readevents.py analysis tool. The data generated by CUSUM can be further calculated using JMP software (SAS Institute, Cary, North Carolina). Other threshold setting methods known in the art for data analysis can also be used.

7. Qualitative Analysis

Qualitative assays can be performed using the methods and procedures described herein. Direct assays can be performed using cleavable connector conjugates, as described in Example 17, with a thiol-based cleavage step, as shown in Figure 25. It should be understood that other cleavable connector schemes for performing such assays may include, but are not limited to, various other methods of cleaving connectors, thereby allowing counting of various tags as described herein. Additionally, aptamers may be employed. For example, in addition to the methods described in Example 17, such alternative cleavage methods and/or reagents may include those described in Examples 16, 18, 19, 20, and 21, as well as other cleavage methods described herein and known to those skilled in the art. It is also understood that although the assay format demonstrated in this example (Example 24) represents a direct assay, other formats such as sandwich immunoassays and/or various competing assay formats (including in-run capture formats, as known to those skilled in the art) can also be used to perform assays using the methods described herein.

For example, the sandwich immunoassay for TSH (thyroid-stimulating hormone) detection, as described in Example 9, demonstrates the ability to perform such assays on a low-cost DMF chip. Furthermore, many different bioconjugation reagents can be synthesized using a variety of heterobifunctional cleavable adapters, such as those described in Examples 1, 2, 3, 4, 5, and 6, and other cleavable adapters known in other ways to those skilled in the art. These can be used to generate immunoconjugates or other active, specific binding members having cleavable adapters. Immunoconjugates suitable for practicing the invention can be synthesized by methods such as those described in Examples 3, 4, 5, and 6, and other methods known to those skilled in the art. Additionally, Example 8 demonstrates the functionality of various fluidic droplet operations on a low-cost chip, facilitating the various steps required for various assay formats, including sandwich and competitive assay formats, and including in-run capture formats, as well as other variations known to those skilled in the art. Example 11 illustrates the fabrication of nanopores that can be used to count cleavable markers in an assay; however, it should be understood that other methods known to those skilled in the art for nanopore fabrication can also be used for this purpose. Example 16 also represents another construct that can be used for the assay, in which cleavage is achieved, resulting in the release of countable markers that can be counted using the nanopore counting method described in this example. This construct, and other constructs that will be appreciated by those skilled in the art, can be used in the assays described herein.

Example 22 illustrates how counting can typically be performed to measure transfer events associated with the presence of multiple markers passing through nanopores. Figure 29 illustrates the concept of a signal threshold, enabling manipulation of the characteristics of data in a counting assay. Figure 28 shows qualitative assay data, representing the type of data that can be used to determine the presence of an analyte using the assay methods described in this example. It is also understood that although dsDNA is used as the marker in this particular example, other markers, such as those described in Examples 5 and/or 22, including, but not limited to, nanobeads, dendritic polymers, etc., can also be utilized. Furthermore, other known markers may also be employed. Such constructs required to produce suitable reagents can be synthesized through the various embodiments described herein or otherwise by methods known to those skilled in the art.

8. Quantitative analysis

Quantitative assays can be performed using the methods and procedures described herein. Direct assays can be performed using cleavable connector conjugates, as described in Example 17, with a thiol-based cleavage step, and as shown in Figure 25. It should be understood that other cleavable connector schemes for performing such assays may include, but are not limited to, various other cleavage methods of the connector, thereby allowing the use of nanopores to count various tags, as described herein. Additionally, aptamers may be employed. For example, in addition to the methods described in Example 17, such other cleavage methods may include, but are not limited to, those described in Examples 18, 19, 20, and 21, as well as other methods described herein and known to those skilled in the art. It is also understood that although the assay format demonstrated in this example (Example 25) represents a direct assay, other formats such as sandwich immunoassays and/or various competing assay formats (including in-run capture formats, as known to those skilled in the art) can also be used for assays.

For example, the sandwich immunoassay for TSH (thyroid-stimulating hormone) detection, as described in Example 9, demonstrates the ability to perform such assays on a low-cost DMF chip. Furthermore, using a variety of heterobifunctional cleavable adapters and conjugates synthesized by methods (such as those described in Examples 1, 2, 3, 4, 5, and 6), as well as other cleavable adapters or conjugates that can be synthesized by methods known to those skilled in the art, many different bioconjugation reagents can be synthesized, which can be used to generate immunoconjugates or other active, specific binding members with cleavable adapters. Additionally, Example 8 demonstrates the functionality of various fluidic microdroplet operations on a low-cost chip, facilitating the various steps required for performing various assays, including sandwich and competitive assays, including in-run capture assays, and other variations thereof known to those skilled in the art. Example 16 also represents another construct that can be used to perform the assay, in which cleavage is achieved, resulting in the release of countable markers that can be counted using the nanopore counting method described in that example. This construct, and other constructs that will be appreciated by those skilled in the art, can be used in the assays described herein.

Example 22 illustrates how counting can typically be performed to measure transfer events associated with the presence of a label passing through a nanopore. Figure 29 illustrates the concept of a signal threshold, enabling manipulation of the characteristics of the data in a counting assay. Figures 31, 32, and 33 show the output of quantitative assay data, representing the data types that can be used to determine the amount of analyte using the type of assay described in this example. Figure 34 shows a standard curve generated from a construct that has been chemically cut. It is also understood that although dsDNA is used as the label in this particular example, other labels, such as those described in Example 5, including, but not limited to, nanobeads, dendritic polymers, etc., can also be used. Furthermore, other known labels can also be employed. Such constructs required to produce suitable reagents can be synthesized as described herein or by methods known to those skilled in the art.

9. Reagent kit and tube

This document also provides kits for performing the methods described above using or without the disclosed apparatus. The kit may include instructions for analyzing the analyte using the disclosed apparatus. The instructions included in the kit may be attached to packaging material or may be included as a package insert. The instructions may be written or printed material, but are not limited thereto. The invention anticipates any medium capable of storing such instructions and delivering them to the end user. Such media include, but are not limited to, electronic storage media (e.g., disks, magnetic tapes, cartridges, chips), optical media (e.g., CD-ROMs), etc. As used herein, “instructions” may include the address of an Internet site providing the instructions.

The kit may include a cartridge comprising a microfluidic module having an embedded nanoporous module, as described above. In some embodiments, the microfluidic and nanoporous modules may be separate components for reversible integration, or may be fully or irreversibly integrated into the cartridge. The cartridge may be disposable. The cartridge may include one or more reagents that can be used to practice the methods disclosed above. The cartridge may include one or more containers that contain the reagents as one or more separate compositions, or optionally, as a mixture, where reagent compatibility would permit. The cartridge may also include other materials that may be desired from a user's perspective, such as buffers, diluents, standards (e.g., calibrators and controls), and/or any other materials that can be used for sample processing, washing, or any other steps of the assay. The cartridge may include one or more of the specific binding members described above.

Alternatively or additionally, the kit may contain calibrators or controls, such as purified and optionally lyophilized target analytes, either in a tube or as a separate liquid, gel, or other form, and/or at least one container (e.g., test tube, microplate, or strip) for use with the apparatus and methods described above, and/or buffers, such as assay buffers or wash buffers, either of which may be provided as concentrated solutions. In some embodiments, the kit contains all the components necessary to perform the assay, i.e., reagents, standards, buffers, diluents, etc. The instructions may also include instructions on preparing a standard curve.

The kit may also include a reference standard for quantifying the target analyte. This reference standard can be used to establish a standard curve to interpolate and/or extrapolate the target analyte concentration. The kit may include reference standards varying in concentration level. For example, the kit may include one or more reference standards with high, medium, or low concentration levels. The concentration range of the reference standard can be optimized based on the assay. Exemplary concentration ranges for reference standards include, but are not limited to, for example: approximately 10 fg/mL, approximately 20 fg/mL, approximately 50 fg/mL, approximately 75 fg/mL, approximately 100 fg/mL, approximately 150 fg/mL, approximately 200 fg/mL, approximately 250 fg/mL, approximately 500 fg/mL, approximately 750 fg/mL, approximately 1000 fg/mL, approximately 10 pg/mL, approximately 20 pg/mL, approximately 50 pg/mL, approximately 75 pg/mL, approximately 100 pg/mL, approximately 150 pg/mL, approximately 200 pg/mL, approximately 250 pg/mL, approximately 500 pg/mL, approximately 750 pg/mL, approximately 1 ng/mL, approximately 5 ng/mL, approximately 10 ng/mL, approximately 12.5 ng/mL, and approximately 15 ng/mL. /mL, approximately 20ng/mL, approximately 25ng/mL, approximately 40ng/mL, approximately 45ng/mL, approximately 50ng/mL, approximately 55ng/mL, approximately 60ng/mL, approximately 75ng/mL, approximately 80ng/mL, approximately 85ng/mL, approximately 90ng/mL, approximately 95ng/mL, approximately 100ng/mL, approximately 125ng/mL, approximately 150ng/mL, approximately 165ng/mL, approximately 175ng/mL, approximately 200ng/mL, approximately 225ng/mL, approximately 250ng/mL, approximately 275ng/mL, approximately 300ng/mL, approximately 400ng/mL, approximately 425ng/mL, approximately 450ng/mL, approximately 465ng/mL, approximately 475ng/mL, approximately 500 ng/mL, approximately 525 ng/mL, approximately 550 ng/mL, approximately 575 ng/mL, approximately 600 ng/mL, approximately 700 ng/mL, approximately 725 ng/mL, approximately 750 ng/mL, approximately 765 ng/mL, approximately 775 ng/mL, approximately 800 ng/mL, approximately 825 ng/mL, approximately 850 ng/mL, approximately 875 ng/mL, approximately 900 ng/mL, approximately 925 ng/mL, approximately 950 ng/mL, approximately 975 ng/mL, approximately 1000 ng/mL, approximately 2 μg/mL, approximately 3 μg/mL, approximately 4 μg/mL, approximately 5 μg/mL, approximately 6 μg/mL, approximately 7 μg/mL, approximately 8 μg/mL, approximately 9 μg/mL, approximately 10 μg/mL, approximately 20 μg/mL mL, approximately 30 μg/mL, approximately 40 μg/mL, approximately 50 μg/mL, approximately 60 μg/mL, approximately 70 μg/mL, approximately 80 μg/mL, approximately 90 μg/mL, approximately 100 μg/mL, approximately 200 μg/mL, approximately 300 μg/mL, approximately 400 μg/mL, approximately 500 μg/mL, approximately 600 μg/mL, approximately 700 μg/mL, approximately 800 μg/mL, approximately 900 μg/mL, approximately 1000 μg/mL, approximately 2000 μg/mL, approximately 3000 μg/mL, approximately 4000 μg/mL, approximately 5000 μg/mL, approximately 6000 μg/mL, approximately 7000 μg/mL, approximately 8000 μg/mL, approximately 9000 μg/mL, or approximately 10000 μg/mL.

Any specific binding member provided in the kit may contain a label or marker, such as a fluorophore, enzyme, aptamer, dendritic polymer, bead, nanoparticle, microparticle, polymer, protein, biotin/antibiotin label, etc., or the kit may include reagents for labeling specific binding members, or reagents for detecting specific binding members and/or for labeling analytes, or reagents for detecting analytes. The kit may contain one or more different labels or markers if desired. The kit may also include components that induce cleavage, such as cleavage-mediating reagents. For example, cleavage-mediating reagents may include reducing agents such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). Specific binding members, calibrators, and/or controls may be provided in separate containers or pre-dispensed in appropriate assay forms or cartridges. The labels can be detected using the disclosed apparatus.

The kit may include one or more specific binding members, for example, to detect one or more target analytes in a sample in a multiplexed assay. The number of different types of specific binding members in the kit may vary widely depending on the intended use of the kit. The number of specific binding members in the kit may range from one to about ten or more. For example, the kit may include 1-10 specific binding members, 1-9 specific binding members, 1-8 specific binding members, 1-7 specific binding members, 1-6 specific binding members, 1-5 specific binding members, 1-4 specific binding members, 1-3 specific binding members, 1-2 specific binding members, 2-10 specific binding members, 2-9 specific binding members, 2-8 specific binding members, 2-7 specific binding members, 2-6 specific binding members, 2-5 specific binding members, 2-4 specific binding members, 3-10 specific binding members, 3-9 specific binding members, 3-8 specific binding members, 3-7 specific binding members, 3-6 specific binding members, 3 -5 specific binding members, 3-4 specific binding members, 4-10 specific binding members, 4-9 specific binding members, 4-8 specific binding members, 4-7 specific binding members, 4-6 specific binding members, 5-10 specific binding members, 5-9 specific binding members, 5-8 specific binding members, 5-7 specific binding members, 5-6 specific binding members, 6-10 specific binding members, 6-9 specific binding members, 6-8 specific binding members, 6-7 specific binding members, 7-10 specific binding members, 7-9 specific binding members, 7-8 specific binding members, 8-10 specific binding members, 8-9 specific binding members, or 9-10 specific binding members. Each of the one or more specific binding members can bind to different target analytes, and each specific binding member can be labeled with different tags and/or aptamers. For example, the kit may include: a first specific binding member binding to a first target analyte, a second specific binding member binding to a second target analyte, a third specific binding member binding to a third target analyte, etc., wherein the first specific binding member is labeled with a first label and/or aptamer, the second specific binding member is labeled with a second label and/or aptamer, the third specific binding member is labeled with a third label and/or aptamer, etc. In addition to one or more specific binding members, the kit may also contain one or more other assay components, such as a suitable buffer medium, etc. The kit may also include means for detecting and measuring the labels and/or aptamers, such as those described above. Finally, the kit may contain instructions for using the specific binding members in the analyte detection method according to the invention, wherein such instructions for use may be present on the kit packaging and/or on the packaging instructions.

Optionally, the kit includes quality control components (e.g., a sensitivity test group, calibrators, and positive controls). The preparation of quality control reagents is well known in the art and is described in inserts of various immunodiagnostic products. The sensitivity test group members are optionally used to establish assay performance characteristics and are further optionally useful indicators of the integrity of the kit reagents and the standardization of the assay.

The kit may optionally include other reagents required for diagnostic assays or convenient quality control evaluations, such as buffers, salts, enzymes, enzyme cofactors, substrates, and assay reagents. Other components (such as buffers and solutions) (e.g., pretreatment reagents) for separating and/or processing test samples may also be included in the kit. The kit may additionally include one or more other controls. One or more components of the kit may be lyophilized; in this case, the kit may further contain reagents suitable for reconstructing the lyophilized components. One or more of the components may be in liquid form.

The various components of the kit may optionally be provided in suitable containers, if necessary. The kit may also include containers for containing or storing samples (e.g., containers or tubes for urine, saliva, plasma, cerebrospinal fluid, or serum samples, or suitable containers for storing, transporting, or processing tissue to prepare tissue aspirates). Where appropriate, the kit may optionally contain reaction containers, mixing containers, and other components to facilitate the preparation of reagents or test samples. The kit may also include one or more sample collection/acquisition devices to assist in obtaining test samples, such as various blood collection/transfer devices such as microsampling devices, microneedles, or other minimally invasive, painless blood collection methods; blood collection tubes; lancets; capillary blood collection tubes; other methods of blood collection by pricking a single fingertip; oral swabs, nasal/throat swabs; 16-gauge or other size needles; circular blades for punch biopsies (e.g., 1-8 mm or other suitable sizes); surgical scalpels or lasers (e.g., particularly handheld); syringes; sterile containers; or cannulas for obtaining, storing, or aspirating tissue samples; etc. The kit may include one or more instruments for assisting joint aspiration, cone biopsy, punch biopsy, fine needle aspiration biopsy, image-guided percutaneous needle aspiration biopsy, bronchoalveolar lavage, endoscopic biopsy, and laparoscopic biopsy.

If the label or detectable marker is or includes at least one acridine compound, the kit may contain at least one acridine-9-carboxamide, at least one aryl acridine-9-carboxylic acid ester, or any combination thereof. If the label or detectable marker is or includes at least one acridine compound, the kit may also contain a source of hydrogen peroxide, such as a buffer, solution, and/or at least one alkaline solution. If desired, the kit may contain a solid phase, such as magnetic particles, beads, membranes, scaffold molecules, thin films, filter paper, disks, or chips.

If desired, the kit may further comprise one or more components, either alone or in combination with the instructions, for measuring the test sample against another analyte, which may be a biomarker, such as a biomarker of a disease state or disorder (such as infectious diseases, heart disease, metabolic diseases, thyroid diseases, etc.).

The present invention has several aspects illustrated by the following non-limiting embodiments.

10. Example

Example 1

Synthesis of optically cleavable 2-nitrobenzylsuccinimide/maleimide bifunctional linkers.

*In the above synthesis, DMF is dimethylformamide.

Synthesis of Compound 2. Synthesis of an optically cleavable sulfosuccinimide/maleimide junction is derived from Agati, et al., J. Am. Chem. Soc., 134(45), 18499-18502, 2012. Briefly, the starting material 4-[4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy]butyric acid (0.334 mmol) was dissolved in anhydrous dichloromethane (DCM) under an argon atmosphere. The flask was cooled to 0 °C by placing it in an ice bath. Compound 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylureon hexafluorophosphate (HBTU) (0.368 mmol) and trimethylamine (TEA) (0.835 mmol) were added to the solution. The reaction mixture was stirred at 0 °C for 5 min, and then N-(2-aminoethyl)maleimide trifluoroacetate (0.368 mmol) was added. After stirring at 0°C for 15 min, the reaction mixture was brought to room temperature (RT) and stirred for 18 h. The reaction mixture was diluted with DCM (45 mL), and the organic phase was washed with water (2 x), saturated NaCl solution (1 x), and dried over sodium sulfate. The organic layer was concentrated under reduced pressure and purified by rapid chromatography using a _SiO2 column (eluent: 100% DCM to 3% methanol in DCM, v/v). Compound 1 (0.024 mmol) was dissolved in anhydrous dimethylformamide (1 mL). N,N'-disulfosuccinimide carbonate (DSC) (0.071 mmol) and TEA (0.096 mmol) were added sequentially to the solution. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was purified by direct loading onto a C18 reversed-phase column (eluent: 5% acetonitrile in water to 95% acetonitrile in water, v/v). The starting materials and other chemicals used in the synthesis can be purchased from Sigma-Aldrich.

Example 2

Synthesis of an optically cleavable sulfosuccinimide/DBCO 2-nitrobenzyl bifunctional linker.

*In the above synthesis, DMF is dimethylformamide.

Synthesis of Compound 4. The synthesis of the optically cleavable sulfosuccinimide/dibenzocyclooctyl (DBCO) ynyl linker is derived from a similar operation described in Agati et al., J. Am. Chem. Soc., 134(45), 18499-18502, 2012. Briefly, the starting material 4-[4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy]butyric acid (0.334 mmol) was dissolved in anhydrous dichloromethane (DCM) under an argon atmosphere. The flask was cooled to 0 °C by placing it in an ice bath. Compound 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylureonium hexafluorophosphate (HBTU) (0.368 mmol) and trimethylamine (TEA) (0.835 mmol) were added to the solution. The reaction mixture was stirred at 0 °C for 5 min, followed by the addition of DBCO-amine (0.368 mmol). After stirring at 0°C for 15 min, the reaction mixture was raised to room temperature and stirred for 18 h. The reaction mixture was diluted with DCM (45 mL), and the organic phase was washed with water (2 x), saturated NaCl solution (1 x), and dried over sodium sulfate. The organic layer was concentrated under reduced pressure and purified by rapid chromatography using a _SiO2 column (eluent: 100% DCM to 3% methanol in DCM, v/v). Compound 3 (0.024 mmol) was dissolved in anhydrous dimethylformamide (1 mL). N,N'-disulfosuccinimide carbonate (DSC) (0.071 mmol) and TEA (0.096 mmol) were added sequentially to the solution. The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was purified by direct loading onto a C18 reversed-phase column (eluent: 5% acetonitrile in water to 95% acetonitrile in water, v/v). Starting materials and other chemicals used in the synthesis were available from Sigma-Aldrich.

Example 3

The antibody-DNA conjugate was coupled and photochemically cleaved using a sulfosuccinimide/maleimide 2-nitrobenzyl bifunctional linker.

Biological conjugation and cleavage of antibodies and DNA. DNA molecules can be conjugated to antibodies using the following protocol. DNA is thiolated at the 5' end by replicating the DNA sequence in a PCR reaction using two PCR primers (one or both of which are 5'-thiol labeled). The labeled DNA (100 μM final concentration) is dissolved in 50 mM HEPES (pH 7.0) with stirring. Compound 2 (2 mM) is added, and the reaction is allowed to proceed at room temperature for 2 hours. After conjugation, excess unreacted maleimide groups are quenched with excess dithiothreitol (DTT). The conjugate is purified by gel filtration on a Sephadex G-25 column or by thorough dialyzing in a suitable conjugate storage buffer at 4°C. The purified DNA-succinimide adapter (50 μM final concentration) is dissolved in 100 mM PBS (pH 7.5) with stirring. Add the natural antibody (50 μM final concentration) and allow the reaction to proceed at room temperature for 2 hours. Purify the Ab-DNA conjugate using a Sephadex column (Sephadex G25) running with 100 mM PBS (pH 7.5) or BioGel P-30 gel filter media.

Prior to nanopore detection, the conjugate can be cleaved by irradiation with a UV lamp at 365 nm. Using the same bioconjugation chemistry, this example can also be applied to DNA dendritic polymers.

Example 4

The antibody-DNA conjugate was coupled and photochemically cleaved using a sulfosuccinimide/DBCO 2-nitrobenzyl bifunctional linker.

Biological conjugation and cleavage of antibody and DNA. DNA molecules can be conjugated to antibodies using the following protocol. DNA is amination at the 5' end by replicating the DNA sequence in a PCR reaction using two PCR primers (one or both of which are 5'-amino labeled). The labeled DNA (100 μM final concentration) is dissolved in 100 mM PBS (pH 7.5) with stirring. Compound 4 (2 mM final concentration) is added and the reaction is allowed to proceed at room temperature for 2 hours. The DNA-DBCO adapter is purified on a gel filter column (Sephadex G-25) or by thorough dialysis in a suitable conjugate storage buffer at 4°C. The purified DNA-DBCO adapter (50 μM final concentration) is dissolved in 50 mM Tris (pH 7.0) with stirring. The DNA-DBCO adapter is conjugated to the antibody using copper-free click chemistry. Add the azide-labeled antibody (Kazane et al., Proc. Natl. Acad. Sci., 109(10), 3731-3736, 2012) (final concentration 25 μM) and allow the reaction to proceed at room temperature for 6–12 hours. Purify the Ab-DNA conjugate using a Sephadex column (Sephadex G25) running with 100 mM PBS (pH 7.5) or BioGel P-30 gel filter media.

Prior to nanopore detection, the conjugate can be cleaved by irradiation with a UV lamp at 365 nm. Using the same bioconjugation chemistry, this example can also be applied to DNA dendritic polymers.

Example 5

Nanoparticle-antibody conjugates for digital immunoassay (nanopore counting).

This example describes the covalent conjugation of an antibody with 26 nm carboxylated polystyrene nanoparticles (NP, PC02N) (such as those available from Bangs Labs (Fishers, IN, USA)). The 26 nm NPs have a surface charge of 528.7 μeq/g and a docking area of 68.4 squared/group (according to manufacturer information).

Activation of carboxylated polystyrene nanoparticles: 1.0 mL (100 mg/mL) of 26 nm carboxylated NP was washed with 10 mL of 0.1 M MES (2-[N-morpholino]ethanesulfonic acid) (pH 4.5-5.0). After washing, the precipitate was resuspended in 100 mL of 0.1 M MES (pH 4.5-5.0) to achieve an NP concentration of 1.0 mg/mL (0.1% solids). 10.0 mL of the nanoparticle suspension (10 mg NP, 5.28 μeq carboxyl) was transferred to a bottle and reacted with 10 μL (5.28 μmol, 1.0 equivalent/ _CO₂H equivalent) of freshly prepared 10 mg/mL LEDC aqueous solution (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and 17 μL (7.93 μmol, 1.5 equivalent/1 equivalent EDC) of 10 mg/mL sulfonyl-NHS aqueous solution (N-hydroxysulfosuccinimide, Sigma, catalog number 56485) at room temperature for 15 min. The reaction suspension was centrifuged at 6,500 g and the solution was discarded. The precipitate was washed with 20 mL of 20 mM PBS/5 mM EDTA (pH 7.5) and precipitated by centrifugation at 6,500 g. The supernatant was removed. The succinimide-activated carboxyl-NP precipitate was resuspended in 50 mM PBS (pH 7.5), and immediately 9.8 μL (52.8 nmol, 0.01 equivalents/1 _CO₂H equivalents) of 1.0 mg/mL pyridine dithioethylamine aqueous solution was added, allowing the reaction to proceed at room temperature for 2–4 h with continuous stirring. The pyridyl-derived carboxyl-NP was washed with 10 mL of 20 mM PBS/5 mM EDTA (pH 7.5) and resuspended in 10.0 mL of the same buffer. The concentration of nanoparticles was determined using a carboxyl-NP calibration curve obtained by UV-Vis spectroscopy (600 nm, scattering). The pyridyl-ligand loading on the NP was determined as follows: a measured amount of NP was reduced with 10 mM TPCEP or DTT, the reducing agent was removed by centrifugation, the pyridyl-activated NP precipitate was resuspended in PBS/EDTA (pH 7.2), and reacted with Ellman's reagent (A412 of the supernatant was measured). If not used for antibody conjugation on the same day, the activated NP should be stored at 4°C.

The range of EDC/NHS and pyridine dithioethylamine molar inputs was evaluated to determine the expected stoichiometry for the preparation of different antibody-nanoparticle conjugates. Reaction parameters (pH, temperature, time) were assessed to achieve the desired NP activation results.

Antibody reduction: Mix 1.0 mL of 10 mg/mL antibody solution (10 mg) with 38 μL of freshly prepared 30 mg/mL 2-MEA solution (10 mM reaction concentration) (2-mercaptoethylamine hydrochloride), cap the mixture, and incubate at 37 °C for 90 min. Allow the solution to reach room temperature, remove excess 2-MEA using a desalting column, and pre-equilibrate in 20 mM PBS/5 mM EDTA (pH 7.5). Determine the concentration of reduced antibody using UV-Vis absorbance at A280 (protein absorbance) and A320 (scatter-corrected). Determine the number of free thiol groups using an Ellman assay. Optimize conditions as needed to produce 2 or 4 free thiol groups (Cys in the antibody hinge region). Immediately use the reduced antibody for conjugation to a pyridyl-derived carboxyl-NP.

Coupling of reduced antibody with activated nanoparticles: Hypotheses are made: (1) the antibody docking area is 45 ^nm² ; (2) the surface area of 26 nm nanoparticles is 2,120 ^nm² ; (3) theoretically 47 antibody molecules can be mounted on the surface of 26 nm NPs.

Procedure: To 10 mL (10 mg) of 0.1% pyridyl-activated carboxyl nanoparticles in 20 mM PBS/5 mM EDTA (pH 7.5), add 0.10 mg (0.66 nmol, 0.10 mL) of reduced antibody (at 1.0 mg/mL in the same EDTA-containing buffer). Allow the mixture to react at room temperature for 2 h under mixed conditions, centrifuge to remove unbound molecules, and aspirate. Wash the precipitate with 10 mL PBS (pH 7.2), centrifuge, and aspirate. Resuspend the antibody-NP conjugate in 10.0 mL PBS (pH 7.2). Determine the NP concentration (% solids) of the conjugate using UV-Vis spectroscopy (600 nm). Examine the particle conjugates by SEM and determine the size/charge distribution using ZetaSizer. Different conjugates can be separated from the potential distribution of the conjugate population using size exclusion chromatography. The antibody-to-NP incorporation ratio can be determined by flow cytometry using fluorescently labeled antigen conjugates or by Micro BCA (uBCA) assays. The range of antibody-to-NP molar inputs can be evaluated along with conjugation temperature and pH to produce homogeneous populations of different conjugates (i.e., NP incorporation ratios of 2 or 4).

Nanopore counting immunoassay

The above scheme explains the nanopore counting assay using reduced antibody-activated nanoparticle conjugates (the preparation of which is described above). The immune complexes formed during the immunoassay can be cleaved by reducing the disulfide bond linkers to form free antibody-analyte-antibody complexes and free nanoparticle tags, which allows for counting of the nanoparticle tags after they have passed through the nanopores.

Example 6

Synthesis of CPSP conjugates

A. CPSP antibody conjugate.

*In the above synthesis, DMF is dimethylformamide.

3-(9-((4-oxo-4-(perfluorophenoxy)butyl)(toluenesulfonyl)carbamoyl)acridin-10-onthium-10-yl)propane-1-sulfonate (2): 3-(9-(((3-carboxypropyl)(toluenesulfonyl)carbamoyl)acridin-10-onthium-10-yl)propane-1-sulfonate (CPSP) (1) (1 mmol), pyridine (5 mmol), and dimethylformamide (10 mL) were packed into a 25 mL round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet. The solution was cooled in an ice bath, and pentafluorophenyl trifluoroacetate (1.3 mmol) was added dropwise. The ice bath was removed, and the reaction mixture was stirred at room temperature for 3 hours. Volatile components were removed from the reaction mixture under vacuum, and the residue was dissolved in methanol and purified by reversed-phase HPLC to obtain the title compound.

CPSP antibody conjugate (3): A solution of 2 (1 μL of 10 mM solution in DMSO) was added to an antibody solution (100 μL of 10 μM aqueous solution) and a sodium bicarbonate aqueous solution (10 μL of 1 M solution). The resulting mixture was stirred at room temperature for 4 hours. Purification of the product was achieved on a spinning column to obtain CPSP antibody conjugate 3. The value of “n” varies in an antibody-dependent manner. Incorporation can be controlled to some extent by increasing or decreasing the concentration of the active ester (i.e., compounds 2, 5, 9, and 13) and/or by increasing or decreasing the pH during the reaction, but always results in a distribution of incorporation values. The average incorporation ratio (“I.R.”) was determined experimentally after the reaction. Typically, “n” is any value between 1 and 10.

B. CPSP antibody conjugates with spacers.

*In the above synthesis, DMF is dimethylformamide.

3-(9-((4-(((5-carboxypentyl)amino)-4-oxobutyl)(toluenesulfonyl)carbamoyl)acridin-10-on-10-yl)propane-1-sulfonate (4): 3-(9-(((3-carboxypropyl)(toluenesulfonyl)carbamoyl)acridin-10-on-10-yl)propane-1-sulfonate CPSP (1) (1 mmol), pyridine (5 mmol), and dimethylformamide (10 mL) were placed in a 25 mL round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet. The solution was cooled in an ice bath, and pentafluorophenyl trifluoroacetate (1.3 mmol) was added dropwise. The ice bath was removed, and the reaction mixture was stirred at room temperature for 3 hours. Then, 6-aminohexanoic acid (1.3 mmol) was added in small portions to the reaction mixture, followed by N,N-diisopropylethylamine (5 mmol), and the reaction mixture was stirred at room temperature for 1 hour. After this time, the volatile components were removed from the reactants under vacuum, and the residue was purified by reversed-phase HPLC to obtain the title compound.

3-(9-((4-oxo-4-((6-oxo-6-(perfluorophenoxy)hexyl)amino)butyl)(toluenesulfonyl)carbamoyl)acridin-10-on-10-yl)propane-1-sulfonate (5): 4 (1 mmol), pyridine (5 mmol), and dimethylformamide (10 mL) were charged into a 25 mL round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet. The solution was cooled in an ice bath, and pentafluorophenyl trifluoroacetate (1.3 mmol) was added dropwise. The ice bath was removed, and the reaction mixture was stirred at room temperature for 3 hours. After this time, volatile components were removed from the reaction mixture under a nitrogen stream, and the residue was purified by reversed-phase HPLC to give the title compound.

CPSP antibody conjugate with spacers (6): A solution of 5 (1 μL of 10 mM solution in DMSO) was added to an antibody solution (100 μL of 10 μM aqueous solution) and an aqueous solution of sodium bicarbonate (10 μL of 1 M solution). The resulting mixture was stirred at room temperature for 4 hours. Purification of the product was achieved on a spin column to obtain CPSP antibody conjugate 6 with spacers. Typically, "n" is any value between 1 and 10.

C.CPSP oligonucleotide-antibody conjugate.

*In the above synthesis, DMF is dimethylformamide.

9-((3-Carboxypropyl)(Toluenesulfonyl)carbamoyl)-10-(Prop-2-yn-1-yl)acridin-10-onium (8): A 100 mL round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet was filled with propynyl alcohol (10 mmol), 2,6-di-tert-butylpyridine (10 mmol), and dichloromethane (50 mL) and cooled to -20 °C. Trifluoromethanesulfonic anhydride was then added dropwise to the solution, and the reaction mixture was stirred at -20 °C for 2 hours. Pentane (25 mL) was added to the reaction mixture, and the resulting precipitate was separated by filtration. The volatile components were evaporated under vacuum, and the residue was redissolved in dichloromethane (25 mL) in a 100 mL round-bottom flask. A small amount of 4-(N-toluenesulfonylacridin-9-formamido)butyric acid (CP-acridin) (7) (1 mmol) was added, and the reaction mixture was stirred at room temperature for 18 hours. The volatile components were evaporated under vacuum, and the residue was dissolved in methanol (5 mL) and purified by reversed-phase HPLC to obtain the title compound.

9-((4-oxo-4-(perfluorophenoxy)butyl)(toluenesulfonyl)carbamoyl)-10-(prop-2-yn-1-yl)acridin-10-onium (9): 8 (1 mmol), pyridine (5 mmol), and dimethylformamide (10 mL) were charged into a 25 mL round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet. The solution was cooled in an ice bath, and pentafluorophenyl trifluoroacetate (1.3 mmol) was added dropwise. The ice bath was removed, and the reaction mixture was stirred at room temperature for 3 hours. Volatile components were removed from the reaction mixture under vacuum, and the residue was dissolved in methanol and purified by reversed-phase HPLC to obtain the title compound.

CPSP antibody conjugate (10): A solution of 9 (1 μL of 10 mM solution in DMSO) was added to an antibody solution (100 μL of 10 μM aqueous solution) and a sodium bicarbonate aqueous solution (10 μL of 1 M solution). The resulting mixture was stirred at room temperature for 4 hours. Purification of the product was achieved on a spin column to obtain CPSP antibody conjugate 10. Typically, "n" is any value between 1 and 10.

CPSP Oligonucleotide-Antibody Conjugate (11): A mixture of oligoazide (10 nmol in 5 μL water), CPSP antibody conjugate 10 (10 nmol in 10 μL water), and freshly prepared 0.1 M “click solution” (3 μL – see below) was shaken at room temperature for 4 hours. The reaction mixture was diluted with 0.3 M sodium acetate (100 μL) and the DNA conjugate was precipitated by adding EtOH (1 mL). The supernatant was removed, and the residue was washed twice with cold EtOH (2 x 1 mL). The residue was dissolved in water (20 μL), and the solution of CPSP oligonucleotide-antibody conjugate 11 was used without further purification. Typically, “n” is any value between 1 and 10.

"Click Solution": Dissolve 1 mg of CuBr in 70 μL of DMSO/t-BuOH 3:1 to prepare a 0.1 M solution (this solution must be freshly prepared and not stored). Dissolve 54 mg of tris(benzyltriazolylmethyl)amine (TBTA) in 1 mL of DMSO/t-BuOH 3:1 to prepare a 0.1 M solution (this solution can be stored at -20°C). Add 1 volume of the 0.1 M CuBr solution to 2 volumes of the 0.1 M TBTA solution to obtain the "click solution".

D. CPSP oligonucleotide-antibody conjugates with spacers.

*In the above synthesis, DMF is dimethylformamide.

9-((4-((5-carboxypentyl)amino)-4-oxobutyl)(toluenesulfonyl)carbamoyl)-10-(prop-2-yn-1-yl)acridin-10-onium (12): 8 (1 mmol), pyridine (5 mmol), and dimethylformamide (10 mL) were charged into a 25 mL round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet. The solution was cooled in an ice bath, and pentafluorophenyl trifluoroacetate (1.3 mmol) was added dropwise. The ice bath was removed, and the reaction mixture was stirred at room temperature for 3 h. Then, 6-aminohexanoic acid (1.3 mmol) was added in small amounts to the reaction mixture, followed by N,N-diisopropylethylamine (5 mmol), and the reaction mixture was stirred at room temperature for 1 h. After this time, the volatile components were removed from the reaction mixture under vacuum, and the residue was purified by reversed-phase HPLC to give the title compound.

9-((4-oxo-4-((6-oxo-6-(perfluorophenoxy)hexyl)amino)butyl)(toluenesulfonyl)carbamoyl)-10-(prop-2-yn-1-yl)acridin-10-onium (13): 12 (1 mmol), pyridine (5 mmol), and dimethylformamide (10 mL) were charged into a 25 mL round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet. The solution was cooled in an ice bath, and pentafluorophenyl trifluoroacetate (1.3 mmol) was added dropwise. The ice bath was removed, and the reaction mixture was stirred at room temperature for 3 hours. After this time, volatile components were removed from the reaction mixture under a nitrogen stream, and the residue was purified by reversed-phase HPLC to give the title compound.

CPSP antibody conjugate with spacers (14): A solution of 13 (1 μL of 10 mM solution in DMSO) was added to an antibody solution (100 μL of 10 μM aqueous solution) and an aqueous solution of sodium bicarbonate (10 μL of 1 M solution). The mixture was stirred at room temperature for 4 hours. Purification of the product was achieved on a spin column to obtain CPSP antibody conjugate 14 with spacers. Typically, "n" is any value between 1 and 10.

CPSP Oligonucleotide-Antibody Conjugate (15): A mixture of oligoazide (e.g., commercially available) (10 nmol in 5 μL water), CPSP antibody conjugate 14 with spacers (10 nmol in 10 μL water), and freshly prepared 0.1 M “click solution” (3 μL—see Example 6.C) was shaken at room temperature for 4 hours. Typically, “n” is any value between 1 and 10. The reactants were diluted with 0.3 M sodium acetate (100 μL), and the DNA conjugate was precipitated by adding EtOH (1 mL). The supernatant was removed, and the residue was washed twice with cold EtOH (2 x 1 mL). The residue was dissolved in water (20 μL), and the solution of CPSP oligonucleotide-antibody conjugate 15 with spacers was used without further purification.

Cleavage of CPSP antibody conjugates with or without spacers and CPSP oligonucleotide-antibody conjugates with or without spacers. The CPSP antibody conjugates with or without spacers and CPSP oligonucleotide-antibody conjugates with or without spacers described are cleaved or “triggered” using an alkaline hydrogen peroxide solution. In the system, an excited-state acridinone intermediate generates photons, which are measured. Additionally, the cleavage products are acridinone and sulfonamide. The conjugates of Examples 6.A-D are used with the disclosed apparatus by counting acridinone and/or sulfonamide molecules.

E. CPSP oligonucleotide conjugates without antibodies.

Antibody-free CPSP oligonucleotide conjugate (16): A mixture of oligoazide (e.g., commercially available) (10 nmol in 5 μL water), propargyl-CPSP 8 (10 nmol in 10 μL water), and freshly prepared 0.1 M “click solution” (3 μL—see Example 6.C) can be shaken at room temperature for 4 hours. The reactants can be diluted with 0.3 M sodium acetate (100 μL) and the DNA conjugate can be precipitated by adding EtOH (1 mL). The supernatant can be removed, and the residue can be washed twice with cold EtOH (2 x 1 mL). The residue can be dissolved in water (20 μL), and the solution of CPSP oligonucleotide-antibody conjugate 16 with spacers can be used without further purification.

Example 7

Manufacturing of low-cost DMF substrate chips.

Using the method described in Lo CY et al., Microelectronic Engineering 86 (2009) 979-983, with some modifications, a low-cost flexible DMF chip was fabricated using roll-to-roll (R2R) flexographic printing combined with a wet lift-off process for electrode patterning. A schematic diagram of the fabrication process is depicted in Figure 10. A 5.0 mil ST506 polyethylene terephthalate (PET) substrate (1) was used as the starting material for DMF electrode printing. A layer of yellow ink (Sun Chemical) was flexographically printed (2) onto the PET substrate at a rate of 10 m/min using a 1.14 mm thick printing plate (Flint MCO3) with an ink transfer volume of 3.8 ml/ ^m² on an Anilox roller assembly. The negative image of the DMF electrode pattern was derived from the flexographic printing step (3). The ink was dried twice in a hot air oven (2 x 100 °C) before metal deposition. A silver metal layer was deposited on a printed PET substrate using an EVAR2R metal evaporator to form a uniform silver coating with a thickness of 80 nm (4). A wet stripping process (6) was performed on the metallized ink-film substrate (5) in an acoustic treatment bath at a speed of 1 m/min using a combination of acetone and ultrasound. This chemical/physical treatment allowed the silver-ink layer to dissolve while maintaining only the silver layer intact. Removal of the ink-silver layer produced a DMF-printed electrode pattern consisting of 80 drive electrodes (2.25 x 2.25 mm) with an electrode gap spacing of 50 or 140 μm (7). As a QC check, a total of 80–90 random chips from a single roll were visually inspected for variations in electrode gap spacing and connector lead width. Typical chip yield (determined to have acceptable gap specifications) was close to 100%. A single fabricated flexible chip is depicted in Figure 11. The fabricated flexible chip measures 3” x 2” and includes electrodes, reservoirs, contact pads, and leads.

The dielectric coating is applied to the electrodes and reservoir using either rotary screen printing or gravure printing. For rotary screen printing, Henkel EDAC PF-455B is used as the dielectric coating by printing with a Gallus NF (400L) screen at a printing speed of 2 m/min and a UV curing rate of 50%. The typical dielectric thickness is 10-15 μm. For gravure printing, the cylinder is designed to print high-viscosity dielectric inks, such as IPD-350 (Inkron), at a speed of 2 m/min using an ink volume of 50 ml/ ^m² . The typical dielectric thickness for gravure printing is 7-8 μm. The final hydrophobic layer is printed using Millidyne Avalon 87 or Cytonix Fluoropel PFC 804UC coatings at a printing speed of 8 m/min using a Gravure cylinder (140-180L) and a printing speed of 8 m/min, followed by four consecutive oven drying steps (4 x 140°C). The typical hydrophobic layer thickness is 40-100 nm.

Alternatively, for small batches of individual chips, the dielectric and hydrophobic coatings can be applied separately using chemical vapor deposition (CVD) and spin coating.

Example 8

Functional testing of low-cost DMF chips

The driving capability was tested on a 3” x 2” PET-based DMF substrate chip manufactured as described in Example 7 above. Figure 12 depicts a 3” x 2” PET-based DMF chip (1) with a 0.7 mm thick glass substrate (3) positioned on it. The glass substrate (3) includes a transparent indium tin oxide (ITO) electrode on the lower surface of the glass substrate and a Teflon coating on the ITO electrode. The DMF chip includes 80 silver driving electrodes with a straight-edge electrode design and 50 μm gaps between the electrodes, as well as 8 buffer reservoirs (see Example 7 above).

A dielectric Parylene-C layer (6-7 μm thick) and a final Teflon coating (50 nm thick) were applied to the bottom electrode via CVD and spin coating, respectively. Approximately 50 μL of PBS buffer (2) containing 0.1% surfactant was transferred into four adjacent reservoirs on the bottom DMF chip. Droplet sizes ranged from 700-1,500 nL (one or two droplets), and vertical and horizontal lateral movements were examined (4), except for a circular scan pattern required for mixing. Droplet actuation was achieved using a voltage of 90 V _rms . Approximately 90% of the driving electrodes on the chip were tested and found to be fully functional.

Example 9

TSH Immunoassay on a Low-Cost DMF Chip

The ability of chemiluminescence detection to perform thyroid-stimulating hormone (TSH) immunoassay was tested on a 3' x 2" PET-based DMF chip covered with a glass substrate, as described in Example 8 above. Dummy samples consisted of TSH calibration material infused with tris-buffered saline (TBS) buffer containing a blocking agent and surfactant. Three samples were tested—0, 4, and 40 μIU/ml. 2 μL of anti-βTSH capture antibody coated on 5 μm magnetic microparticles (3 x ^10⁸ particles/ml) was dispensed from the microparticle reservoir into the center of the DMF electrode array. The electrode array was then constructed by attaching neodymium magnet strips (3 inches x 1/2 inches x 1/4 inches thick, relative permeability _μr = 1.05, residual field strength _Br) under the DMF chip. =1.32T), the magnetic microparticles were separated from the buffer (Fig. 13A). 5 μL of sample was transferred to a microparticle slug, and the microparticle suspension was then mixed on a 4-electrode square configuration (Fig. 13B) for 5 min. The microparticles were separated from the sample by magnetism, and the supernatant was transferred to the waste tank (Figs. 13C and 13D). 2 μL of 1 μg/mL horseradish peroxidase (HRP) conjugated anti-TSH detection antibody was transferred to the microparticle slug and mixed for 2 min. The microparticles were separated by magnetism, and the supernatant was transferred to the waste tank. The microparticles containing the immunoassay sandwich complex were washed four times with 4 x 2 μL of PBS wash buffer containing 0.1% surfactant. The wash buffer from each wash step was transferred to the waste tank after the steps were completed. 1 μL of SuperSignal _H₂O was added. A chemiluminescent substrate consisting of ₂ μL of luminol (ThermoFisherScientific) was transferred to a microparticle block and then mixed for 6 minutes. The chemiluminescence signal was measured using an integrated Hamamatsu H10682-110 PMT with a 5VDC source at 427 nm emission (347 nm excitation). Dose-response curves were plotted against relative luminescence (see Figure 13E).

Example 10

Nanoporous module manufacturing

Nanoporous modules were fabricated using a standard soft lithography method with integrated coupling to commercially available silicon nitride (SiN_{_x}) films embedded in a TEM window (Norcada). The modules consist of four separate PDMS layers—top and bottom PDMS substrates containing transfer microchannels, and two optional intermediate PDMS layers to seal the TEM window.

SU8 Master Mold Fabrication: A clean, dry glass substrate is spin-coated with photoresist (SU8-50) to the desired thickness. The coated areas are then selectively exposed to near-ultraviolet light using a photomask. The photomask exposes the photoresist to ultraviolet light only in areas where the transfer microchannels and reservoir shape are to be preserved. After exposure, baking is performed to crosslink the exposed photoresist areas. The remaining, unexposed photoresist is then removed from the substrate using SU8 developer. The final product is the master mold—a glass substrate with patterned transfer microchannels and a hard photoresist reservoir.

Fabrication of the intermediate PDMS layer: To fabricate the intermediate PDMS layer, a solution containing PDMS monomer and its curing agent (Sylgard 184 silicone elastomer) (at a 7:1 PDMS monomer:curing agent ratio) was spin-coated onto a glass slide, followed by heating on a hot plate at 70°C for 30 minutes. The PDMS layer was peeled from the glass substrate, and a 1.25 mm cutout was punched through the PDMS layer to provide an opening allowing access to the TEM window. The surface of the PDMS layer was made hydrophilic by plasma treatment at a distance of 8 mm for 30 seconds using a corona processor. A second plasma treatment (5 seconds) was used to treat the surface of the PDMS layer and the TEM window, and then a SiN _x TEM window was bonded between the two intermediate PDMS layers.

Fabrication of Top and Bottom PDMS: As shown in Figure 14B, the top and bottom PDMS substrates containing microchannels are fabricated as follows: PDMS monomer and curing agent are mixed in a 7:1 ratio and poured onto a glass containing an SU8 patterned mold (6) patterned with transfer microchannels and reservoirs (see the above description of SU8 master mold fabrication). The microchannels are measured to be approximately 110-135 μm wide and 50 μm deep. After degassing for 15 minutes, the SU8 mold is heated on a hot plate at 70°C for 60 minutes (7). After curing, the PDMS substrate is peeled off the SU8 mold (8) and cut to produce a rectangular PDMS substrate of approximately 30 mm length x 20 mm width x 3 mm depth. Access holes (1.25 mm diameter) are punched through the PDMS substrate to allow subsequent insertion of electrodes into the microchannels. The final components are shown in Figure 14A and, from bottom to top, include: a bottom PDMS substrate containing a microchannel (1), a first intermediate PDMS layer (2) containing a cutout portion positioned above the microchannel, a SiN _x film in a TEM window (3) containing a second intermediate PDMS layer (4) also containing a cutout portion, and a top PDMS substrate (5) containing a second microchannel.

Alignment of the top and bottom PDMS substrates: The PDMS bottom substrate (prepared as described in “ Top and Bottom PDMS Fabrication ” above) was plasma-treated for 30 seconds, followed by the attachment of the first intermediate PDMS layer (prepared as described in “ Intermediate PDMS Layer Fabrication ” above) to the PDMS bottom substrate. Similarly, the PDMS top substrate was plasma-treated for 30 seconds, followed by the attachment of the second intermediate PDMS layer to the PDMS top substrate. The cutouts in the intermediate layers were aligned with the microchannels. The top and bottom PDMS sheets were plasma-treated for 30 seconds, followed by the arrangement of the SiNx film window between the top and bottom sheets and alignment with the cutouts in the intermediate PDMS layers. The top sheet aligned with the SiNx film (which is aligned with the bottom sheet) was pressed together until all bubbles were released. The final nanoporous PDMS assembly was heated on a hot plate at 100°C for 30 minutes and plasma-treated for 5 minutes. The final module assembly shown in Figure 14C(9a) contains two channels (one straight channel and one “L-shaped” channel), each terminating in a reservoir of solution (e.g., buffer solution). The TEM window containing the SiNx film is located at the intersection of two vertical microchannels (Fig. 14C, 9b).

Example 11

Nanopore manufacturing

Nanopore fabrication is accomplished as follows: a SiN _x TEM window housed between two PDMS layers is potential-biased until dielectric breakdown occurs, thereby opening a small-diameter pore in the film. This allows for in-situ pore formation within a microfluidic device prior to analyte detection. Nanopore formation via dielectric breakdown has previously been demonstrated for the rapid fabrication of small-diameter pores in solid dielectric films (H. Kwok, K. Briggs, V. Tabard-Cossa, PLoS-One, 9(3), 2014).

A commercially available SiN _x film, used as a transmission electron microscope (TEM) window (Norcada), was embedded in an assembled PDMS module (as described in Example 10 above) and used to fabricate nanopores. A cross-sectional area (50 μm x 50 μm) of the SiN _x TEM window was exposed to a salt solution (1 M KCl) placed on opposite sides (cis and anti) of the film at the vertical microchannel confluence. An Ag/AgCl electrode was placed in each microchannel, entering approximately 3 mm into the pore flushed through the PDMS substrate from the center of the SiN _x TEM window. The cis and anti microchannels were filled using a syringe with a blunt needle by adding ethanol to both reservoirs until liquid was observed emerging from the channel openings at the module edge. Resistance was measured to check for proper sealing and to ensure the integrity of the TEM-SiN _x film. Resistance in the MΩ range indicates a good seal and an intact, undamaged film. The ethanol was flushed out of the microchannels with deionized water and replaced with 1 M KCl solution by injecting it into both reservoirs. Resistance was measured again to check for proper sealing.

A constant voltage of 4.4V was applied to the membrane module, and the leakage current was monitored in real time. The real-time measured leakage current is plotted in Figure 15A. Figure 15A shows the leakage current before nanopore establishment (1). A threshold of >5nA was used as the cutoff value, indicating that the pores had established. An increase in leakage current was observed after approximately 10 minutes (2). The voltage was immediately turned off after the increase in leakage current was detected. The diameter of the established pores was 6.9 nm, as determined by the following relationship:

Where G = electrical conductivity, σ = volumetric conductivity (12.35 S/m measured with KCl), L = film thickness (10 nm), and d = pore diameter (S. Kowalczyk, A. Grosberg, Y. Rabin, C. Dekker, Nanotech., 22, 2011).

After the pores were established, current-voltage (I-V) curves (see Figure 15B) were used to verify that the nanopores exhibited ohmic properties, indicating that the nanopores were symmetrical in shape and that their resistance was independent of the applied voltage or current. The same 1 MkCl solution was used for pore fabrication and I-V curves.

Example 12

Dry microchannel filling

To test its ability to spontaneously fill high-salt solutions from the DMF electrode assembly, capillary conduits (Figures 16A-16C) contained in an assembled PDMS module (i.e., an integrated device including a DMF module and a nanoporous module) were tested. Filling was accomplished via spontaneous capillary flow (SCF). No nanoporous membrane was included to allow for better observation of the microchannels. Referring to Figure 16A, a microdroplet (2) containing 3.6M LiCl, 0.05% Brij 35, and blue dye (for observation) was moved using a glass DMF chip (3” x 2” x 0.0276”) with 80 driving electrodes (1) (2.25 mm x 2.25 mm, Cr-200 nm thickness). The PDMS module (3) contains two openings (4) facing the DMF electrode array, two reservoirs (5), and two microchannels—one straight channel (6) and one L-shaped channel (7). The module assembly was placed on the DMF glass surface such that the two channel openings faced the interior of the DMF electrode array. Since no top-ground electrode chip was used, the microdroplet movement was achieved by generating a driving potential using a coplanar bottom electrode.

A 10 μL droplet of blue LiCl salt solution was placed on the electrode at the center of the DMF electrode array. The droplet was moved to a transfer electrode near the opening of the straight microchannel using a voltage of 100 V _rms (10 kHz). As shown in Figure 16B, after the droplet contacted the PDMS surface (8), the time required to fill the 130 μm diameter straight channel (9) and reach the reservoir was measured. As shown in Figure 16C, after approximately 30 seconds, the droplet volume appeared smaller (10) and the channel was half-filled (11). The total time required to fill the entire dry microchannel (130 μm diameter) was 53 seconds.

Wet microchannel filling: A 10 μL droplet of blue LiCl salt solution was placed on the central electrode of the DMF electrode array. The droplet was moved to a transfer electrode near the opening of a straight microchannel using a voltage of 100 V _rms (10 kHz). The channel was pre-filled with ethanol to mimic a pre-wetted channel. After the droplet contacted the PDMS surface, filling the channel into the reservoir took less than 1 second. This is significantly faster than a dry channel, suggesting that pre-wetting with a hydrophilic solution improves the microchannel filling rate.

Example 13

DMF droplet transfer in integrated silicon NP devices

Besides flexible substrates such as PDMS, rigid substrates (e.g., silicon) can be used to fabricate nanopore modules. Figure 17 shows a digital microfluidic (DMF) chip (1) containing a driving electrode (4), from which droplets are transferred to a silicon microfluidic chip containing a nanopore sensor (2). The droplets are transferred between the two component chips through an access port (3) on the top surface of the microfluidic chip containing the nanopore sensor. The access port is connected to the nanopore sensor (5) via a microfluidic channel (6). The droplets move from the access port through the microfluidic channel by capillary force, and the movement can be assisted by a passive paper pump fabricated from a micropillar array (7) (Figure 18). The passive pump can also remove fluid from the microchannel, allowing different fluid solutions to be used sequentially without contamination (e.g., between solutions used for nanopore formation and nanopore sensing).

Fabrication of silicon nanopore modules can include the use of standard CMOS photolithography and etching methods. Figure 18 shows an embodiment of a silicon nanopore module design, where the approximate mold size is 10 mm x 10 mm, with front (forward) and rear (reverse) channels for filling the nanopore buffer. The front channel has a width and depth of 30 μm and a length of 11 mm. The rear channel has a width of 50 μm, a depth of 200 μm, and a length of 11 mm. The micropillar dimensions are a 30 μm pillar diameter, a 30 μm spacing, and a depth of 200 μm.

The DMF and nanopore module can be connected using an interface made from molded plastic or by direct bonding (Figures 19 and 20). Microdroplets positioned on electrodes (4) within the DMF chip aligned with the access port are transferred by capillary force and accelerated by the interposer (7). Alternatively, the top electrode (8) of the DMF chip can be modified to further facilitate this process by introducing a hole connecting the drive electrode (4) to the interposer (8) (Figure 20).

Example 14

Microdroplet transfer between DMF and nanoporous modules via capillary forces

The ability to move a high-salt transfer buffer from a DMF chip to a module containing a suitable nanoporous membrane was tested in a silicon microfluidic chip. A tortuous microchannel was tested for its ability to passively move microdroplets of 1M KCl (pH=8) using spontaneous capillary flow (SCF) as the sole driving force. The entire microchannel was fabricated in silicon and served as a model for fluid transfer in a CMOS-based silicon environment. The tortuous microchannel was designed with two access ports (for fluid loading). The channel size was measured to be 160 μm in diameter with an approximate length of 2.5 cm. Microdroplets suitable for solutions forming nanopores through dielectric breakdown were demonstrated to fill the silicon microfluidic structure using passive capillary forces.

Referring to Figure 21, individual droplets of 1M KCl solution (pH = 8.0) were placed in one of the inlet ports (1) connected to the transport microfluidic channel (2), resulting in a 2.5 cm long tortuous channel (3). The channel terminates (4) at a port exposed to atmospheric pressure (not shown). A magnified image of the tortuous channel is shown in Figure 22. Capillary filling was monitored using an sCMOS camera coupled with an optical microscope. Deposition of the salt solution in the inlet port resulted in spontaneous filling of the microchannel at a rate of several mm/s by passive capillary forces, thus confirming the ability to transfer fluid to the nanoporous membrane within the microchannel.

As another test of the transfer rate, the channel was emptied of KCl solution and dried under a nitrogen stream. Additional droplets of 1M KCl solution (pH = 8.0) were placed in the inlet port of the dried microchannel, and capillary filling was monitored using an optical microscope. A faster filling rate was observed compared to the “dried” channel (i.e., compared to the first time KCl solution was introduced into the channel), indicating that pre-filling of the silicon microchannel with aqueous solutions enhances subsequent fluid filling.

Example 15

Fabrication of integrated nanopore sensors with fluid microchannels

Integrated nanopore sensors with fluid microchannels were fabricated by modifying silicon-on-oxide (SOI) wafers on insulators using photolithography and etching methods (Figures 23A-23B).

The SOI wafer (1) is photolithographically and etched (2) to produce a structure (3) suitable for the movement of small fluid volumes, which has a width of 30 μm and a channel depth of 10-30 μm.

Silicon nitride (SiN) material (5) is deposited on a patterned SOI wafer by evaporation (4).

An oxide material layer (7) is deposited on top of silicon nitride (5) by evaporation (6). The underlying silicon (1) is exposed by selectively removing the superimposed oxide and nitride material (6) covering one of the microstructures using a combination of photolithography and etching. This structure will form microchannels for driving fluids in small volumes.

By using a combination of photolithography and etching to remove only the superimposed oxide layer, the underlying silicon nitride within the second microstructure is selectively exposed (8).

The exposed microstructures are permanently bonded to the carrier wafer (9), and the structures are inverted for further processing (10). The back side of each microstructure is exposed using a combination of photolithography and etching, and the oxide material on the reverse side of the SOI wafer is selectively patterned (11).

Example 16

Synthesis of cleavable DNA-biotin constructs

Synthesis of non-biotinylated double-stranded DNA (NP1): Two single-stranded 50-mers were synthesized using standard aminophosphite chemistry (IntegratedDNA Technologies). Oligomeric NP1-1S consisted of a 50-nucleotide DNA sequence containing an amino group at the 5' end, separated from the DNA by a C-12 carbon spacer (SEQ ID NO:1) (1, MW = 15,522.3 g/mol, ε = 502,100 ^{M⁻¹ cm⁻¹} ). Oligomeric NP1-2AS consisted of a 50-nucleotide DNA sequence complementary to NP1-1S (SEQ ID NO:2) (2, MW = 15,507.1 g/mol, ε = 487,900 ^{M⁻¹ cm⁻¹} ). Both oligonucleotides were quantified and lyophilized before further processing.

Synthesis of non-biotinylated double-stranded 50-bp DNA constructs: NP1-2AS (1.44 mg, 93.4 nmol) was reconstructed in 0.5 mL of distilled water to obtain a 187 μM solution. NP1-1S (1.32 mg, 85.3 nmol) was reconstructed in 0.5 mL of 50 mM phosphate, 75 mM sodium chloride buffer (pH 7.5) to obtain a 171 μM solution. Double-stranded constructs (3) (SEQ ID NO: 1 - forward strand (top); SEQ ID NO: 2 - reverse strand (bottom)) were prepared by annealing 60 μL of NP1-1S solution (10.2 μmol) together with 40 μL of NP1-2AS solution (7.47 μmol). The mixture was placed in a heating block at 85 °C for 30 min, followed by slow cooling to room temperature over 2 hours. The double-stranded material was purified by injecting the entire annealing volume (100 μL) onto a TosoH G3000SW column (7.8 mm x 300 mm) equilibrated with 10 mM PBS buffer (pH 7.2). Column elution was monitored at 260 and 280 nm. The double-stranded material (3) eluted at 7.9 min (approximately 20 min). The DNA was concentrated to 150 μL using a 0.5 mL Amicon filter concentrator (MW cutoff 10,000 Da). The final DNA concentration was calculated to be 40.5 μM, as determined by A ₂₆₀ absorbance.

Biotinylation of single-chain 5'-amino oligomers: A 100 mM solution of sulfonyl-NHS-SS-biotin (4, ThermoFisher Scientific) was prepared by dissolving 6 mg of the powder in 0.099 mL of anhydrous DMSO (Sigma-Aldrich). The solution was vortexed and immediately used for biotinylation of 5'-amino-DNA. Approximately 100 μL of ssDNA (1,171 μM, 17.1 μmol, 0.265 mg) (SEQ ID NO: 1) in 50 mM PBS (pH 7.5) was mixed with 3.4 μL of 0.1 mM biotinylation reagent in DMSO (34.1 μmol, a 20-fold molar excess relative to ssDNA). The mixture was combined and allowed to react at room temperature for 2 hours. Two 0.5 mL Zeba spin desalting columns (MW cutoff 7,000 Da, ThermoFisher Scientific) were equilibrated in 10 mM PBS (pH 7.2). The crude biotinylated ssDNA solution was added to a Zeba column and eluted at 4,600 rpm for 1.3 min. The eluent was transferred to a second Zeba column and eluted as described. The concentration of purified NP1-1S-SS-Biotin(5) (SEQ ID NO:1) (2.03 mg/ml, 131 μM) was determined by measuring _A260 absorbance.

Formation of biotinylated double-stranded DNA: Approximately 60 μL of NP1-1S-SS-biotin solution (5, 7.85 μmol, 131 μM, 2.03 mg/mL) was mixed with 42 μL of NP1-2AS solution (2, 7.85 μmol, 187 μmol/L) (SEQ ID NO:2). The solution was placed in a heating block at 85 °C for 30 min, followed by slow cooling to room temperature over 2 h. The double-stranded product was purified using 10 mM PBS (pH 7.2) on a TosoH G3000SW column (7.8 mm x 300 mm) by injecting the entire annealing volume (approximately 100 μL). The double-stranded biotinylated material eluted at 7.9 min (20 min run time), as monitored by A ₂₆₀ absorbance. The elution volume was reduced to 480 μL using a 0.5 mL Amicon filter concentrator (MW cutoff 10,000 Da). The final concentration of NP1-dithio-biotin (6) (SEQ ID NO:1-forward chain (top); SEQ ID NO:2-reverse chain (bottom)) was calculated to be 16.3 μM, as determined by A ₂₆₀ absorbance.

Example 17

Alternative Synthesis of Cleavable DNA-Biotin Constructs

Complementary DNA sequences (NP-31a and NP-31b): Two single-stranded 60-mers were synthesized using standard aminophosphite chemistry (Integrated DNA Technologies). Oligomeric NP-31a consists of a 60-nucleotide DNA sequence containing an amino group at the 5' end, separated from the DNA by a C-6 carbon spacer (SEQ ID NO:3) (1, MW = 18,841.2 g/mol, 1.7 μM/OD). Oligomeric NP-31b consists of a 60-nucleotide DNA sequence complementary to NP-31a (SEQ ID NO:4) (2, MW = 18,292.8 g/mol, 1.8 μM/OD). Both oligonucleotides were quantified and lyophilized before further processing.

Biotinylation of the single-chain 5'-amino oligomer NP-31a: A 10 mM NHS-SS-dPEG ₄ -biotin (4, MW = 751.94 g/mol, QuantaBioDesign, Ltd.) solution was prepared by dissolving 15.04 mg of the powder in 2.0 mL of dimethylformamide (Sigma Aldrich). The solution was vortexed and immediately used for biotinylation of 5'-amino-DNA. Approximately 100 μL of ssDNA (1,100 μM, 0.01 μmol, 0.188 mg) (SEQ ID NO:3) in 10 mM phosphate-buffered saline (PBS, pH 7.4) was mixed with 10 μL of a 10 mM biotinylation reagent in DMF (0.1 μmol, a 10-fold molar excess relative to the ssDNA). The mixture was combined and allowed to react at room temperature for 2 hours. Two 0.5 mL Zeba spin desalting columns (MW cutoff 7,000 Da, ThermoFisher Scientific) were equilibrated in 10 mM PBS (pH 7.2). A crude biotinylated ssDNA solution was added to one Zeba column and eluted at 4,600 rpm for 2 min. The eluent was transferred to a second Zeba column and eluted as described. The concentration of purified NP-31-SS-biotin (5) (SEQ ID NO:3) (1.45 mg/mL, 77 μM) was determined by measuring _A260 absorbance.

Formation of biotinylated double-stranded DNA: Approximately 60 μL of NP-31-SS-biotin solution (5.77 μM) (SEQ ID NO:3) was mixed with 50 μL of NP-31b solution (2.100 μM). The solution was placed in an 85°C heating block for 30 minutes, followed by slow cooling to room temperature over 2 hours. The double-stranded products (SEQ ID NO:3 - forward strand (top); SEQ ID NO:4 - reverse strand (bottom)) were purified on a TosoH G3000SW column (7.8 mm x 300 mm) using 10 mM PBS (pH 7.2) by injecting the entire annealing volume (approximately 100 μL). The double-stranded biotinylated material eluted at 7.57 min, as monitored by A ₂₆₀ absorbance. The elution volume was reduced using a 0.5 mL Amicon filter concentrator (MW cutoff 10,000 Da). The final concentration of dsNP-31-SS-biotin(6) (SEQ ID NO:3-forward chain (top); SEQ ID NO:4-reverse chain (bottom)) was calculated to be 11 μM, as determined by A ₂₆₀ absorbance.

Example 18

Synthesis of cleavable DNA-biotin (thiol-mediated cleavage construct)

Binding and chemical cleavage (TCEP or DTT) of ssNP-31-SS-Biotin-coated paramagnetic microparticles (SA-MP) : Chemical cleavage experiments were performed on the magnetic microparticles as follows (see Figure 25). 100 μL of a 77 μM solution of the modified oligonucleotide ssNP-31-SS-Biotin in PBS (pH 7.2) was incubated with 1 μL of 0.1% paramagnetic microparticles at room temperature for 30 min. Excess oligonucleotides were removed by attracting the particles to a magnet and washing 10 times with PBST buffer (pH 7.4). The oligonucleotide-bound particles were incubated for 15 min with solutions of different concentrations of DTT or TCEP in PBS (pH 7.4). The microparticles were washed 10 times with PBST buffer (pH 7.4) to remove any cleaved oligonucleotides. The complementary sequence NP-31c (SEQ ID NO:5) (7, MW = 7494.6 g/mol, 5.2 μM/OD) containing the fluorophore was incubated with the microparticles in PBS (pH 7.4) for 30 min to bind any uncleaved ssNP-31-SS-biotin remaining intact on the microparticles. The microparticles were attracted to a magnet and washed 10 times with PBST buffer (pH 7.4) to remove any excess NP-31c fragments. The washed but unchemically cleaved coated microparticles prepared as above served as controls. The fluorescence signal on the microparticles was measured by fluorescence microscopy. Maximum cleavage efficiencies were measured at 79% and 93% for DTT and TCEP, respectively, as shown in Table 1.

Table 1

<![CDATA[<u>DTT(mM)</u>]]> <![CDATA[<u>fluorescence signal on the particle (relative optical units)</u>]]> <![CDATA[<u>cutting efficiency</u>]]> 50 3579 79% 25 7417 57% 12.5 11642 32% 0.78125 17052 0% 0 17059 (Comparison) <![CDATA[<u>TCEP(mM)</u>]]> <![CDATA[<u>fluorescence signal on the particle (relative optical units)</u>]]> <![CDATA[<u>cutting efficiency</u>]]> 250 460 93% 222 448 94% 187 474 93% 142 512 92% 83 477 93% 45 453 93% 19 452 93% 0 5023 (Comparison)

.

Example 19

Synthesis of cleavable DNA-biotin (photocleavage construct)

Evaluation of photocleavable DNA sequences and cleavage efficiency on microparticles: Photocleavable single-stranded DNA sequences were synthesized using standard aminophosphite chemistry (Integrated DNA Technologies). The oligonucleotides consisted of 48 nucleotides forming two oligomeric segments (oligomeric 8-1 (SEQ ID NO:6) and oligomeric 8-2 (SEQ ID NO:7)) separated by two photocleavable portions (8, MW = 15,430.1 g/mol, 441800 ^{Lmol⁻¹ cm⁻¹} ). The 5' end contained an amino group separated from the DNA by a C-6 carbon spacer. A fluorescently labeled complementary strand to oligomeric 8-2 (9, MW = 7738.8 g/mol, 212700 ^{Lmol⁻¹ cm⁻¹} ) (SEQ ID NO:8) was synthesized. Both oligonucleotides were quantified and lyophilized before further processing.

Oligo 8-1 (SEQ ID NO: 6): 5'AAA AAA GGT CCG CAT CGA CTG CAT TCA3'

Oligo 8-2 (SEQ ID NO: 7): 5'CCC TCG TCC CCA GCT ACG CCT3'

NP-8(8) (oligomers 8-1 (SEQ ID NO: 6) and 8-2 (SEQ ID NO: 7) connected by two optically slicable portions (“PC”):

H ₂ N-5'AAAAAAGGTCCGCATCGACTGCATTCA-PC-PC-CCCTCGTCCCCAGCTACGCCT3'

NP-9(9) (SEQ ID NO: 8): AlexaFluor546-5'AGG CGT AGC TGG GGA CGA GGG3'

Photocutting experiments were performed on magnetic microparticles using the following method (see Figures 26A and 26B). NP-8 was covalently attached to an antibody to generate an Ab-oligomeric complex (prepared by Biosynthesis Inc.). 100 μL of the 33 nM antibody-oligomeric complex was incubated with 1 μL of 0.1% solids of goat anti-mouse microparticles at room temperature for 30 min. Excess antibody-oligomeric complex was removed by attracting the particles to a magnet and washing 10 times with PBST buffer (pH 7.4). The microparticle complex solution was irradiated with UV light (300–350 nm wavelength) for 5 min. The microparticles were attracted to a magnet and washed 10 times with PBST buffer (pH 7.4) to remove any cleaved oligomeric segments. After resuspending the particles in PBST buffer (pH 7.4), fluorescently labeled oligo 9 (SEQ ID NO: 8) was added to the irradiated microparticles and incubated at room temperature for 30 min. The washed but un-UV-irradiated coated microparticles (uncut oligomers 8-2) prepared above served as a control. Fluorescence signals on the particles were imaged using a fluorescence microscope (546). The cleavage efficiency at 74% binding with paramagnetic microparticles is shown in Table 2.

Table 2

Fluorescence signal on the particle (relative light units) Irradiation 3660 No irradiation (control) 13928 Cutting efficiency 74%

.

Example 20

Heat-cut joints

This embodiment describes thermally cuttable connectors and their cutting. Such thermally cuttable connectors can be used in, for example, DMF chips, droplet-based microfluidic chips, SAW chips, etc., as described herein.

Thermally cleavable adapters are cleaved by raising the temperature above a threshold, as in the thermal separation of double-stranded DNA. The temperature rise in a DMF chip can be photothermally achieved by transferring energy from light to an absorption target. In one method, a light source (such as a laser) having a wavelength of about 980 nm (ranging from about 930 nm to about 1040 nm) is applied to a region of the DMF chip containing a fluid sample. The light can be absorbed by water molecules in the fluid, resulting in a temperature rise and adapter cleavage. The level and duration of heating can be controlled by pulse length, pulse energy, number of pulses, and pulse repetition rate. Photothermal heating using the light-absorbing bands of water is described, for example, in U.S. Patent 6,027,496.

Photothermal heating can also be achieved by coupling a light source with a target containing dye or pigment. In this case, the target area of a DMF chip is printed with a light-absorbing dye or pigment (e.g., carbon black). When a fluid comes into contact with the target, the light source (e.g., a commercially available laser diode) is directed at the light-absorbing target, resulting in a localized increase in temperature and cutting of the junction. The level and duration of heating can be controlled by the light absorption properties of the target, the wavelength of light, the pulse length, the pulse energy, the number of pulses, and the pulse repetition rate. For example, photothermal heating using a light source combined with a light-absorbing target is described in U.S. Patent 6,679,841.

In a third method of photothermal heating, a light-absorbing dye or pigment can be introduced into a fluid within a DMF chip. Light then propagates through the DMF chip, transferring energy to the dissolved or suspended light-absorbing material, resulting in a localized increase in temperature and the cutting of the junction. The level and duration of heating are controlled by the light-absorbing properties of the target material, the wavelength of light, the pulse length, the pulse energy, the number of pulses, and the pulse repetition rate. In one embodiment of this method, the light-absorbing target is a suspension of magnetic microparticles used in the device. For example, photothermal heating using suspended nanoparticles in fluid droplets is described in Walsh et al., Analyst, 140(5), 1535-42 (2015). The references of Walsh et al. also confirm some controls that can be achieved in photothermal applications.

Example 21

Thermal cutting achieved through microwave-induced particle overheating

This embodiment describes the use of microwave-induced particle overheating to promote thermal denaturation (such as dsDNA denaturation, reverse-Michael reaction, reverse-Dils-Alder, and other eliminations) to release countable moieties via heat-sensitive cleavable adapters, as the use of low-power microwave radiation can accelerate immunoassay detection. Such heat-sensitive cleavable adapters can be used, for example, in DMF chips, as described herein.

In this embodiment, the formation of orthogonally functionalized short dsDNA segments (such as 15 bp sequences having double-stranded T _m in the 40-55°C range) acts as a thermal release agent. The dsDNA segments can react with antibodies (via attachment chemistry such as thiol-maleimide interaction) and 26 nm carboxylated polystyrene nanoparticles (NPs) (such as those available from Bangs Labs (Fishers, IN, USA), via attachment chemistry such as amine-activated carboxylic acid chemistry). The 26 nm NPs have a surface charge of 528.7 μeq/g and a docking area of 68.4 squared/unit (according to manufacturer information). The antibody and nanoparticles bind via the dsDNA segments, which form thermally triggered, releaseable linkers. The thermal linkers can be cleaved using techniques such as microwave radiation to trigger particle overheating and localized temperature gradients.

DNA sequence 1(10) (SEQ ID NO: 9): H2N-5’CAAGCCCGGTCGTAA3’

DNA sequence 1b(11) (SEQ ID NO: 10): maleimide-5’TTACGA CCG GGC TTG3’

dsDNA sequence (12) (SEQ ID NO: 9 - forward strand (top); SEQ ID NO: 10 - reverse strand (bottom)):

H ₂ N-5'CAA GCC CGG TCG TAA3'

3’GTT’CGG GCC AGCATT5’-maleimide.

Annealing of orthogonally functionalized complementary DNA sequence complexes : A solution of approximately 100 μM DNA sequence 1 (SEQ ID NO: 9) in PBS (pH 7.5) was mixed with 1.0 molar equivalent of DNA sequence 1b (theoretical T _m 51.6 °C, according to Integrated DNA Technologies Oligomer Analyzer tool) (SEQ ID NO: 10) in PBS (pH 7.5) and placed in a heating block at 60 °C for 30 min, followed by slow cooling to room temperature over 2 h. The resulting dsDNA product was purified by injecting the entire annealing volume into a TosoH G3000SW column (7.8 mm x 300 mm) using 10 mM PBS (pH 7.2). The elution volume was reduced using a 0.5 mL Amicon filter concentrator. The final dsDNA concentration was determined by A260 absorbance. The reaction protocol is described below (“Mal” stands for maleimide).

Activation of carboxy-polystyrene nanoparticles and addition of double-stranded DNA: Carboxy-based nanoparticles were pre-activated as described in the “Activation of Carboxy-Polystyrene Nanoparticles” section of Example 5. The DNA loading on the NPs was determined by thermal denaturation of the bound DNA strands, particle washing, annealing of fluorescently labeled complementary DNA sequences (such as AlexaFluor546-5'-TTACGACCG GGC TTG3' (SEQ ID NO: 11)), and quantified using fluorescence microscopy.

Antibody Reduction and Conjugation to the NP-dsDNA Complex : The antibody was reduced as described in the "Antibody Reduction" section of Example 5. The reduced antibody could be immediately used for conjugation to the NP-dsDNA complex. The resulting conjugate was centrifuged at 6,500 g, and the supernatant was removed by decantation. The washing operation was repeated 5 times with PBS (pH 7.5) to remove any free antibody from the nanoparticles. The active antibody to nanoparticle incorporation ratio could be quantified using a fluorescently labeled antigen with a given antibody. The NP concentration (% solids) of the conjugate was determined using UV-Vis spectroscopy (600 nm). The particle conjugates were examined by SEM, and the size/charge distribution was determined using ZetaSizer.

Microwave-Induced Particle Overheating and Nanopore Counting Immunoassay : The above protocol explains a nanopore counting assay utilizing a thermally denatured antibody-nanoparticle conjugate (the preparation of which is described above). A sandwich immunoassay can be prepared using magnetic microparticles coated with an analyte trapping agent, wherein a blood analyte is incubated with the magnetic microparticles, washed, and then incubated with the antibody-nanoparticle conjugate described above. Particle overheating can be induced by using microwave radiation to create a local temperature gradient near the particle surface. Particle overheating methods such as those reviewed in Dutz and Hergt (Nanotechnology, 25:45-2001 (2014)) can be used. The adaptation of these techniques to local thermal denaturation in the immunoassay context can provide a method for releasing counting fractions (such as nanoparticles). After the magnetic microparticles are removed, the counting fractions (nanoparticles) are counted after passing through the nanopores.

Example 22

Nanopore counting data

This example describes nanopore counting data for various tags, such as ssDNA hybrid molecules with polyethylene glycol (DNA-star), dsDNA, dsDNA labeled with DBCO, and PAMAM succinamide dendritic polymers. The application of these different tags with nanopores of varying sizes was demonstrated to provide nanopore optimization. Different molecular polymer tags were suspended in appropriate salt buffers and detected using standard fluidic grid cell units.

Current-voltage (i-V) records (voltage measurement data) and current-time (i-t) records were recorded using homemade instruments. The acquired data was processed using a computer software program called CUSUM, which detected events based on user-input thresholds. By detecting as many events as possible and filtering them later for specific purposes, any influence of subjectivity in the evaluation was minimized.

Preliminary experiments were performed using a label added to the symmetric side of the membrane. A 200 mV electrical bias was applied to the label solution, and current blocking was monitored using an Axopatch 200B amplifier and CUSUM software.

Small molecules are known to pass through nanopores very rapidly unless the pore size restricts their passage. Current blocking of rapid events can be distorted due to the limited bandwidth of the system. Certain systems may even fail to detect faster molecules at all.

In our study, only larger polymers and molecules labeled with large group modifiers were detected. Experimental conditions and the number of detectable events are shown in Table 3.

Table 3

.

These data confirm that DNA dendrimers, polymers, and PAMAM dendrimers can be used as detection markers for solid-state nanopore sensors.

Example 23

Nanopore differentiation of biomolecules

In this embodiment, nanopores are used to distinguish biomolecules (e.g., dsDNA stars, DBCO-modified dsDNA, and conventional dsDNA). This method can be used for multiplexing using different tag types.

This embodiment uses: a 50bp oligonucleotide containing a branch point at the center (base pair number 25), with a single-stranded oligonucleotide (DNA star) covalently linked at the branch point; a double-stranded 50bp oligonucleotide containing a dibenzylcyclooctynyl (DBCO) modified at the center (base pair number 25); and a 5'-thiol modified double-stranded DNA oligonucleotide.

These different modified DNA molecules were analyzed using three different SiNx nanopores in 3.6M LiCl buffer. DNA-star molecules were analyzed using 4.0 nm diameter pores; DBCO-modified DNA was analyzed using 3.7 nm diameter pores; and thiol-modified DNA was analyzed using 4.2 nm diameter pores. Current blocking levels (pA) were plotted relative to nanopore duration (μsec) to demonstrate the ability of the nanopores to distinguish the three different biomolecules. At the population level, the three different labels appeared to be distinguishable, as evidenced by the differences in the patterns in the scatter plots (Figs. 24A-24C). Real-time identification of individual events requires additional levels of blocking level and time information as a way to distinguish signals from noise. The ability to distinguish different nanopore labels confirms that nanopores can be used for multiplexing in different assays.

Example 24

Qualitative analysis

The following examples describe a method for performing qualitative assays. Essentially, in this example, a construct is used to validate the principle of the assay on a DMF chip. The construct is cleaved, and the label is released. Nanopore counting is then used to generate a signal as it transfers through the nanopores, thus indicating the binding of two specific binding member pairs (streptococcus and biotin). This cleavage and subsequent counting of the dsDNA label are associated with the specific binding that has already occurred during the assay. Furthermore, appropriate control experiments are performed to confirm that the signal generated from the label (based on its counting during nanopore transfer measurement) is due to a specific binding event that has already occurred during the assay, and not associated with the presence of a thiol-cleaving reagent introduced into the assay flow. Details of the experiments are as follows.

Thiol-mediated cleavage using DMF : Biotin-labeled double-stranded DNA containing cleavable disulfide bonds (Example 17(6)) was used as the target for nanopore detection/counting. The binding assay involved binding biotinylated DNA to streptavidin magnetic microparticles on a DMF chip, followed by a thiol-mediated chemical cleavage step (see Figure 25). The reagent arrangement on the DMF chip is shown in Figure 27. The cleaved DNA target (separated from the material bound to the streptavidin magnetic particles) was transferred to a nanopore fluid grid cell containing a solid silicon nitride (SiN _{x )} film with pre-drilled nanopores established by controlled dielectric breakdown (H. Kwok et al., PLoS, 9(3), 2014). The DNA target material was counted and analyzed using the open-source CUSUM software analysis package (NIST).

Appropriate reagents were loaded onto a glass DMF chip (3” x 2” x 0.0276”) containing eight reagent reservoirs. Each reservoir, except for the waste reservoir, contained approximately 5 μL of each reagent. The reagent concentrations were as follows: 11 μM biotin-SS-DNA in PBS (pH 7.2); 10 mg/mL (w/v) M-270 2.7 μm streptavidin-coated magnetic microparticles (Life Technologies); PBS wash buffer (pH 7.2) + 0.05% ETKT (ethylenetetra-KIS(ethoxy-block-propoxy-tetra) tetra), 50 mM tris-(2-carboxyethyl)phosphine (TCEP). The approximate size of the dispensing DMF droplets was 1.5 μL.

One droplet of M-270 streptavidin-coated microparticles was dispensed and mixed with one droplet of dsNP-31-SS-biotin for approximately 40 minutes. Mixing was completed by combining the two droplets and moving them in a circular pattern (3x4) on a DMF chip with 12 electrodes. The bottom magnet was engaged to collect the microparticles, and the supernatant was transferred to a waste reservoir. Next, two droplets of PBS/ETKT buffer were dispensed and transferred to the microparticle mass, which was then resuspended in solution. The suspension was mixed for 5 minutes, then the magnet was re-engaged, and the supernatant was removed to a waste reservoir. The particle washing step was repeated a total of 11 times, with the mixing time gradually increased to 45 minutes. The supernatant from the final wash was transferred to an empty reservoir. Five more droplets of PBS/ETKT were transferred to the same reservoir. For nanopore analysis preparation, the wash buffer and PBS/ETKT in the reservoir were removed using a 34-AWG non-metallic syringe (Microfil 34-AWG) and transferred to a 1.5 mL Eppendorf tube. The cutting process was initiated by transferring two microdrops of TCEP reagent to the microparticle block and mixing for 45 minutes. The bottom magnet was engaged, and the supernatant (containing the cut DNA) was transferred to an empty reservoir. Five more microdrops of PBS/ETKT wash buffer were transferred to the same reservoir. For nanopore analysis preparation, the final extract was removed from the DMF chip using a 34-AWG non-metallic syringe and transferred to a 1.5 mL Eppendorf tube. The cutting eluent was centrifuged for 30 seconds and placed in a magnetic holder for 1 minute to remove any trace particles.

Nanopore Analysis: Nanopore fabrication was achieved using controlled dielectric breakdown (CBD) of a 10 nm thick SiN _x film (NorcadaNT0052, low-stress SiN _x ) embedded in a TEM window (0.05 μm x 0.05 μm). This method enables the production of small-diameter solid-state pores with high precision and minimal cost. The TEM-SiN _x film was placed in a polytetrafluoroethylene (PTFE) fluid grid cell containing two buffered compartments and sealed with two silicone elastomer gaskets. The fluid grid cell contained a 16 μL volume channel in the bottom of the grid cell, which connected the salt solution in the upper compartment to the nanopore membrane. For nanopore fabrication, the fluid grid cell was first filled with degassed ethanol, exchanged with degassed deionized water, then filled with degassed 0.5 M KCl, and buffered to pH 10 with sodium bicarbonate in 18 MΩ deionized water. Fabrication was performed using an amplifier with an 8 V bias voltage. Silver/silver chloride wires were used to connect the two sides of the fluid grid cell. As described by Kwok et al., when a fixed voltage of 8V is set, the current exhibits capacitance in real time (current decreases). As the current increases, power is removed from the grid cells. The sampling rate used for manufacturing is 25kHz. The increase in leakage current indicates the formation of the aperture, thus shutting off the voltage. The aperture diameter is determined from the following conductance-based equation:

Where G = electrical conductivity, σ = volumetric conductivity (12.35 S/m for KCl), L = membrane thickness (10 nm), and d = pore diameter (S. Kowalczyk, A. Grosberg, Y. Rabin, C. Dekker, Nanotech., 22, 2011). The nanopores were examined for ohmic performance by generating I-V curves. The measured nanopore diameter was determined to be 4.4 nm and was subsequently used to detect the cleaved ds-SS-DNA target.

The salt buffer was replaced with 3.6M LiCl, which was used as the sensing buffer for detecting transfer events. The head section was placed between the Axopatch 200B amplifier and the silver/silver chloride (which is connected to the fluid grid unit containing the nanoporous membrane).

Approximately 0.2 μL of TCEP-cut ds-DNA target was diluted with 1.8 μL of PBS buffer (representing a 10-fold dilution of the TCEP-cutting eluent) and the entire volume was loaded and mixed into a nanopore grid cell containing approximately 30 μL of 3.6 M LiCl salt solution. The final DMF wash eluent was used as a negative cutting control (undiluted). The number of DNA transfers was measured at 23 and 65 min for both the TCEP eluent and the negative control, and converted to throughput ( ^s⁻¹ ). The results depicted in Figure 28 confirm successful cleavage of the ds-SS-DNA target from M-270 streptavidin particles using DMF, and detection was performed using solid-state nanopores as a detector. The SNR was determined to be 21.9, as measured from the nanopore throughput.

Data Analysis : First, the expected current changes observed in double-stranded DNA transfer events were calculated under experimental test conditions using the following equations mentioned in Kwok et al., “Nanopore Fabrication by controlled Dielectric Breakdown” Supplementary Information Section 8 and Kwok, H.; Briggs, K.; and Tabard-Cossa, V., “Nanopore Fabrication by Controlled Dielectric Breakdown” - PLoS ONE 9(3):e92880 (2014), to determine the number of transfer events.

Using the expected current blocking value, the binary data of the experimental nanopore output is visually or manually scanned for acceptable expected current blocking events. Using these events, the threshold and hysteresis parameters required by the CUSUM nanopore software are applied and executed. The output from the software is further analyzed using the cusumtools readevents.py software, filtering blocking events greater than 1000 pA (as determined from the initial calculation). Flux events, time intervals between events, and other calculations are determined from the readevents.py analysis tool. Additional calculations are performed on the data generated by CUSUM using JMP software (SAS Institute, Cary, North Carolina). It should be understood that this threshold setting method is one approach for data analysis and threshold setting, and the invention is not limited to this method; other such methods known to those skilled in the art can also be used.

Summary : This embodiment describes a qualitative assay achieved by performing the steps described herein. A direct assay is performed using a cleavable linker conjugate, as described in Example 17, with a thiol-based cleavage step, as shown in Figure 25. It should be understood that other cleavable linker schemes for performing such assays may include, but are not limited to, various other linker cleavage methods, thereby allowing the counting of multiple tags as described herein. For example, in addition to the method described in Example 17, such alternative cleavage methods and/or reagents may include those described in Examples 16, 18, 19, 20, and 21, as well as other cleavage methods described herein and known to those skilled in the art. It is also understood that although the assay format demonstrated in this embodiment (Example 24) represents a direct assay, other formats such as sandwich immunoassays and/or various competing assay formats (as known to those skilled in the art) can also be implemented for assays using the method described herein.

For example, the sandwich immunoassay for TSH (thyroid-stimulating hormone) detection, as described in Example 9, demonstrates the ability to perform such assays on a low-cost DMF chip. Furthermore, many different bioconjugation reagents can be synthesized using a variety of heterobifunctional cleavable adapters, such as those described in Examples 1, 2, 3, 4, 5, and 6, and other cleavable adapters known in other ways to those skilled in the art. These can be used to generate immunoconjugates or other active, specific binding members having cleavable adapters. Immunoconjugates suitable for practicing the present invention can be synthesized by methods such as those described in Examples 3, 4, 5, and 6, and by methods known to those skilled in the art. Additionally, Example 8 demonstrates the functionality of various fluidic microdroplet operations on a low-cost chip, which can facilitate the various steps required for performing various assays, including sandwich and competitive assays, and other variations thereof known to those skilled in the art. Example 11 illustrates the fabrication of nanopores that can be used to count cleavable markers in an assay; however, it should be understood that other methods known to those skilled in the art for fabricating nanopores can also be used for this purpose. Example 16 also represents another construct that can be used for measurement, in which cleavage is achieved, resulting in the release of countable markers, which can then be counted using the nanopore counting method described in this example.

Example 22 illustrates how counting can typically be performed to measure transfer events associated with the presence of multiple markers passing through nanopores. Figure 29 illustrates the concept of a signal threshold, enabling manipulation of the characteristics of data in a counting assay. Figure 28 shows qualitative assay data, representing the type of data that can be used to determine the presence of an analyte using the assay methods described in this example. It is also understood that although dsDNA is used as the marker in this particular example, other markers, such as those described in Examples 5 and/or 22, can also be utilized, including, but not limited to, nanobeads, dendritic polymers, etc. Such constructs required to produce suitable reagents can be synthesized through the various embodiments described in this application or otherwise through methods known to those skilled in the art.

Example 25

Quantitative analysis

The following examples describe a method for performing quantitative assays. Essentially, in this example, and as an extension of Example 24, a standard curve is generated to demonstrate that the increased amount of the counting marker (in this case, the countable marker is a dsDNA molecule) is correlated on the standard curve with the amount of specific binder already bound in the assay (binding) step (which in turn is correlated with the amount of analyte present in the original sample). The standard curve for this particular experiment can be seen in Figures 31, 32, 34 (based on various different data analysis methods) or Figure 34 (which depends on throughput to generate the standard curve). In the latter case, the measurement method shown in Figure 34 is based on event/time (throughput of counting events); however, it should be understood that other measurement methods can also be used to generate a standard curve relating to the amount of analyte concentration measured in a given sample. Details of the experiments performed are as follows.

Nanopore fabrication: Nanopore fabrication was achieved using controlled dielectric breakdown (CBD) of a 10 nm thick SiN _x film (NorcadaNT0052, low-stress SiN _x ) embedded in a TEM window (0.05 μm x 0.05 μm), as this method enables the production of small-diameter solid-state pores with high precision and minimal cost. The TEM-SiN _x film was placed in a polytetrafluoroethylene (PTFE) fluid grid cell containing two buffered compartments and sealed with two silicone elastomer gaskets. The fluid grid cell contained a 16 μL volume channel in the bottom of the grid cell, which connected the salt solution in the upper compartment to the nanopore membrane. For nanopore fabrication, the fluid grid cell was first filled with degassed ethanol, exchanged with degassed deionized water, then filled with degassed 0.5 M KCl, and buffered to pH 10 with sodium bicarbonate in 18 MΩ deionized water. Fabrication was performed using an amplifier with an 8 V bias voltage. Silver/silver chloride wires were used to connect the two sides of the fluid grid cell. As described by Kwok et al., when a fixed voltage of 8V is set, the current exhibits capacitance in real time (current decreases). As the current increases, power is removed from the grid cells. The sampling rate used for fabrication is 25kHz. A sudden increase in leakage current indicates the formation of an orifice, thus shutting off the voltage. The 0.5M KCl buffer was replaced with 3.6M LiCl (pH = 8.3).

The aperture diameter is determined from the following conductivity-based equation:

Where G = electrical conductivity, σ = volumetric conductivity (16.06 S/m for LiCl), L = film thickness (10 nm), and d = pore diameter (S. Kowalczyk, A. Grosberg, Y. Rabin, C. Dekker, Nanotech., 22, 2011). The nanopores were examined for ohmic performance by generating I-V curves. The measured nanopore diameter was determined to be 4.8 nm and subsequently used to determine the DNA calibration standard.

DNA Dose-Response : DNA standards were used as calibrators to observe the dose-response curve by determining the change in nanopore flux rate as DNA concentration increased. This generated a standard curve that can be used to quantify cleaved DNA tags in immunoassays. 2 μl of 1.5 μM 100 bp DNA standard (ThermoScientific) was transferred into a PTFE fluid grid cell containing 30 μl of 3.6 M LiCl salt solution to obtain a final concentration of 94 nM DNA. The reagents were mixed by aspirating the solution several times before nanopore analysis. A +200 mV DC bias was applied to the grid cell, and current blocking was monitored for 60 minutes. The electrical signal and counting rate were characterized using CUSUM analysis software. This procedure was repeated twice to obtain two additional points on the standard curve: 182 nM and 266 nM. The current blocking time was shown for all three standards at different time periods—41 seconds for 94 nM (Figure 30A); 24 seconds for 182 nM (Figure 30B); and 8 seconds for 266 nM (Figure 30C). Baseline noise was empirically estimated at approximately 900 pA, 900 pA, and 1,000 pA for Figures 30A, 30B, and 30C, respectively.

Data from the run were used to generate three different types of dose-response curves: the number of events over a fixed time period (5 minutes) (Figure 31); the time required to reach a fixed number of events (200 events) (Figure 32); and the number of events per unit time (Figure 33). Each of these curves can be used as a standard curve for quantitative nanopore-based immunoassays, where DNA is used as a tag. Similarly, other tags can be used to quantify various analytes, such as dendritic polymers, polymers, nanoparticles, etc.

Seq31-SS-Biotin DNA Dose-Response : A synthetic DNA construct, Seq31-SS-Biotin, was used as the source material to generate dose-response curves (Figure 47). This target can be used to quantify the cleaved label NP-Seq31-SS-Biotin, which is cleaved from streptavidin beads in a qualitative assay. Because this material has approximately the same molecular weight and charge density as the cleaved label Seq31-SS-Biotin, it can be used in calibration curves to quantify the cleaved target from streptavidin particles using TCEP and/or DTT.

Data Analysis: First, the expected current changes observed in double-stranded DNA transfer events were calculated under experimental test conditions using the following equations mentioned in Kwok et al., “Nanopore Fabrication by controlled Dielectric Breakdown” Supplementary Information Section 8 and Kwok, H.; Briggs, K.; and Tabard-Cossa, V., “Nanopore Fabrication by Controlled Dielectric Breakdown” - PLoS ONE 9(3):e92880 (2014), to determine the number of transfer events.

Using the expected current blocking value, the binary data of the experimental nanopore output is visually or manually scanned for acceptable expected current blocking events. Using these events, the threshold and hysteresis parameters required by the CUSUM nanopore software are applied and executed. The output from the software is further analyzed using the cusumtools readevents.py software, filtering blocking events greater than 1000 pA (as determined from the initial calculation). Flux events, time intervals between events, and other calculations are determined from the readevents.py analysis tool. Additional calculations are performed on the data generated by CUSUM using JMP software (SAS Institute, Cary, North Carolina). It should be understood that this threshold setting method is one approach for data analysis, and the invention is not limited to this method; other such methods known to those skilled in the art can also be used.

Summary : This embodiment describes a quantitative assay achieved by performing the steps described herein. A direct assay is performed using a cleavable connector conjugate, as described in Example 17, with a thiol-based cleavage step, as shown in Figure 25. It should be understood that other cleavable connector schemes for performing such assays may include, but are not limited to, various other connector cleavage methods, thereby allowing the use of nanopores to count multiple tags, as described herein. For example, in addition to the method described in Example 17, such other cleavage methods may include, but are not limited to, those described in Examples 18, 19, 20, and 21, as well as other methods described herein and known to those skilled in the art. It is also understood that although the assay format demonstrated in this embodiment (Example 25) represents a direct assay, other formats such as sandwich immunoassays and/or various competing assay formats (such as those known to those skilled in the art) can also be used for assays.

For example, the sandwich immunoassay for TSH (thyroid-stimulating hormone) detection, as described in Example 9, demonstrates the ability to perform such an assay on a low-cost DMF chip. Furthermore, using a variety of heterobifunctional cleavable linkers and conjugates synthesized by methods (such as those described in Examples 1, 2, 3, 4, 5, and 6), as well as other cleavable linkers or conjugates that can be synthesized by methods known to those skilled in the art, many different bioconjugation reagents can be synthesized, which can be used to generate immunoconjugates or other active, specific binding members with cleavable linkers. Additionally, Example 8 demonstrates the functionality of various fluidic microdroplet operations on a low-cost chip, which can facilitate the various steps required for performing various assays, including sandwich and competitive assays, as well as other variations thereof known to those skilled in the art. Example 16 also represents another construct that can be used to perform an assay, in which cleavage is achieved, resulting in the release of countable markers that can be counted using the nanopore counting method described in that example.

Example 22 illustrates how counting can typically be performed to measure transfer events associated with the presence of a label passing through a nanopore. Figure 29 illustrates the concept of a signal threshold, enabling manipulation of the characteristics of the data in a counting assay. Figures 31, 32, and 33 show the output of quantitative assay data, representing the data types that can be used to determine the amount of analyte using the assay methods described in this example. Figure 34 shows a standard curve generated from a construct that has been chemically cut. It is also understood that although dsDNA is used as the label in this particular example, other labels, such as those described in Example 5, can also be utilized, including, but not limited to, nanobeads, dendritic polymers, etc. Such constructs can be synthesized using methods known to those skilled in the art.

Example 26

Nanopore electric field simulation

A series of COMSOL simulation runs were performed on the proposed nanoporous membrane design for use in a silicon module to investigate the effect of the size of the _SiO2 via on counter ion concentration and electroosmotic flow rate through a theoretically 10 nm diameter nanopore. The _SiO2 top layer serves several purposes: 1) to provide an insulating layer for the _SiNx film, thereby reducing the capacitive noise of the nanopores; 2) to increase the robustness and strength of the _SiNx film within the silicon substrate; and 3) to reduce the size of the SiNx area exposed to solution, thereby improving the localization of the pores on the film from the controlled dielectric breakdown (CBD) process. Electric field simulations were used to determine the effect of the SiO2 layer on local counter ion concentration and electroosmotic flow through the pores.

Referring to Figure 35, a silicon substrate (1) is etched to create cis and anti-cells located above and below the SiN _x film. A SiN _x film (50 μm x 50 μm) (2) lies between a 300 μm thick SiO ₂ underlayer and a 300 μm thick SiO ₂ toplayer (3). The toplayer is fabricated to form a SiO ₂ via (4), which allows for the formation of nanopores during the CBD process. The optimal diameter of the SiO ₂ via is determined through simulation.

COMSOL Simulation Results : The COMSOL electric field simulation uses a physical model based on material, electrostatic, molecular transport, and laminar flow properties. The electric potential is based on the Poisson equation; the ion flux is based on the Nernst-Planck equation; and the fluid velocity is based on the Stokes equation. The physical parameters used in the simulation are defined in Table 1 and shown in Figure 36.

COMSOL results for counterbalancing ion concentration gradients near the pores are shown in Figure 37, indicating that ion concentration has minimal to negligible effect when the _SiO2 via diameter is >50 nm. Below 50 nm, this leads to net charge accumulation near the pores. The most severe effect is observed at a diameter of 25 nm, where a large ion gradient forms near the pores. These results demonstrate a considerable influence on the _SiO2 surface when the nanopores are less than 25–50 nm from the _SiO2 wall.

Simulating the electroosmotic flow rate of counter-ions through the pores was used as a way to determine any potential influence of the _SiO2 layer on nanopore sensing (Figure 38). Higher electroosmotic flow rates occurred with larger via diameters (100–4,500 nm). A decrease in flow rate through the pores was observed for 50 nm _SiO2 vias, followed by a significant decrease for 25 nm vias.

As shown in Figure 39, the conductivity measurements of the through-hole relative to the via diameter show a saturation curve above 100 nm, with the conductivity decreasing as the via diameter decreases from 100 nm to 25 nm.

Example 27

Integrating nanopore modules into digital microfluidics (DMF) modules

The nanopore module is positioned on one side of the DMF module. Pores exist within the DMF module to allow liquid to be transported from the DMF module to the nanopore module for pore formation and analyte detection (see, for example, Figure 40).

One electrode from the nanopore module terminates within the fluid volume of the nanopore module. Other electrodes terminate within the fluid volume of the DMF module. This electrode passes through a second pore in the DMF module. To demonstrate that the liquid can move through the pores within the DMF module, a flat piece of paper is pressed onto the outer surface of the chip after the liquid has moved into place. The wetting of the paper indicates that the liquid can move from the DMF module to another module located above the pore via capillary forces (Figure 41).

Referring to Figure 42, an Ag/AgCl electrode is provided to the DMF module for controlled nanopore fabrication. In this scheme, the liquid volume on the nanopore module is an open-air droplet of LiCl. This liquid is directly dispensed onto the nanopore module, and the electrode tip is suspended within the droplet.

The sample is moved into the pores within a DMF module using DMF technology. The sample passively migrates through the pores to become exposed to the nanopore module used for nanopore fabrication. The nanopore module is then sealed to the DMF module (e.g., using PDMS, pressure, wax, etc.), separating the liquid volume retained within each module. Figure 45 shows the current changing over time during nanopore fabrication.

Once the nanopores are established, a conditioning process (changing the voltage over time) is used to physically alter the nanopores and the cleaning signal. This process improves the symmetry in the I-V curves. The I-V curves before and after are shown in Figures 46A and 46B, respectively.

Example 28

Counting markers and pore size analysis

A series of experiments were run using double-stranded DNA under different condition groups to analyze and confirm certain properties related to pore size and counting label size. In these experiments, a variety of parameters were investigated, including detection voltage, DNA length, DNA concentration, salt concentration and composition, membrane material, membrane thickness, nanopore diameter, and other factors.

The dataset was then analyzed in relation to the signal-to-noise ratio, and this factor was compared with various pore sizes associated with the count marker size (estimated molecular diameter). For example, some factors, such as membrane material and thickness, remained constant in this set of experiments, while others varied.

Analysis of the comprehensive dataset shows the average ratio of the counted label's average diameter to the nanopore size plotted against the SNR (signal-to-noise ratio) determined experimentally (Figure 47). Figure 47 generally confirms that useful counting data can be derived from many such ratios, and in this particular dataset, from approximately 0.4–0.8 of such ratios—approximately assuming a molecular diameter of approximately 2.0 nm for dsDNA. It is known from the literature that straight-chain dsDNA is near this molecular diameter, and the analysis assumes that the DNA passes through the pores in its straight-chain conformation. Table 4 shows the calculated data.

Table 4

The ratio of average pore-labeled molecule diameter to pore size SNR 0.645 27.5 0.556 53.7 0.476 12 0.714 23.7 0.803 17 0.645 22.5 0.8 20.2 0.588 17 0.69 55 0.476 23.5

.

As previously mentioned in this embodiment, when conditions are changed, the general range in the dataset indicates that count data with a reasonable signal-to-noise ratio can be obtained within this range. Furthermore, it should be noted that those skilled in the art will recognize that other ratios of the counting marker molecule diameter to the nanopore diameter can be used to achieve a reasonable SNR. Additionally, those skilled in the art will recognize that, generally, the marker should be smaller than the size of the nanopore in at least one dimension of its molecular diameter so that it can pass through the pore; or in other words, the ratio of the marker molecule diameter to the nanopore diameter should generally be less than 1 so that the marker can pass through the pore, except where conditions such as those described in a technique known as nanopore force spectroscopy are used, in which energy is applied to the system to promote a conformational change in the marker and thus allow it to deform to a level that allows such a transfer event to occur before passing through the pore.

Those skilled in the art will also understand that other markers can be used to count other dsDNAs described in this embodiment, and they may have behaviors different from those shown in the figure. Furthermore, it should be understood that acceptable SNRs can also be obtained from other molecular diameter to nanopore ratios to achieve molecular counting of such markers, and current blocking can be correlated with the molecular diameter of such counting markers, as described in the following equations.

It can be found in the following references: Kwok et al., “Nanopore Fabrication by controlled Dielectric Breakdown” Supplementary Information Section 8 and/or Kwok, H.; Briggs, K.; and Tabard-Cossa, V.; “Nanopore Fabrication by Controlled Dielectric Breakdown” - PLoS ONE 9(3):e92880 (2014). This equation can be used for gating or thresholding signals, as described in Examples 24 and 25 of this document.

Some specific conditions that were altered in this comprehensive set of nanopore counting experiments include:

• Ionic strength -3 or 3.6M

DNA length - 10kbp, 50bp, or 1kbp

• The ionic salt used is either LiCl or KCl.

• Membrane material - SiNx (constant throughout the dataset)

• Film thickness -10 nm (constant throughout the dataset)

DNA concentration - varies between 3 nM and approximately 306 nM

• Voltage variation, including increments between 50-600mV

• Nanopore diameter - Multiple pore sizes, including 8.0, 1.1, 3.6, 4.2, 2.8, 2.5, 7.7, 3.1, 2.7, 2.6, 2.9 and 4.2 (all in nanometers).

It can be concluded that various conditions (including, but not limited to, these) indicate that when a voltage is applied in the amount calculable according to the equation mentioned by Kwok et al. in this embodiment [Kwok et al., “Nanopore Fabrication by controlled Dielectric Breakdown” Supplementary Information Section 8 and/or Kwok, H.; Briggs, K.; and Tabard-Cossa, V.; “Nanopore Fabrication by Controlled Dielectric Breakdown” - PLoS ONE 9(3):e92880(2014)], the conditions available when the countable mark is smaller than the pore diameter can cause a blockage in the flux of the ion current across the pore.

It is also understood that these conditions can be applied to show the ratio of the diameter to the pore diameter of the counting marker molecule that will function with a reasonable signal-to-noise ratio for markers other than dsDNA, including, but not limited to, dendritic polymers, semi-dendritic polymers, nanobeads, anionic or cationic polymers, denatured linearized aptamers, negatively or positively charged peptides or other charged polymers or countable molecular entities, etc.

Finally, although different aspects and features of the invention have been described with respect to different embodiments and specific examples herein, they can all be conventionally made or implemented, and it should be understood that the invention is protected within the full scope of the appended claims.

It should be understood that the foregoing detailed description and accompanying embodiments are merely exemplary and should not be used as a limitation on the scope of the invention, which is defined only by the appended claims and their equivalents.

Those skilled in the art will understand that different variations and modifications can be made to the disclosed embodiments. Such variations and modifications can be made without departing from its spirit and scope, including but not limited to those relating to chemical structures, substituents, derivatives, intermediates, synthesis, compositions, formulations, or methods of use of the invention.

For the sake of completeness, different aspects of the invention are set forth in the following numbered clauses:

Clause 1. A method for measuring or detecting an analyte present in a biological sample, the method comprising: (a) contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support, and wherein the first binding member specifically binds the analyte; (b) contacting the analyte with a second binding member, wherein the second binding member specifically binds the analyte, and wherein the second binding member includes a severable tag attached thereto; (c) removing the second binding member from the analyte not bound to the first binding member; (d) slicing the tag attached to the second binding member, the second binding member binding to the analyte bound to the first binding member; (e) transferring the tag through one or more nanopores in a layer; and (f) evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tags transferred through the layer detects the analyte present in the sample.

Clause 2. The method of Clause 1, wherein each tag transferred across the layer is a transfer event, and measuring the number of transfer events measures the amount of analyte present in the sample, wherein the amount of analyte present in the sample is determined as follows: i) counting the number of transfer events over a set time period and correlating the number of transfer events with a control; ii) measuring the time amount of time that a set number of transfer events occurs and correlating it with a control; or iii) measuring the average time between the occurrence of transfer events and correlating it with a control, wherein the control is a reference standard comprising a calibration curve, a standard addition, or a digital polymerase chain reaction.

Clause 3. The method of Clause 2, wherein the standard curve in subsection i) is determined by measuring the number of transfer events for the control concentration of the analyte within a set time period; wherein the standard curve in subsection ii) is determined by the time required for the control concentration of the analyte to occur for the set number of transfer events; and wherein the standard curve in subsection iii) is determined by the average time between the transfer events for the control concentration of the analyte.

Clause 4. The method of any one of Clauses 1-3, wherein the method involves counting individual molecules.

Clause 5. The method of any one of Clauses 1-4, wherein the label is selected from anionic polymers, cationic polymers, dendritic polymers and nanoparticles.

Clause 6. The method of any of Clauses 1 or 5, wherein the label is substantially spherical or hemispherical.

Clause 7. The method of any one of Clauses 1-6, wherein the label is substantially spherical and contains nanoparticles.

Clause 8. The method of any one of Clauses 1-7, wherein the label is substantially spherical or hemispherical and comprises a dendritic polymer.

Clause 9. The method of Clause 8, wherein the dendritic polymer is positively or negatively charged.

Clause 10. The method of Clause 5 or 7, wherein the nanoparticles comprise positively charged nanoparticles.

Clause 11. The method of Clause 10, wherein the nanoparticles comprise negatively charged nanoparticles.

Clause 12. The method of any one of Clauses 1-11, wherein the first binding member and the second binding member are antibodies or receptors.

Clause 13. The method of any one of Clauses 1-12, wherein the first binding member is a receptor and the second binding member is an antibody, or wherein the first binding member is an antibody and the second binding member is a receptor.

Clause 14. The method of any one of Clauses 1-12, wherein the first binding member is a first antibody and the second binding member is a second antibody.

Clause 15. The method of any one of Clauses 1-14, wherein the tag is negatively charged, and the transfer comprises applying a positive potential across the layer thereby transferring the tag through the layer.

Clause 16. The method of any one of Clauses 1-14, wherein the tag is positively charged, and the transfer comprises applying a negative potential across the layer thereby transferring the tag through the layer.

Clause 17. The method of any one of Clauses 1-16, wherein at least steps (a)-(d) are performed in a device that is: a microfluidic device, a droplet-based microfluidic device; a digital microfluidic device (DMF), a surface acoustic wave-based microfluidic device (SAW), a fully integrated DMF and nanopore device, or a fully integrated SAW and nanopore device.

Clause 18. The method of Clause 17, wherein the DMF element and the nanoporous element are operatively coupled in the fully integrated DMF and nanoporous device, or the SAW element and the nanoporous element are operatively coupled in the fully integrated SAW and nanoporous device.

Clause 19. The method of Clause 17, wherein the DMF device or the SAW device is manufactured by a roll-to-roll based printed electronics method.

Clause 20. The method of Clause 18, wherein the DMF element or the SAW element is manufactured by a roll-to-roll based printed electronics method.

Clause 21. The method of Clause 17, wherein the fully integrated DMF and nanopore device or the fully integrated SAW and nanopore device comprises a microfluidic conduit.

Clause 22. The method of Clause 21, wherein the microfluidic conduit couples the DMF element to the nanoporous element, and the microfluidic conduit contains a fluid flow induced by a dynamic or active force.

Clause 23. The method of any one of Clauses 1-22, wherein the nanopore is a solid nanopore or a biological nanopore.

Clause 24. The method of any one of Clauses 1-23, wherein measuring the number of tags transferred through the layer includes observing changes in current induced by the interaction of the tags with the nanopores.

Clause 25. The method of Clause 24, wherein the analyte is present in the sample when the change in current has an order of magnitude exceeding a threshold level.

Clause 26. The method of Clause 23, wherein the method further comprises: transporting a microdroplet containing a tag obtained in step (d) to a nanopore device, and placing the microdroplet across a nanopore layer present in the nanopore device such that the microdroplet is segmented by the nanopore layer and connected through nanopores present in the nanopore layer, wherein the tag is present in the microdroplet on two sides of the nanopore layer.

Clause 27. The method of Clause 26, wherein the method comprises: transferring a tag present on a first side of the nanoporous layer across the nanopore to a second side of the nanoporous layer, thereby collecting the tag in a segmented droplet on the second side of the nanoporous layer.

Clause 28. The method of Clause 26, the method further comprising: transferring the tag to a first side of the nanoporous layer, and determining the number of tags present in the droplets.

Clause 29. The method of any of Clauses 1-28, wherein the label comprises a cutable connector.

Clause 30. A method for measuring or detecting a target analyte present in a biological sample, the method comprising: (a) contacting the sample with a solid support, a first specific binding member, and a second specific binding member, wherein the solid support comprises an immobilization reagent, the first specific binding member comprises a ligand of the immobilization reagent and specifically binds the target analyte, and the second specific binding member comprises a cleavable tag and specifically binds the target analyte, wherein a solid support/first specific binding member/target analyte/second specific binding member complex is formed; (b) (c) Remove the second specific binding member that is not bound to the solid support/first specific binding member/analyte/second specific binding member complex; (d) cut the tag attached to the labeled analyte that is bound to the second specific binding member in the solid support/first specific binding member/target analyte/second specific binding member complex; (e) transfer the tag through one or more nanopores in the layer; and (f) evaluate the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tags transferred through the layer detects the analyte present in the sample.

Clause 31. A method for measuring or detecting an analyte present in a biological sample, the method comprising: (a) contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support, and wherein the first binding member specifically binds the analyte; (b) contacting the analyte with a second binding member, wherein the second binding member specifically binds the analyte, and wherein the second binding member comprises an aptamer; (c) removing aptamers not bound to the analyte bound to the solid substrate; (d) dissociating the aptamers bound to the analyte; (e) transferring the dissociated aptamers through one or more nanopores in a layer; and (f) evaluating the aptamers transferred through the layer, wherein measuring the number of aptamers transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the aptamers transferred through the layer detects the analyte present in the sample.

Clause 32. The method of Clause 31, wherein each aptamer transferred across the layer is a transfer event, and measuring the number of transfer events measures the amount of analyte present in the sample, wherein the amount of analyte present in the sample is determined as follows: i) counting the number of transfer events over a set time period and correlating the number of transfer events with a control; ii) measuring the time amount of time during which the set number of transfer events occurs and correlating it with a control; or iii) measuring the average time between the occurrence of transfer events and correlating it with a control, wherein the control is a reference standard comprising a calibration curve, a standard addition, or a digital polymerase chain reaction.

Clause 33. The method of Clause 32, wherein the standard curve in subsection i) is determined by measuring the number of transfer events for the control concentration of the analyte within a set time period; wherein the standard curve in subsection ii) is determined by the time required for the control concentration of the analyte to occur for the set number of transfer events; and wherein the standard curve in subsection iii) is determined by the average time between the transfer events for the control concentration of the analyte.

Clause 34. The method of any one of Clauses 31-33, wherein the method relates to counting individual molecules.

Article 35. The method of any one of Articles 31-34, wherein the aptamer is a DNA aptamer.

Article 36. The method of any one of Articles 31-34, wherein the aptamer is an RNA aptamer.

Clause 37. The method of any of Clauses 31-36, wherein the first binding member is an antibody.

Clause 38. The method of any one of Clauses 31-36, wherein the analyte is a ligand and the first binding member is a receptor.

Clause 39. The method of any one of Clauses 31-38, wherein at least steps (a)-(d) are performed in a microfluidic device, a droplet-based microfluidic device, a digital microfluidic device (DMF), a surface acoustic wave-based microfluidic device (SAW), a fully integrated DMF and nanopore device, or a fully integrated SAW and nanopore device.

The methods of Clause 40 and Clause 39, wherein the DMF element and the nanoporous element are operatively coupled in the fully integrated DMF and nanoporous device, or the SAW element and the nanoporous element are operatively coupled in the fully integrated SAW and nanoporous device.

The method of Clause 41. Clause 39, wherein the DMF device or the SAW device is manufactured by a roll-to-roll based printed electronics method.

Clause 42. The method of Clause 40, wherein the DMF element or the SAW element is manufactured by a roll-to-roll based printed electronics method.

The method of Clause 43. Clause 39, wherein the fully integrated DMF and nanopore device or the fully integrated SAW and nanopore device comprises a microfluidic conduit.

The method of Clause 44. Clause 43, wherein the microfluidic conduit couples the DMF element to the nanoporous element, and the microfluidic conduit contains a fluid flow induced by a dynamic or active force.

Clause 45. The method of any one of Clauses 31-44, wherein the nanopore is a solid nanopore or a biological nanopore.

Clause 46. An integrated digital microfluidic nanopore device comprising: a first substrate including an electrode array; a second substrate spaced apart from the first substrate; and a nanopore layer disposed between the first and second substrates, wherein the electrode array is configured to position a droplet across the nanopore layer such that the droplet is divided into a first portion and a second portion by the nanopore layer, wherein at least two electrodes of the electrode array are positioned across the nanopore layer, wherein the two electrodes form an anode and a cathode and operate to drive a current through nanopores in the nanopore layer when the liquid droplet is positioned across the nanopore layer.

The apparatus of Clause 47. Clause 46, wherein the nanoporous layer is attached to the first substrate and the second substrate.

The apparatus of Clause 48. Clause 46, wherein the nanoporous layer is attached to the first substrate or the second substrate.

Clause 49. The apparatus of any of Clauses 46-48, wherein the electrode is transparent.

The apparatus of any one of Clauses 50 and 46-49, wherein the electrodes are arranged in a grid pattern.

The apparatus of any one of Clauses 51.46-50, wherein at least two electrodes of the electrode array positioned across the nanopore layer are side-mounted to the nanopore layer and are not positioned across the nanopore layer.

Clause 52. Any of the devices in Clauses 46-51, wherein the electrodes are intersecting.

The apparatus of any one of Clauses 53.46-51, wherein the electrode array is configured to be activated by a power source, wherein the power source activates the electrodes in a sequential manner.

Clause 54. Clause 53 of the apparatus, wherein the sequential manner includes turning one or more electrodes on or off.

Clause 55. An apparatus of any of Clauses 52-54, wherein activation of the electrode array by a power source is controlled by a set of instructions executed by a processor that controls the power source.

Clause 56. An integrated digital microfluidic nanopore device comprising: a first substrate including an electrode array; a second substrate spaced apart from the first substrate; and a nanopore layer disposed between the first and second substrates, wherein the electrode array is configured to position a droplet across the nanopore layer such that the nanopore layer divides the droplet into a first portion and a second portion, wherein at least one electrode of the electrode array contacts the first portion of the droplet positioned across the nanopore layer, and an electrode in the second substrate is positioned to contact the second portion of the droplet positioned across the nanopore layer, wherein the two electrodes form an anode and a cathode and operate to drive a current through nanopores in the nanopore layer when the liquid droplet is positioned across the nanopore layer.

The apparatus of Clause 57 and Clause 56, wherein the nanoporous layer is attached to the first substrate.

The apparatus of any one of Clauses 58, 56, or 57, wherein the nanoporous layer is attached to the second substrate.

Clause 59. Any of the provisions of Clauses 56-58, wherein the first substrate and/or the second substrate is transparent.

The apparatus of any one of Clauses 60 and 56-59, wherein the electrode array is transparent.

Clause 61. A kit comprising the apparatus of any of Clauses 46-60, or for use in any of the methods of Clauses 1-45.

The kit of Clause 62. Clause 61, the kit further comprising additional reagents, wherein at least one reagent comprises a tag detectable by transfer through a nanoporous layer of the device.

Clause 63. The method of using the apparatus of any one of Clauses 46-60, or the method of any one of Clauses 1-45, for measuring or detecting an analyte present in a biological sample or for diagnosing a patient or screening a blood supply.

Clause 64. The method of any of Clauses 1-45, wherein at least steps (a)-(d) are performed using the apparatus of any of Clauses 46-60.

Clause 65. The use of any of the apparatuses in Clauses 46-60, or any of the methods in Clauses 1-45, in the diagnosis of a patient or screening of blood supply, or for the measurement or detection of an analyte present in a biological sample.

Clause 66. A method for measuring or detecting an analyte present in a biological sample, the method comprising: (a) contacting the sample with a binding member, wherein the binding member is immobilized on a solid support and wherein the binding member specifically binds the analyte; (b) contacting the sample with a labeled analyte, wherein the labeled analyte is labeled with a severable tag; (c) removing the labeled analyte not bound to the binding member; (d) severing a tag attached to the labeled analyte bound to the binding member; (e) transferring the tag through one or more nanopores in a layer; and (f) evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tags transferred through the layer detects the analyte present in the sample.

Clause 67. A method for measuring or detecting an analyte present in a biological sample, the method comprising: (a) contacting the sample with a binding member, wherein the binding member is immobilized on a solid support, and wherein the binding member specifically binds the analyte; (b) contacting the sample with a labeled analyte, wherein the labeled analyte comprises an aptamer; (c) removing the labeled analyte not bound to the binding member; (d) dissociating the aptamer bound to the labeled analyte and transferring the dissociated aptamer through one or more nanopores in a layer; and (e) evaluating the aptamers transferred through the layer, wherein measuring the number of aptamers transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the aptamers transferred through the layer detects the analyte present in the sample.

Clause 68. A method for measuring or detecting an analyte present in a biological sample, the method comprising: (a) contacting the sample with a binding member, wherein the binding member specifically binds the analyte, and the binding member being labeled with a severable tag; (b) contacting the sample with an immobilized analyte, wherein the immobilized analyte is immobilized on a solid support; (c) removing binding members not bound to the immobilized analyte; (d) severing a tag attached to a binding member bound to the immobilized analyte; (e) transferring the tag through one or more nanopores in a layer; and (f) evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tags transferred through the layer detects the analyte present in the sample.

Clause 69. A method for measuring or detecting an analyte present in a biological sample, the method comprising: (a) contacting the sample with a binding member, wherein the binding member specifically binds the analyte and the binding member comprises an aptamer; (b) contacting the sample with an immobilized analyte, wherein the immobilized analyte is immobilized on a solid support; (c) removing binding members not bound to the immobilized analyte; (d) dissociating the aptamer bound to a binding member bound to the immobilized analyte, and transferring the dissociated aptamer through one or more nanopores in a layer; and (e) evaluating the aptamers transferred through the layer, wherein measuring the number of aptamers transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the aptamers transferred through the layer detects the analyte present in the sample.

The method of Clause 70, Clause 66, or 68, wherein each tag transferred across the layer is a transfer event, and measuring the number of transfer events measures the amount of analyte present in the sample, wherein the amount of analyte present in the sample is determined as follows: i) counting the number of transfer events over a set time period and correlating the number of transfer events with a control; ii) measuring the time amount at which the set number of transfer events occurs and correlating it with a control; or iii) measuring the average time between the occurrence of transfer events and correlating it with a control, wherein the control is a reference standard comprising a calibration curve, a standard addition, or a digital polymerase chain reaction.

Clause 71. The method of Clause 67 or 69, wherein each aptamer transferred across the layer is a transfer event, and measuring the number of transfer events measures the amount of analyte present in the sample, wherein the amount of analyte present in the sample is determined as follows: i) counting the number of transfer events over a set time period and correlating the number of transfer events with a control; ii) measuring the time amount at which the set number of transfer events occurs and correlating it with a control; or iii) measuring the average time between the occurrence of transfer events and correlating it with a control, wherein the control is a reference standard comprising a calibration curve, a standard addition, or a digital polymerase chain reaction.

Clause 72. The method of Clause 70 or 71, wherein the standard curve in subsection i) is determined by measuring the number of transfer events for the control concentration of the analyte within a set time period; wherein the standard curve in subsection ii) is determined by the time required for the control concentration of the analyte to occur for the set number of transfer events; and wherein the standard curve in subsection iii) is determined by the average time between the transfer events for the control concentration of the analyte.

Clause 73. The method of any one of Clauses 66-72, wherein said method involves counting individual molecules.

Clause 74. The method of any of Clauses 66, 68, 70, 72 or 73, wherein at least steps (a)-(d) are performed using the apparatus of any of Clauses 46-60.

Clause 75. The method of any of Clauses 67, 69, 71, 72 or 73, wherein at least steps (a)-(d) are performed using the apparatus of any of Clauses 46-60.

Clause 76. The method of any one of Clauses 66, 68, 70, 72, 73 or 74, wherein the label is selected from anionic polymers, cationic polymers, dendritic polymers and nanoparticles.

Clause 77. The method of any of Clauses 66, 68, 70, 72, 73 or 74, wherein the label is substantially spherical or hemispherical.

Clause 78. The method of any of Clauses 66, 68, 70, 72, 73 or 74, wherein the label is substantially spherical and contains nanoparticles.

Clause 79. The method of any one of Clauses 66, 68, 70, 72, 73 or 74, wherein the label is substantially spherical or hemispherical and comprises a dendritic polymer.

The method of Clause 80 and Clause 79, wherein the dendritic polymer is positively or negatively charged.

The method of Clause 81 and Clause 79, wherein the nanoparticles comprise positively charged nanoparticles.

The method of Clause 82, Clause 76 or 78, wherein the nanoparticles comprise negatively charged nanoparticles.

Clause 83. The method of any one of Clauses 66, 68, 70, 72, 73, 74 or 76-82, wherein the binding member is an antibody or a receptor.

Clause 84. The method of any one of Clauses 66, 68, 70, 72, 73, 74 or 76-83, wherein the tag is negatively charged and the transfer comprises applying a positive potential across the layer thereby causing the tag to transfer across the layer.

Clause 85. The method of any one of Clauses 66, 68, 70, 72, 73, 74 or 76-84, wherein the tag is positively charged and the transfer comprises applying a negative potential across the layer thereby causing the tag to transfer across the layer.

Clause 86. The method of any one of Clauses 66, 68, 70, 72, 73, 74 or 76-85, wherein measuring the number of tags transferred through the layer includes observing the current blocking effect of the tags on the nanopores.

Clause 87. The method of Clause 86, wherein the analyte is present in the sample when the current blocking effect is above a threshold level.

Article 88. The method of any one of Articles 67, 69, 71, 72, 73 or 75, wherein the aptamer is a DNA aptamer.

Article 89. The method of any one of Articles 67, 69, 71, 72, 73 or 75, wherein the aptamer is an RNA aptamer.

Article 90. The method of any one of Articles 67, 69, 71, 72, 73, 75, 88 or 89, wherein the binding member is an antibody.

Clause 91. The method of any one of Clauses 67, 69, 71, 72, 73, 75, 88 or 89, wherein the analyte is a ligand and the binding member is a receptor.

Clause 92. The method of any one of Clauses 67, 69, 71, 72, 73, 75 or 88-91, wherein the method further comprises: transporting a microdroplet containing the tag to a nanoporous device, and placing the microdroplet across a nanoporous layer present in the nanoporous device such that the microdroplet is segmented by the nanoporous layer and connected by nanopores present in the nanoporous layer, wherein the tag is present in the microdroplet on two sides of the nanoporous layer.

Clause 93. The method of Clause 92, wherein the method comprises: transferring a tag present on a first side of the nanoporous layer across the nanopore to a second side of the nanoporous layer, thereby collecting the tag in a segmented droplet on the second side of the nanoporous layer.

Clause 94. The method of Clause 92, the method further comprising: transferring the tag to a first side of the nanoporous layer, and determining the number of tags present in the droplets.

Clause 95. The method of any one of Clauses 67, 69, 71, 72, 73, 75 or 88-94, wherein the method further comprises: transporting a droplet containing the aptamer to a nanopore device, and placing the droplet across a nanopore layer present in the nanopore device such that the droplet is segmented by the nanopore layer and connected through nanopores present in the nanopore layer, wherein the aptamer is present in droplets on two sides of the nanopore layer.

Clause 96. The method of Clause 95, wherein the method comprises: transferring an aptamer present on a first side of the nanopore layer across the nanopore to a second side of the nanopore layer, thereby collecting the aptamer in a segmented droplet on the second side of the nanopore layer.

The method of Clause 97.95, the method further comprising: transferring the aptamer to a first side of the nanoporous layer, and determining the number of aptamers present in the droplets.

The method of any of the provisions of Clause 98 or Clauses 66-97, wherein the nanopore is a solid nanopore or a biological nanopore.

Article 99. The method of any one of Articles 1-45 or 63-98, wherein the second combining member further comprises a spacer.

The methods of Clause 100 and Clause 99, wherein the spacer comprises nitrobenzyl, dithioethylamino, a 6-carbon spacer, a 12-carbon spacer, or 3-(9-((3-carboxypropyl)(toluenesulfonyl)carbamoyl)acridin-10-on-10-yl)propane-1-sulfonate.

Clause 101. The method of Clause 100, wherein the spacer comprises nitrobenzyl and the tag is a DNA molecule.

Clause 102. The method of Clause 100, wherein the spacer is dithioethylamino and the tag is carboxylated nanoparticles.

Clause 103. The method of Clause 100, wherein the spacer is 3-(9-((3-carboxypropyl)(toluenesulfonyl)carbamoyl)acridin-10-on-10-yl)propane-1-sulfonate, and the tag is an oligonucleotide.

Clause 104. The method of Clause 103, wherein the spacer comprises a 6-carbon spacer or a 12-carbon spacer, and the label is biotin.

Clause 105. The method of Clause 104, wherein the second binding member comprises a nucleic acid comprising a nucleotide sequence shown in any one of SEQ ID NO: 1-11.

Clause 106. An integrated digital microfluidic nanopore device comprising a microfluidic module and a nanopore module; the microfluidic module comprising an electrode array wherein the electrode array transports at least one droplet of fluid to a first transfer location in the electrode array, wherein the first transfer location is at an interface between the microfluidic module and the nanopore module; the nanopore module comprising: a first capillary channel; and a second capillary channel; wherein at least the first capillary channel extends to the interface and is adjacent to the first transfer location, and is positioned to receive a fluid droplet positioned at the first transfer location; wherein the first capillary channel intersects with the second capillary channel, wherein a nanopore layer is positioned between the first capillary channel and the second capillary channel at the location where the first capillary channel and the second capillary channel intersect.

The apparatus of Clause 107. Clause 106, wherein the electrode array transports at least one microdroplet of fluid to a second transfer location in the electrode array, wherein the second transfer location is at an interface between the microfluidic module and the nanopore module, wherein a second capillary channel extends to the interface and is adjacent to the second transfer location and is positioned to receive the fluid microdroplet located at the second transfer location.

The apparatus of Clause 108 and Clause 106, wherein the second capillary channel extends between an outlet or reservoir at one or both ends of the second capillary channel.

The apparatus of Clause 109 and Clause 108, wherein the second capillary channel is connected to a first reservoir at one end and a second reservoir at the other end.

The apparatus of Clause 110 and Clause 109, wherein the first reservoir and/or the second reservoir contains fluid, the fluid being positioned within the second capillary channel at the intersection, the fluid facilitating the operation of the nanopore layer to drive an electric current through the nanopores of the nanopore layer.

The apparatus of Clause 111. Clause 109, wherein the first capillary channel and/or the second capillary channel vary in cross-sectional width over the length of the capillary channel such that the width is reduced at the intersection compared to the width on either side of the intersection.

The apparatus of Clause 112. Clause 106, wherein the first capillary channel includes a first pair of electrodes and the second capillary channel includes a second pair of electrodes, wherein the first pair of electrodes is positioned in the first capillary channel and laterally connected to a nanopore in the nanopore layer, and wherein the second pair of electrodes is positioned in the second capillary channel and laterally connected to a nanopore in the nanopore layer.

The apparatus of any one of Clauses 113.107-112, wherein the microdroplet is a microdroplet containing molecules to be counted by transporting through the nanopores in the nanopore layer.

The apparatus of Clause 114. Clause 107, wherein the fluid droplets have different compositions and are a first droplet and a second droplet, the first droplet containing molecules to be counted by transport across the nanopore layer through the nanopore, and the second droplet containing a conductive fluid lacking the molecules, wherein the conductive fluid facilitates the transport of the molecules across the nanopore layer via the nanopore.

The apparatus of any one of Clauses 115.106-114, wherein the first capillary channel includes a first electrode positioned at a proximal end of the nanoporous layer, and the second capillary channel includes a second electrode positioned at a proximal end of the nanoporous layer, wherein each of the first and second electrodes is exposed in the capillary channel such that it comes into contact with a fluid present in the capillary channel, and wherein when the liquid is positioned to traverse the nanoporous layer in the first and second capillary channels, the first and second electrodes operate to drive a current through the nanopores in the nanoporous layer.

The apparatus of any one of Clauses 116 to 115, wherein the first transfer position and the first capillary channel are substantially in the same plane, and wherein the fluid droplet is aligned with the opening of the first capillary channel.

Clause 117. An apparatus of any of Clauses 106-115, wherein the first transfer position is at a plane higher than the first capillary channel, and wherein the apparatus is configured to have a vertical port for transferring the fluid droplet downward to the opening of the first capillary channel.

The apparatus of Clause 118. Clause 117, wherein a first surface of the first substrate includes a first region on which the electrode array is disposed and a second region in which the first microchannel is formed, wherein the electrode array is on a plane that is higher than the plane on which the first microchannel is formed.

The apparatus of Clause 119. Clause 117, wherein the second substrate includes a groove located at a side edge of the interface, wherein the groove is aligned above the first capillary channel and provides a vertical port for transporting a microdroplet located at the transfer electrode to an opening of the first capillary channel.

The apparatus of any one of Clauses 120 and 106-119, the apparatus further comprising a single electrode spaced apart from the electrode array, wherein the single electrode extends over at least a portion of the electrode array at the first transfer position and is biplane configured with respect to at least a portion of the electrode array at the first transfer position.

Clause 121. An apparatus of any of Clauses 107-119, the apparatus further comprising a single electrode spaced apart from the electrode array.

Clause 122. An apparatus of any one of Clauses 106-119, the apparatus further comprising a single electrode spaced apart from the electrode array, wherein the single electrode does not extend over the first transfer position and is not biplanar with respect to the electrode array, wherein the fluid droplet is moved to the first transfer position by using a coplanar electrode.

The apparatus of any one of Clauses 123.106-119, the apparatus further comprising a single electrode spaced apart from the electrode array, wherein the single electrode does not extend over the first transfer position and is not biplanar with respect to the electrode array, wherein the fluid droplet is moved to the transfer position by using a coplanar electrode.

Clause 124. A method using the apparatus of any of Clauses 106-123, or the method of any of Clauses 66-105, for measuring or detecting an analyte present in a biological sample or for diagnosing a patient or screening a blood supply.

Clause 125. The method of any one of Clauses 1-45 or 66-105, wherein at least steps (a)-(d) are performed using the apparatus of any one of Clauses 106-123.

Clause 126. The use of the apparatus of any of Clauses 106-123, or the use of the method of any of Clauses 1-45, in the diagnosis of a patient or screening of blood supply, or for the measurement or detection of an analyte present in a biological sample.

Clause 127. A method for measuring an analyte present in a biological sample, the method comprising: (a) contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support, and wherein the first binding member specifically binds the analyte; (b) contacting the analyte with a second binding member, wherein the second binding member specifically binds the analyte, and wherein the second binding member includes a cleavable tag attached thereto; (c) removing the second binding member from the analyte not bound to the first binding member; (d) cleaving the tag attached to the second binding member, the second binding member binding to the analyte bound to the first binding member; (e) transferring the tag through one or more nanopores in a layer; and (f) evaluating the tags transferred through the layer, wherein each tag transferred through the layer is a transfer event, wherein the transfer is measured. The number of events measures the amount of analyte present in the sample, wherein the amount of analyte present in the sample is determined as follows: i) counting the number of transfer events within a set time period and correlating the number of transfer events with a control; ii) measuring the time required for the set number of transfer events to occur and correlating it with a control; or iii) measuring the average time between the occurrence of transfer events and correlating it with a control, wherein the control is a reference standard comprising a calibration curve, a standard addition, or a digital polymerase chain reaction, wherein the standard curve in sub-item i) is determined by measuring the number of transfer events for a control concentration of the analyte within the set time period; wherein the standard curve in sub-item ii) is determined by measuring the time required for the set number of transfer events to occur for a control concentration of the analyte; and wherein the standard curve in sub-item iii) is determined by measuring the average time between the occurrence of transfer events for a control concentration of the analyte.

Clause 128. A method for measuring an analyte present in a biological sample, the method comprising: (a) contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support, and wherein the first binding member specifically binds the analyte; (b) contacting the analyte with a second binding member, wherein the second binding member specifically binds the analyte, and wherein the second binding member comprises an aptamer; (c) removing aptamers not bound to the analyte bound to the solid substrate; (d) dissociating the aptamers bound to the analyte; and (e) transferring the dissociated aptamers through one or more nanopores in a layer; and (f) evaluating the aptamers transferred through the layer, wherein each aptamer transferred through the layer is a transfer event, and wherein measuring the number of transfer events measures the analyte present in the sample. The amount of analyte present in the sample is determined by: i) counting the number of transfer events within a set time period and correlating the number of transfer events with a control; ii) measuring the time required for the set number of transfer events to occur and correlating it with a control; or iii) measuring the average time between the occurrence of transfer events and correlating it with a control, wherein the control is a reference standard comprising a calibration curve, a standard addition, or a digital polymerase chain reaction, wherein the standard curve in sub-item i) is determined by measuring the number of transfer events for a control concentration of the analyte within a set time period; wherein the standard curve in sub-item ii) is determined by measuring the time required for the set number of transfer events to occur for a control concentration of the analyte; and wherein the standard curve in sub-item iii) is determined by measuring the average time between the occurrence of transfer events for a control concentration of the analyte.

Clause 129. A method for measuring an analyte present in a biological sample, the method comprising: (a) contacting the sample with a binding member, wherein the binding member is immobilized on a solid support, and wherein the binding member specifically binds the analyte; (b) contacting the sample with a labeled analyte, wherein the labeled analyte is labeled with a severable tag; (c) removing the labeled analyte not bound to the binding member; (d) severing a tag attached to the labeled analyte bound to the binding member; (e) transferring the tag through one or more nanopores in a layer; and (f) evaluating the tags transferred through the layer, wherein each tag transferred through the layer is a transfer event, wherein measuring the number of transfer events measures the amount of analyte present in the sample, wherein as... The amount of analyte present in the sample is determined by: i) counting the number of transfer events within a set time period and correlating the number of transfer events with a control; ii) measuring the time required for the set number of transfer events to occur and correlating it with a control; or iii) measuring the average time between the occurrence of transfer events and correlating it with a control, wherein the control is a reference standard comprising a calibration curve, a standard addition, or a digital polymerase chain reaction, wherein the standard curve in sub-item i) is determined by measuring the number of transfer events for a control concentration of the analyte within the set time period; wherein the standard curve in sub-item ii) is determined by measuring the time required for the set number of transfer events to occur for a control concentration of the analyte; and wherein the standard curve in sub-item iii) is determined by measuring the average time between the occurrence of transfer events for a control concentration of the analyte.

Clause 130. A method for measuring an analyte present in a biological sample, the method comprising: (a) contacting the sample with a binding member, wherein the binding member is immobilized on a solid support, and wherein the binding member specifically binds the analyte; (b) contacting the sample with a labeled analyte, wherein the labeled analyte comprises an aptamer; (c) removing the labeled analyte not bound to the binding member; (d) dissociating the aptamer bound to the labeled analyte and transferring the dissociated aptamer through one or more nanopores in a layer; and (e) evaluating the aptamer transferred through the layer, wherein each aptamer transferred through the layer is a transfer event, wherein measuring the number of transfer events measures the amount of analyte present in the sample, wherein the presence of analyte in the sample is determined as follows: The amount of analyte in the sample: i) counting the number of transfer events within a set time period and correlating the number of transfer events with a control; ii) measuring the time required for the set number of transfer events to occur and correlating it with a control; or iii) measuring the average time between the occurrence of transfer events and correlating it with a control, wherein the control is a reference standard comprising a calibration curve, a standard addition, or a digital polymerase chain reaction, wherein the standard curve in sub-item i) is determined by measuring the number of transfer events for a control concentration of the analyte within a set time period; wherein the standard curve in sub-item ii) is determined by measuring the time required for the set number of transfer events to occur for a control concentration of the analyte; and wherein the standard curve in sub-item iii) is determined by measuring the average time between the occurrence of transfer events for a control concentration of the analyte.

Clause 131. A method for measuring an analyte present in a biological sample, the method comprising: (a) contacting the sample with a binding member, wherein the binding member specifically binds the analyte, and the binding member is labeled with a severable tag; (b) contacting the sample with an immobilized analyte, wherein the immobilized analyte is immobilized on a solid support; (c) removing binding members not bound to the immobilized analyte; (d) severing a tag attached to a binding member bound to the immobilized analyte; (e) transferring the tag through one or more nanopores in a layer; and (f) evaluating the tags transferred through the layer, wherein each tag transferred through the layer is a transfer event, and measuring the number of transfer events measures the amount of analyte present in the sample, wherein the following Determine the amount of analyte present in the sample by: i) counting the number of transfer events within a set time period and correlating the number of transfer events with a control; ii) measuring the time required for the set number of transfer events to occur and correlating it with a control; or iii) measuring the average time between the occurrence of transfer events and correlating it with a control, wherein the control is a reference standard comprising a calibration curve, a standard addition, or a digital polymerase chain reaction, wherein the standard curve in sub-item i) is determined by measuring the number of transfer events for a control concentration of the analyte within the set time period; wherein the standard curve in sub-item ii) is determined by measuring the time required for the set number of transfer events to occur for a control concentration of the analyte; and wherein the standard curve in sub-item iii) is determined by measuring the average time between the occurrence of transfer events for a control concentration of the analyte.

Clause 132. A method for measuring an analyte present in a biological sample, the method comprising: (a) contacting the sample with a binding member, wherein the binding member specifically binds the analyte and the binding member comprises an aptamer; (b) contacting the sample with an immobilized analyte, wherein the immobilized analyte is immobilized on a solid support; (c) removing binding members not bound to the immobilized analyte; (d) dissociating the aptamer bound to the binding member bound to the immobilized analyte, and transferring the dissociated aptamer through one or more nanopores in a layer; and (e) evaluating the aptamers transferred through the layer, wherein each aptamer transferred through the layer is a transfer event, wherein measuring the number of transfer events measures the amount of analyte present in the sample, wherein the following determines... The amount of analyte present in the sample is determined by: i) counting the number of transfer events within a set time period and correlating the number of transfer events with a control; ii) measuring the time required for the set number of transfer events to occur and correlating it with a control; or iii) measuring the average time between the occurrence of transfer events and correlating it with a control, wherein the control is a reference standard comprising a calibration curve, a standard addition, or a digital polymerase chain reaction, wherein the standard curve in sub-item i) is determined by measuring the number of transfer events for a control concentration of the analyte within the set time period; wherein the standard curve in sub-item ii) is determined by measuring the time required for the set number of transfer events to occur for a control concentration of the analyte; and wherein the standard curve in sub-item iii) is determined by measuring the average time between the occurrence of transfer events for a control concentration of the analyte.

Clause 133. A method for measuring or detecting an analyte present in a biological sample, the method comprising: (a) contacting the sample with a binding member, wherein the binding member is immobilized on a solid support, the binding member comprising a cleavable tag attached thereto, and the binding member specifically binding the analyte; (b) removing binding members not bound to the analyte; (c) cleaving the tag, the tag being attached to a binding member bound to the analyte; (d) transferring the tag through one or more nanopores in a layer; and (e) evaluating the tags transferred through the layer, wherein each tag transferred through the layer is a transfer event, wherein measuring the number of transfer events measures the amount of analyte present in the sample, wherein the amount of analyte present in the sample is determined as follows: i) counting the number of transfer events over a set time period and correlating the number of transfer events with a control; ii) measuring the time amount at which a set number of transfer events occur and correlating it with a control; or iii) measuring the average time between the occurrence of transfer events and correlating it with a control, wherein the control is a reference comprising a calibration curve, a standard addition, or a digital polymerase chain reaction.

Clause 134. The method of Clause 133, wherein the standard curve in subsection i) is determined by measuring the number of transfer events for the control concentration of the analyte within a set time period; wherein the standard curve in subsection ii) is determined by the time required for the control concentration of the analyte to occur for the set number of transfer events; and wherein the standard curve in subsection iii) is determined by the average time between the transfer events for the control concentration of the analyte.

Clause 135. An integrated digital microfluidic nanopore realization device, comprising: a microfluidic module and a nanopore realization module; the microfluidic module comprising an electrode array spaced from a single electrode, the single electrode being sized to overlap at least a portion of the electrode array, wherein the electrode array and the single electrode transport at least one microdroplet of fluid to a transfer electrode in the electrode array, wherein the transfer electrode is positioned at an interface between the microfluidic module and the nanopore realization module; the nanopore realization module comprising: a first microchannel positioned on a first surface of a first substrate; a second microchannel positioned on a first surface of a second substrate; wherein the first surface of the first substrate contacts the first surface of the second substrate, thereby closing the first microchannel and the second microchannel to provide a first capillary channel and a second capillary channel, respectively. A capillary channel, wherein at least the first capillary channel extends to the interface between the microfluidic module and the nanopore realization module and is adjacent to the transfer electrode, and is positioned to receive fluid droplets positioned on the transfer electrode; wherein the first capillary channel intersects with the second capillary channel, wherein a layer is positioned between the first substrate and the second substrate at the intersection of the first capillary channel and the second capillary channel, wherein the layer lacks nanopores and separates the ionic liquid present in the first capillary channel and the second capillary channel, wherein the first capillary channel and the second capillary channel are electrically connected to an electrode for driving a voltage from the first capillary channel to the second capillary channel (or vice versa) to establish a nanopore in the layer at the intersection of the first capillary channel and the second capillary channel.

The apparatus of Clause 136. Clause 135, wherein the ionic liquid is an aqueous solution.

The apparatus of Clause 137 and Clause 136, wherein the aqueous solution is a salt solution.

Clause 138. An apparatus of any one of Clauses 135-137, wherein the ionic liquid contains a target analyte, wherein the apparatus is configured to detect the presence or absence of the analyte in the ionic liquid.

Clause 139. A method for generating nanopores in an integrated digital microfluidic nanopore realization device, the method comprising: providing an integrated digital microfluidic nanopore realization device of any one of Clauses 135-138; applying a voltage in a first capillary channel and a second capillary channel to drive a current through the layer; measuring the conductance across the layer; and terminating the application of the voltage after detecting conductance indicative of nanopore generation in the layer.

Clause 140. An integrated digital microfluidic nanopore device comprising: a first substrate including an array of electrodes; a second substrate spaced apart from the first substrate; an opening in the first or second substrate, the opening being in fluid communication with a nanopore layer including nanopores; and a pair of electrodes configured to apply an electric field through the nanopores, wherein the electrode array is configured to transport at least one microdroplet of fluid to the opening.

The apparatus of Clause 141. Clause 140, wherein the opening is a capillary channel.

Clause 142. The apparatus of Clause 141, wherein the capillary channel has an opening on a first side of the first substrate or the second substrate, the opening being wider than the opening on a second side of the first substrate or the second substrate.

The apparatus of Clause 143. Clause 142, wherein the detection electrode pair comprises a first detection electrode that is a single electrode.

Clause 144. The apparatus of Clause 142 or 143, wherein the detection electrode pair includes a second detection electrode disposed on the second side.

Clause 145. A pair of integrated digital microfluidic nanopore devices, comprising:

According to Clause 142, a first integrated digital microfluidic nanopore device, wherein the single electrode is a first single electrode and the capillary channel is a first capillary channel; and a second integrated digital microfluidic nanopore device comprising: a third substrate having a fifth side and a sixth side opposite to the fifth side, wherein the fifth side comprises an electrode array; a fourth substrate spaced from the third substrate, wherein the fourth substrate has a seventh side facing the fifth side of the third substrate and an eighth side opposite to the seventh side, wherein the seventh side comprises a second single electrode, and wherein the nanopore layer is disposed on the eighth side, wherein the fourth substrate includes a second capillary channel extending from the seventh side of the fourth substrate to the eighth side, wherein the nanopore layer is positioned over the opening of the capillary channel, wherein the nanopore layer is interposed between the second substrate and the fourth substrate such that the nanopore provides an electroosmotic conduit between the first capillary channel and the second capillary channel, wherein the detection electrode pair includes a second detection electrode, which is the second single electrode.

Clause 146. An integrated digital microfluidic nanopore realization device comprising: a first substrate having a first side and a second side opposite to the first side, wherein the first side includes an electrode array; a second substrate spaced from the first substrate, wherein the second substrate includes a third side facing the first side of the first substrate and a fourth side opposite to the third side; a nanopore realization layer lacking nanopores and disposed on an outer surface of the device, wherein the outer side is selected from the second side or the fourth side, wherein one of the first substrate or the second substrate including the outer side includes a capillary channel extending from the first side of the first substrate to the second side or from the third side of the second substrate to the fourth side, wherein the nanopore realization layer is positioned over an opening of the capillary channel; and a pair of electrodes configured to apply an electric field across the nanopore realization layer, wherein the electrode array is configured to transport at least one microdroplet of fluid to the capillary channel.

Clause 147. A method for generating nanopores in an integrated digital microfluidic nanopore realization device, the method comprising: providing the integrated digital microfluidic nanopore realization device of Clause 143; immersing two sides of the nanopore realization layer in an ionic liquid such that the ionic liquid on each side of the layer makes electrical contact with any one of the detection electrode pairs; applying a voltage between the detection electrode pairs to drive a current through the layer; measuring the conductance across the layer; and terminating the application of the voltage after detecting conductance indicating the generation of a nanopore in the layer.

The method of Clause 148 and Clause 147, wherein the ionic liquid is a salt solution.

Clause 149. The method of Clause 147 or 148, wherein the ionic liquid contains a target analyte, wherein the device is configured to detect the presence or absence of the analyte in the ionic liquid.

The method of any one of Clause 150, Clause 139, or Clauses 147-149, wherein the method further comprises conditioning the generated nanopores.

Clause 151. The method of Clause 150, wherein the conditioning comprises: alternately applying a first voltage having a first polarity and a second voltage having a second polarity opposite to the first polarity across the nanopore membrane, wherein the first voltage and the second voltage are each applied at least once; and measuring electroosmotic properties in relation to the size of the nanopore.

Clause 152. The method of Clause 150 or 151, the method further comprising measuring electroosmotic properties in relation to the size of the nanopores prior to the conditioning.

Clause 153. A composition comprising a binding member, a label, and a spacer.

The compositions of Clause 154 and Clause 153, wherein the spacer comprises nitrobenzyl, dithioethylamino, a 6-carbon spacer, a 12-carbon spacer, or 3-(9-((3-carboxypropyl)(toluenesulfonyl)carbamoyl)acridin-10-on-10-yl)propane-1-sulfonate.

The composition of Clause 155 and Clause 154, wherein the spacer comprises nitrobenzyl and the tag is a DNA molecule.

The composition of Clause 156 and Clause 154, wherein the spacer is dithioethylamino and the label is carboxylated nanoparticles.

The composition of Clause 157 and Clause 154, wherein the spacer is 3-(9-((3-carboxypropyl)(toluenesulfonyl)carbamoyl)acridin-10-on-10-yl)propane-1-sulfonate, and the tag is an oligonucleotide.

The composition of Clause 158 and Clause 154, wherein the spacer comprises a 6-carbon spacer or a 12-carbon spacer, and the label is biotin.

The composition of Clause 159. Clause 158, wherein the second binding member comprises a nucleic acid comprising a nucleotide sequence shown in any one of SEQ ID NO: 1-11.

Compositions of any of the clauses 160, 153-159, wherein the label comprises a cutable connector.

Clause 161. The composition of Clause 160, wherein the cuttable connector is selected from optically cuttable connectors, chemically cuttable connectors, thermally cuttable connectors, heat-sensitive cuttable connectors, and enzyme-cuttable connectors.

The composition of Clause 162. Clause 161, wherein the cutable connector is a photocutable connector, wherein the photocutable connector comprises a photocutable portion derived from the following

The composition of Clause 163. Clause 161, wherein the cuttable joint is a heat-cuttable joint and is cut using a localized temperature rise.

The composition of Clause 164 and Clause 163, wherein the local temperature rise is generated photothermally or by microwave radiation.

Compositions of Clause 165 and Clause 164, wherein energy from light is transferred to an absorbing target.

Compositions of Clause 166 and Clause 165, wherein the absorbent target comprises a dye, pigment, or water.

Clause 167. A composition of any one of Clauses 163-166, wherein the cleavable adapter comprises double-stranded DNA.

The composition of Clause 168 and Clause 161, wherein the cuttable joint is a chemically cuttable joint and the cutting is mediated by a thiol group.

The method of any one of Clauses 169, 29, 66, 68, 70, 72-86, 92-94, 98-105, 127, 129, 131, 133 and 134, wherein the label comprises a cutable connector.

The methods of Clause 170 and Clause 169, wherein the cuttable connector is selected from optically cuttable connectors, chemically cuttable connectors, thermally cuttable connectors, heat-sensitive cuttable connectors, and enzyme-cuttable connectors.

Clause 171. The method of Clause 170, wherein the cutable connector is a photocutable connector, and the photocutable connector comprises a photocutable portion derived from the following

Clause 172. The method of Clause 170, wherein the cutable joint is a thermally cutable joint and is cut using a localized temperature rise.

Clause 173. The method of Clause 172, wherein the local temperature rise is generated photothermally or by microwave radiation.

Clause 174. The method of Clause 173, wherein energy from light is transferred to an absorbing target.

The method of Clause 175. Clause 174, wherein the absorbed target comprises a dye, pigment or water.

Clause 176. The method of any one of Clauses 172-175, wherein the cleavable adapter comprises double-stranded DNA.

Clause 177. The methods of Clauses 30 and 170, wherein the cuttable connector is a chemically cuttable connector and is thiol-cut.

Clause 178. The method of any one of Clauses 2-30, 31-45, 64, 70-105, 125, 127-134 and 169-177, wherein one or more transfer events correspond to the binding events of the binding member and the analyte.

Clause 179. The method of Clause 178, wherein one of the transfer events corresponds to the combination event of the member and the analyte.

The methods of Clause 180 and Clause 178, wherein two or more transfer events correspond to the binding events of the binding member and the analyte.

Clause 181. The method of Clause 180, wherein each binding member is incorporated with two or more tags, and two or more transition events represent the binding of the binding member to the analyte.

Clause 182. The method of any one of Clauses 1-45, 63-105, 124, 125, 127-134 and 169-181, wherein at least two or more nanopores are in said layer.

Clause 183. The method of Clause 182, wherein the at least two or more nanopores are present in parallel or in series.

Clause 184. An integrated digital microfluidic nanopore device comprising: a first substrate including an electrode array; a second substrate spaced apart from the first substrate; and a nanopore layer having a first surface and a second surface disposed between the first substrate and the second substrate, wherein the electrode array is configured to position a first microdroplet at the first surface of the nanopore layer, wherein at least two electrodes of the electrode array are positioned transversely across the nanopore layer, wherein the two electrodes form an anode and a cathode and operate to drive a current through nanopores in the nanopore layer when the liquid microdroplet is at the first surface of the nanopore layer.

The electrode array of Clause 185 and Clause 173 is further configured to position the second microdroplet on the second surface of the nanoporous layer.

Clause 186. An integrated digital microfluidic nanopore device comprising a microfluidic module and a nanopore module; the microfluidic module comprising an electrode array, wherein the electrode array transports at least one microdroplet of fluid to a transfer location in the electrode array, wherein the transfer location is at an interface between the microfluidic module and the nanopore module; the nanopore module comprising: a first capillary channel extending from the transfer location to a nanopore layer.

Clause 187. An integrated digital microfluidic nanopore device comprising: a first substrate having an array of electrodes; a second substrate spaced apart from the first substrate; a first nanopore layer having one or more nanopores therein; a second nanopore layer having one or more nanopores therein; and at least two electrodes for establishing an electric field to drive a tag through the nanopores in the first and second nanopore layers.

The method of Clause 188. Clause 30, wherein the immobilization agent comprises biotin or streptavidin.

The method of Clause 189 and Clause 188, wherein the immobilization agent comprises biotin and the ligand comprises streptavidin.

The methods of Clause 190 and Clause 188, wherein the immobilization agent comprises streptavidin and the ligand comprises biotin.

Clause 191. The method of any one of Clauses 30 and 188-190, wherein the solid support, the first binding member and the second binding member are added to the sample sequentially or simultaneously.

Clause 192. The method of any one of Clauses 1-45, 63-105, 124, 125, 127-134, 169-181, 183 and 188-191, wherein the ratio of the size of the hole to the size of the label is or less than 1.0.

Clause 193. A method for measuring or detecting a target analyte present in a biological sample, the method comprising: (a) contacting the sample with a solid support, a binding member, and a labeled analyte labeled with a cleavable tag, wherein the solid support comprises an immobilization reagent, the binding member comprises a ligand of the immobilization reagent, and the binding member specifically binds the target analyte to form a solid support/binding member/target analyte complex or a solid support/binding member/labeled analyte complex; (b) removing the labeled analyte that is not bound to a binding member in the solid support/binding member/labeled analyte complex; (c) cleaving the tag, the tag being attached to the labeled analyte bound to a binding member in the solid support/binding member/labeled analyte complex; (d) transferring the tag through one or more nanopores in the layer; and (e) evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tags transferred through the layer detects the analyte present in the sample.

The method of Clause 194 and Clause 193, wherein the immobilization agent comprises biotin or streptavidin.

The method of Clause 195. Clause 194, wherein the immobilization agent comprises biotin and the ligand comprises streptavidin.

The method of Clause 196. Clause 194, wherein the immobilization agent comprises streptavidin and the ligand comprises biotin.

Clause 197. The method of any of Clauses 193-196, wherein the solid support, the binding member and the labeled analyte are added to the sample sequentially or simultaneously.

Clause 198. A method for measuring or detecting a target analyte present in a biological sample, the method comprising: (a) contacting the sample with a solid support, a binding member, and an exogenous analyte, wherein the solid support comprises an immobilization reagent, the exogenous analyte comprises a ligand of the immobilized reagent and binds to the solid support to form a solid support/immobilized analyte complex, and the binding member comprises a cleavable tag and specifically binds to the target analyte to form a solid support/target analyte/binding member complex or a solid support/immobilized analyte/binding member complex; (b) Remove binding members not bound to the solid support/immobilized analyte/binding member complex or the solid support/target analyte/binding member complex; (c) Cut a tag attached to a binding member in the solid support/immobilized analyte/binding member complex; (d) Transfer the tag through one or more nanopores in the layer; and (e) Evaluate the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tags transferred through the layer detects the analyte present in the sample.

The method of Clause 199. Clause 198, wherein the immobilization agent comprises biotin or streptavidin.

The method of Clause 200 and Clause 199, wherein the immobilization agent comprises biotin and the ligand comprises streptavidin.

The method of Clause 201. Clause 199, wherein the immobilization agent comprises streptavidin and the ligand comprises biotin.

Clause 202. The method of any of Clauses 198-201, wherein the solid support, the binding member and the exogenous analyte are added to the sample sequentially or simultaneously.

Clause 203. The method of any of Clauses 198-201, wherein the solid support/immobilized analyte/binding member complex is separated prior to cutting the label in step (c).

Clause 204. The method of Clause 203, wherein separation may be accomplished using a magnetic field.

Clause 205. A method for measuring or detecting a target analyte present in a biological sample, the method comprising: (a) contacting the sample with a solid support, a binding member, and a labeled analyte labeled with an aptamer, wherein the solid support comprises an immobilization reagent, the binding member comprises a ligand of the immobilization reagent, and the binding member specifically binds the target analyte to form a solid support/binding member/target analyte complex or a solid support/binding member/labeled analyte complex; (b) removing unbound analytes from the solid support/binding member/target analyte complex. (c) Dissociating the aptamer attached to the labeled analyte bound to the binding member in the solid support/binding member/labeled analyte complex; (d) Transferring the dissociated aptamer through one or more nanopores in the layer; and (e) Evaluating the aptamers transferred through the layer, wherein measuring the number of aptamers transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the aptamers transferred through the layer detects the analyte present in the sample.

The method of Clause 206. Clause 205, wherein the immobilization agent comprises biotin or streptavidin.

The method of Clause 207. Clause 206, wherein the immobilization agent comprises biotin and the ligand comprises streptavidin.

The method of Clause 208. Clause 206, wherein the immobilization agent comprises streptavidin and the ligand comprises biotin.

Clause 209. The method of any of Clauses 205-208, wherein the solid support, the binding member and the labeled analyte are added to the sample sequentially or simultaneously.

Clause 210. A method for measuring or detecting a target analyte present in a biological sample, the method comprising: (a) contacting the sample with a solid support, a binding member, and an exogenous analyte, wherein the solid support comprises an immobilization reagent, the exogenous analyte comprises a ligand of the immobilized reagent and binds to the solid support to form a solid support/immobilized analyte complex, and the binding member comprises an aptamer and specifically binds to the target analyte to form a solid support/target analyte/binding member complex or a solid support/immobilized analyte/binding member complex; (b) (c) Remove binding members not bound to the solid support/immobilized analyte/binding member complex or the solid support/target analyte/binding member complex; (d) dissociate the aptamer bound to the binding member in the solid support/immobilized analyte/binding member complex; (e) transfer the tag through one or more nanopores in the layer; and (f) evaluate the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tags transferred through the layer detects the analyte present in the sample.

Clause 211. The method of Clause 210, wherein the immobilization agent comprises biotin or streptavidin.

Clause 212. The method of Clause 211, wherein the immobilization agent comprises biotin and the ligand comprises streptavidin.

Clause 213. The method of Clause 211, wherein the immobilization agent comprises streptavidin and the ligand comprises biotin.

Clause 214. The method of any of Clauses 210-213, wherein the solid support, the binding member and the exogenous analyte are added to the sample sequentially or simultaneously.

Clause 215. The method of any of Clauses 210-213, wherein the solid support/immobilized analyte/binding member complex is separated prior to dissociation of the aptamer in step (c).

Clause 216. The method of Clause 215, wherein separation may be accomplished using a magnetic field.

Clause 217. A method for measuring or detecting an analyte present in a biological sample, the method comprising:

I. (a) Contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support, and wherein the first binding member specifically binds the analyte; (b) Contacting the analyte with a second binding member, wherein the second binding member specifically binds the analyte, and wherein the second binding member comprises an aptamer; (c) Removing aptamers not bound to the analyte bound to the solid substrate; (d) Dissociating the aptamers bound to the analyte and transferring the dissociated aptamers through or across one or more nanopores in the layer; and (e) Evaluating the aptamers transferred through the layer, wherein measuring the number of aptamers transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the aptamers transferred through the layer detects the analyte present in the sample.

II. (a) Contacting the sample with a binding member, wherein the binding member is immobilized on a solid support and wherein the binding member specifically binds the analyte; (b) Contacting the sample with a labeled analyte, wherein the labeled analyte is labeled with a severable tag; (c) Removing the labeled analyte that is not bound to the binding member; (d) Cutting a tag attached to the labeled analyte bound to the binding member; (e) Transferring the cut tag through or across one or more nanopores in the layer; and (f) Evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or wherein detecting the tags transferred through the layer detects the presence of analyte in the sample.

III. (a) Contacting the sample with a binding member, wherein the binding member is immobilized on a solid support, and wherein the binding member specifically binds the analyte; (b) Contacting the sample with a labeled analyte, wherein the labeled analyte comprises an aptamer; (c) Removing the labeled analyte not bound to the binding member; (d) Dissociating the aptamer bound to the labeled analyte bound to the binding member and transferring the dissociated aptamer through or across one or more nanopores in the layer; and (e) Evaluating the aptamers transferred through the layer, wherein measuring the number of aptamers transferred through the layer measures the amount of analyte present in the sample, or detecting the aptamers transferred through the layer detects the analyte present in the sample.

IV. (a) Contacting the sample with a binding member, wherein the binding member specifically binds the analyte, and the binding member is labeled with a severable tag; (b) Contacting the sample with an immobilized analyte, wherein the immobilized analyte is immobilized on a solid support; (c) Removing binding members not bound to the immobilized analyte; (d) Cutting a tag attached to a binding member bound to the immobilized analyte; (e) Transferring the cut tag through or across one or more nanopores in the layer; and (f) Evaluating the tags transferred through the layer, wherein measuring the number of tags transferred through the layer measures the amount of analyte present in the sample, or detecting the tags transferred through the layer detects the presence of analyte in the sample; and

V. (a) Contacting the sample with a binding member, wherein the binding member specifically binds the analyte and the binding member comprises an aptamer; (b) Contacting the sample with an immobilized analyte, wherein the immobilized analyte is immobilized on a solid support; (c) Removing binding members not bound to the immobilized analyte; (d) Dissociating the aptamer bound to the binding member bound to the immobilized analyte and transferring the dissociated aptamer through or across one or more nanopores in the layer; and (e) Evaluating the aptamers transferred through the layer, wherein measuring the number of aptamers transferred through the layer measures the amount of analyte present in the sample, or detecting the aptamers transferred through the layer detects the analyte present in the sample.

Clause 218. The method of any of Clauses 193-217, wherein the ratio of the size of the hole to the size of the label is or is less than 1.0.

Claims (11)

1. An integrated digital microfluidic nanopore device, comprising: A first substrate, which contains an electrode array; A second substrate spaced apart from the first substrate; An opening in the first or second substrate, the opening being in fluid communication with a nanoporous layer containing nanopores; and A pair of electrodes, the electrodes being configured to apply an electric field through the nanopore, The electrode array is configured to transport at least one microdroplet of fluid to the opening. 2. The apparatus of claim 1, wherein the nanopore is a solid nanopore or a biological nanopore. 3. The apparatus of claim 1, wherein at least one of the first and second substrates is substantially transparent. 4. The apparatus of claim 1, wherein the electrodes in the array are arranged in a grid pattern. 5. The apparatus of claim 1, wherein the electrodes in the array are intersected. 6. The apparatus of claim 1, wherein the opening is a capillary channel. 7. The device of claim 6, wherein the opening is wider on a first side of the first or second substrate than on a second side of the first or second substrate. 8. The apparatus of claim 1, wherein a first electrode of a pair of electrodes forms an anode and a second electrode of a pair of electrodes forms a cathode to drive current through the nanopore. 9. The apparatus of claim 8, wherein the nanoporous layer is attached to at least one of the first substrate and the second substrate, and wherein the first electrode and the second electrode are side-mounted to the nanoporous layer. 10. The apparatus of claim 1, further comprising at least one reagent disposed on the microfluidic device and configured to be carried by the microdroplets. 11. The apparatus of claim 10, wherein the reagent is a severable tag comprising anionic polymers, cationic polymers, dendritic polymers, or nanoparticles.