HK1246395B - Nanopore detection of small molecules through competition assays - Google Patents

Description

Nanopore Detection of Small Molecules via Competition Assays

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 62/131,969, filed March 11, 2016, the disclosure of which is incorporated herein by reference in its entirety.

Background Art

Small molecules play an important role in many biological functions. Because small molecules can, for example, change the function of a protein by binding to a protein and inhibiting or activating its normal function, they can disrupt/destroy the system in which the protein works. In turn, this type of destruction can trigger a disease or accelerate the progression of a disease. On the other hand, by controlling the dosage of small molecules, it is possible to reveal details about the effects of proteins in these systems (Stockwell, Brent R. "Exploring biology with small organic molecules." Nature 432.7019 (2004): 846-854). Detecting the presence and concentration of small molecules can also be used to assess metabolic processes such as drug metabolism, which may facilitate and accelerate clinical trials and drug efficacy studies. In addition to human and animal diagnosis, small molecules can also be used to assess the state of the environment, including for monitoring agricultural plant stress and water quality. Therefore, the ability to detect and accurately quantify small molecules is valuable in many biotechnology fields.

There are a variety of available methods for measuring small molecules; some are economical but lack precision and sensitivity, while other methods are very sensitive but expensive and complex. More economical high-throughput methods generally involve binding a dye to a target molecule of interest and measuring the overall fluorescence or absorbance of the sample. This technique can be hampered by the non-specificity of the dye or interfering molecules, which produces high false positives and/or high false negatives. More complex analytical methods, including high performance liquid chromatography and mass spectrometry, can accurately measure target small molecules, but they are complex and expensive and require specialized laboratory space and skilled personnel. Therefore, what is needed is a method for detecting small molecules that is economical (similar to dye binding assays), provides high specificity, sensitivity and precision, and can be performed on portable devices. Preferred analytical tests for detecting and/or quantifying small molecules are not limited to specialized laboratories and do not require specialized equipment or skilled personnel. In addition, preferred analytical devices are low-cost and highly reproducible. Therefore, there is a need for methods for providing accurate and low-cost analytical results on low-cost devices that tolerate a wide range of nanopore geometries and/or larger nanopores.

Summary of the Invention

Disclosed herein are competition assays for detecting small molecules using nanopores.

Target molecules of sufficient size (>20 kDa) cause changes in current impedance, translocation time, or other measurable parameters when passing through the solid-state nanopore. In cases where the target molecule is not large enough and therefore does not cause a significant change, additional molecules/corrosive agents can be used to aid detection. Such detection agents will bind small molecules or "capture ligand-molecule complexes" (e.g., peptide detection is aided by monoclonal antibodies (mAbs) that recognize peptide/aptamer complexes).

However, there may still be small molecules (e.g., drug compounds) for which this strategy is less than ideal. For example, due to the reduction in available surface area for binding to additional detection molecules, it may be difficult to produce detection reagents for the target small molecule during its capture. Therefore, we have designed a new strategy that enables the detection of small molecule compounds and has the ability to measure binding affinity and kinetics (K _on , K _off , K _d , K _m , etc.). This method is also suitable for studying complex aggregates (e.g., HIV Env protein monomers, dimers, or trimers).

In some embodiments, provided herein is a method for detecting the presence or absence of a target molecule suspected of being present in a sample, the method comprising: providing a device comprising a layer, wherein the layer separates an interior space of the device into a first volume and a second volume, wherein the layer comprises a nanopore connecting the first volume and the second volume, and wherein the device comprises a sensor configured to identify an object passing through the nanopore; providing a surrogate molecule, a fusion molecule, and a polymer backbone, the fusion molecule comprising a polymer backbone binding domain adapted to bind to the polymer backbone to form a backbone/fusion molecule complex, and the fusion molecule comprising a target molecule binding domain adapted to bind to the surrogate molecule or the target molecule; performing a competition assay by combining the surrogate molecule and the fusion molecule with the sample, wherein, if the target molecule is present in the sample, the target molecule competes with the surrogate molecule for binding to the target molecule binding domain; loading the sample into the first volume; applying a voltage across the nanopore, wherein the first volume comprises the polymer backbone, the fusion molecule, the surrogate molecule, and the sample suspected of containing the target molecule, wherein the polymer backbone hybridizes to the fusion molecule, and wherein the fusion molecule hybridizes to the surrogate molecule or the target molecule.

In some embodiments, the applied voltage induces the scaffold/fusion molecule complex bound to the target molecule or the surrogate molecule to translocate from the first volume through the nanopore to produce a change in the electrical signal detected by the sensor. In some embodiments, the method further comprises recording the detected change in the electrical signal as a function of time. In further embodiments, the method further comprises analyzing the detected and recorded change in the electrical signal as a function of time to determine the presence or absence of the target small molecule in the sample.

In some embodiments, a competition assay is performed after loading the sample into the first volume. In some embodiments, a competition assay is performed before loading the sample into the first volume.

In some embodiments, the surrogate molecule comprises maleimide polyethylene glycol. In some embodiments, the surrogate molecule comprises a chemically reactive group selected from the group consisting of ketones, aldehydes, isocyanates, amines, carboxylic acids, halides, esters, maleimides, thiols, dicyclocarbimides, pyridyls, pyridyldisulfides, and acetyl groups. In some embodiments, the surrogate molecule comprises a strong or weak nucleophile or electrophile. In some embodiments, the surrogate molecule comprises a peptide, a dendrimer, a nucleic acid, a nano- or micron-bead or particle, a quantum dot, a protein, a polynucleotide, a liposome, an antibody, or an antibody fragment. In some embodiments, the surrogate molecule comprises a payload binding site adapted to bind a payload molecule. In further embodiments, the payload molecule is selected from the group consisting of a dendrimer, a double-stranded DNA, a single-stranded DNA, a DNA aptamer, a fluorophore, a protein, an antibody, a polypeptide, a nanobead, a nanorod, a nanotube, a nanoparticle, a fullerene, a PEG molecule, a liposome, or a cholesterol-DNA hybrid. In some embodiments, the payload molecule comprises a charge. In some embodiments, the charged payload molecule is selected from the group consisting of a peptide, an amino acid, a charged nanoparticle, a synthetic molecule, a nucleotide, a polynucleotide, a metal, or an ion. In some embodiments, the surrogate molecule is bound to the payload molecule via an interaction selected from the group consisting of a covalent bond, a hydrogen bond, an ionic bond, a van der Waals force, a hydrophobic interaction, a cation-pi interaction, a planar stacking interaction, a metal bond, and a biotin-avidin interaction.

In some embodiments, the target molecule comprises a small molecule having a size of less than 30,000 Da, 20,000 Da, 10,000 Da, 5,000 Da, 2,000 Da, 1,000 Da, 500 Da, 200 Da, 100 Da, 50 Da, 20 Da, or 10 Da. In some embodiments, the target molecule comprises a peptide, insulin, oxytocin, an amino acid, a protein or protein domain, a nucleotide, an oligomer, DNA, RNA, PNA, LNA, BNA, a hormone, a lipid, cholesterol, a metabolite, a carbohydrate, a glycan, a peptidoglycan, a polyglycan, a phospholipid, a steroid, a chemically synthesized agonist and antagonist, a synthetic derivative of a polynucleic acid, a polycyclic aromatic hydrocarbon, a carbon decomposition byproduct, a dioxin, a cyclohexamide, a vitamin, adenosine triphosphate and ATP analogs, a neurotransmitter, dopamine, L-dopa, serotonin, a metal, an electrolyte, an organometallic, an anesthetic and an anesthetic derivative, hyaluronic acid, or retinol.

In some embodiments, the fusion molecule comprises a peptide nucleic acid. In some embodiments, the fusion molecule comprises a cysteine-tagged bis peptide nucleic acid. In some embodiments, the fusion molecule comprises a bridging nucleic acid, a locked nucleic acid, biotin, streptavidin, a streptavidin derivative, a zinc finger protein, a ZFP binding domain, a CRISPR domain, a TALEN, a DNA, a PNA, or an RNA oligomer.

In some embodiments, the polymer backbone comprises a negatively or positively charged polymer adapted to translocate from the first volume to the second volume through the nanopore upon application of an electrical potential across the nanopore. In some embodiments, the polymer backbone comprises a molecule selected from the group consisting of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), a DNA/RNA hybrid, and a polypeptide.

In some embodiments, the sensor is configured to identify an object that passes through only a single nanopore. In some embodiments, the sensor is an electrical sensor. In some embodiments, the sensor detects a current passing through the nanopore when a voltage is applied across the nanopore.

In some embodiments, analysis of changes in the electrical signal as a function of time comprises separating events resulting from translocation of the scaffold/fusion complex bound to the surrogate molecule through the nanopore from events resulting from translocation of the scaffold/fusion complex bound to the target molecule through the nanopore. In some embodiments, the methods of target small molecule detection provided herein provide a confidence level of greater than 90%, 95%, 98%, or 99% for detection of the target small molecule. In some embodiments, the sample is not purified prior to loading into the first volume.

In some embodiments, the nanopore diameter is at least 5 nm, 10 nm, 20 nm, 30 nm, 40 nm, or 50 nm. In some embodiments, the device comprises at least two nanopores in series, and wherein the amplicon bound to the payload molecule is simultaneously in the at least two nanopores during translocation.

Also provided herein is a kit comprising a device comprising a nanopore, wherein the device comprises a layer that separates an interior space of the device into a first volume and a second volume, wherein the layer comprises a nanopore passing through the layer connecting the first volume and the second volume, and wherein the device comprises a sensor configured to identify an object passing through the nanopore; a surrogate molecule, a fusion molecule, and a polymer backbone, the fusion molecule comprising a polymer backbone binding domain adapted to bind to the polymer backbone to form a backbone/fusion molecule complex, and the fusion molecule comprising a target molecule binding domain adapted to bind to the surrogate molecule or the target molecule; and instructions for detecting the presence or absence of the target small molecule by observing the results of a competition assay in the device.

In some embodiments, the surrogate molecule of the test kit comprises maleimide polyethylene glycol. In some embodiments, the surrogate molecule of the test kit comprises a chemically reactive group selected from the group consisting of ketones, aldehydes, isocyanates, amines, carboxylic acids, halides, esters, maleimides, thiols, dicyclocarboimides, pyridyls, pyridine disulfides, and acetyl groups. In some embodiments, the surrogate molecule of the test kit comprises a weak or strong nucleophile or electrophile. In some embodiments, the surrogate molecule of the test kit comprises a peptide, a dendrimer, a nucleic acid, a nano or micron bead or particle, a quantum dot, a protein, a polynucleotide, a liposome, or an antibody. In some embodiments, the surrogate molecule of the test kit comprises a payload binding site adapted to bind to a payload molecule.

In some embodiments, the payload molecule of the kit is selected from the group consisting of a dendrimer, double-stranded DNA, single-stranded DNA, a DNA aptamer, a fluorophore, a protein, an antibody, a polypeptide, a nanobead, a nanorod, a nanotube, a nanoparticle, a fullerene, a PEG molecule, a liposome, or a cholesterol-DNA hybrid. In some embodiments, the payload molecule of the kit comprises an electric charge. In some embodiments, the charged payload molecule of the kit is selected from the group consisting of a peptide, an amino acid, a charged nanoparticle, a synthetic molecule, a nucleotide, a polynucleotide, a metal, or an ion. In some embodiments, the surrogate molecule of the kit binds to the payload molecule via an interaction selected from the group consisting of a covalent bond, a hydrogen bond, an ionic bond, a van der Waals force, a hydrophobic interaction, a cation-pi interaction, a planar stacking interaction, a metallic bond, and a biotin-avidin interaction.

In some embodiments, the target molecule comprises a small molecule having a size of less than 30,000 Da, 20,000 Da, 10,000 Da, 5,000 Da, 2,000 Da, 1,000 Da, 500 Da, 200 Da, 100 Da, 50 Da, 20 Da, or 10 Da. In some embodiments, the target molecule of the kit comprises a molecule selected from the group consisting of N-ethylmaleimide, peptides, insulin, oxytocin, amino acids, proteins or protein domains, nucleotides, oligomers, DNA, RNA, hormones, lipids, cholesterols, metabolites, carbohydrates, glycans, peptidoglycans, polysaccharides, phospholipids, steroids, chemically synthesized agonists and antagonists, synthetic derivatives (PNA, LNA, BNA), polycyclic aromatic hydrocarbons (PAH), carbon decomposition byproducts, dioxins, cyclohexamide, vitamins, adenosine triphosphate and ATP analogs, neurotransmitters, dopamine, L-dopa, serotonin, metals, electrolytes, organometallics, anesthetics and anesthetic derivatives, hyaluronic acid, or retinol.

In some embodiments, the fusion molecule of the kit comprises a peptide nucleic acid. In some embodiments, the fusion molecule of the kit comprises a cysteine-tagged bis peptide nucleic acid. In some embodiments, the fusion molecule of the kit comprises a bridging nucleic acid, a locked nucleic acid, biotin, streptavidin (or a streptavidin derivative), a zinc finger protein or a zfp binding domain, a CRISPR domain, a TALEN, a DNA or RNA oligomer fused to a domain that binds a target small molecule.

In some embodiments, the polymer backbone of the kit comprises a negatively or positively charged polymer adapted to translocate from the first volume to the second volume through the nanopore upon application of an electrical potential to the nanopore. In some embodiments, the polymer backbone of the kit comprises a molecule selected from the group consisting of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), a DNA/RNA hybrid, and a polypeptide.

In some embodiments, the sensor of the device in the kit is configured to identify an object that passes through only a single nanopore. In some embodiments, the sensor of the device in the kit is an electrical sensor. In some embodiments, the sensor of the device in the kit detects an electric current passing through the nanopore when a voltage is applied across the nanopore.

Also provided herein is a method for quantifying the amount of a target small molecule present in a sample, the method comprising: providing a device comprising a layer, wherein the layer divides an interior space of the device into a first volume and a second volume, wherein the layer comprises a nanopore connecting the first volume and the second volume, and wherein the device comprises a sensor configured to identify an object passing through the nanopore; providing a surrogate molecule, a fusion molecule, and a polymer scaffold, the fusion molecule comprising a polymer scaffold-binding domain adapted to bind to the polymer scaffold to form a scaffold/fusion molecule complex, and the fusion molecule comprising a target molecule-binding domain adapted to bind to the surrogate molecule or the target molecule; and performing competitive quantification of the surrogate molecule by combining the surrogate molecule and the fusion molecule with the sample. The method comprises the steps of: loading the sample into the first volume; applying a voltage across the nanopore; wherein the first volume comprises the polymer backbone, the fusion molecule, the surrogate molecule, and the sample suspected of comprising the target molecule, wherein the polymer backbone is hybridized to the fusion molecule, and wherein the fusion molecule is hybridized to the surrogate molecule or the target molecule; and comparing the capture rate of the backbone/fusion molecule bound to the target small molecule in the nanopore and the capture rate of the backbone/fusion molecule bound to the surrogate molecule in the nanopore to quantify the amount of the target small molecule in the test sample.

In some embodiments of the method for quantifying the amount of a target small molecule present in a sample, the voltage induces translocation of the scaffold/fusion molecule complex bound to the target molecule or the surrogate molecule from the first volume through the nanopore to produce a change in the electrical signal detected by the sensor. In some embodiments, the method for quantifying the amount of a target small molecule present in a sample further comprises recording the change in the electrical signal as a function of time. In some embodiments, the method for quantifying the amount of a target small molecule present in a sample further comprises analyzing the change in the electrical signal as a function of time to determine the presence or absence of the target small molecule in the sample.

In some embodiments of the method for quantifying the amount of a target small molecule present in a sample, the competition assay is performed after loading the sample into the first volume. In some embodiments of the method for quantifying the amount of a target small molecule present in a sample, the competition assay is performed before loading the sample into the first volume.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features, and advantages will be apparent from the following description of specific embodiments of the present invention as illustrated in the accompanying drawings in which like reference characters indicate the same parts in the different views. The drawings are not necessarily to scale, emphasis instead being placed on illustrating the principles of the various embodiments of the present invention. Data graphics are also provided as embodiments of the present disclosure, which are illustrated by way of example only and not limitation.

Figure 1 depicts a schematic diagram of a competition assay in which a small molecule of interest competes with a surrogate small molecule with an attached payload for binding to the small molecule binding domain of a scaffold/fusion complex.

Figure 2 depicts the above backbone/fusion-target and backbone/fusion-surrogate molecule types, captured one at a time into the nanopore.

Figure 3 depicts idealized nanopore current event characteristics for backbone//fusion-target and backbone/fusion-surrogate molecule types.

Figure 4 shows single-molecule detection using a nanopore device. (a) Representative current excursion events induced by a 3.2 kb dsDNA backbone passing through a 27 nm diameter nanopore at V = 100 mV (1 M LiCl). Events were quantified by conductance excursion depth (δG = δI/V) and duration. (b) Scatter plot of δG versus duration for 744 events recorded over 10 minutes.

Figure 5 shows the characterization by RP-HPLC of a single reactant used as a benchmark control for subsequent competition reactions.

Figure 6 shows the results of RP-HPLC characterization of the products of incubation of cysteine-labeled PNA molecules with varying concentrations of excess PEG-maleimide and/or NEM to confirm complex formation and evaluate competitive binding to PNA molecules.

Figure 7 shows a gel comparing the electrophoretic mobility of backbone/fusion-target (lane 4) and backbone/fusion-surrogate complexes (lane 9) tested using a nanopore, where NEM is the target and PEG is the surrogate, each competing for binding sites on PNA bound to the DNA backbone.

Figure 8 shows representative events recorded for backbone/fusion-target and backbone/fusion-surrogate complexes, where NEM is the target and PEG is the surrogate. Increasing the payload size of the surrogate produces deeper and longer lasting event signatures.

Figure 9 shows a scatter plot of the mean δG relative duration of all scaffold/fusion-target events and then all scaffold/fusion-replacement events recorded on the same pore.

Figure 10 shows the evolution of the percentage of events with a duration of less than 100 μs recorded over time in the population from Figure 7. The error bars indicate the uncertainty in the measured percentage.

Figure 11 shows a gel comparing the electrophoretic mobility of backbone/fusion-target and backbone/fusion-surrogate complexes with 1:1 (lane 3) and 4:1 (lane 9) surrogate:target competition ratios, both tested on a nanopore. As before, NEM was the target and PEG was the surrogate, each competing for binding to PNA bound to the DNA backbone.

Figure 12 shows a scatter plot of the relative duration of delta G for 100% backbone/fusion-target and 100% backbone/fusion-surrogate complexes and backbone/fusion following incubation at 4:1 and 1:1 surrogate:target (PEG:NEM) competition ratios.

Figure 13 shows the evolution of the percentage of events with a duration of less than 100 μs recorded over time in the population from Figure 10. Error bars quantify the uncertainty in the measured percentages. An increase in events faster than 100 μs is an indication of an increase in the fraction of the target small molecule in complex with the scaffold/fusion compared to the surrogate molecule.

FIG14 shows the results of RP-HPLC characterization of the products of incubation of cysteine-labeled PNA molecules with a fixed concentration of PEG-maleimide and varying concentrations of NEM to verify complex formation and evaluate competitive binding to PNA molecules.

Figure 15 shows a gel showing the electrophoretic mobility of backbone/fusion-target ((a), lane 4) and backbone/fusion-surrogate complexes ((b), lane 4) tested using a nanopore, where NEM is the target and PEG is the surrogate, each competing for binding sites on PNA bound to the DNA backbone.

Figure 16 shows a scatter plot of the relative duration of delta G for all scaffold/fusion-target events and then all scaffold/fusion-replacement events recorded on the same pore.

Figure 17 shows the evolution of the percentage of events with a duration less than 70 μs recorded over time in the population from Figure 13. Error bars quantify the uncertainty in the measured percentage.

Figure 18 shows a gel showing the electrophoretic mobility of backbone/fusion-target and backbone/fusion-surrogate complexes with surrogate:target competition ratios of 50:1 ((a), lane 4), 10:1 ((b), lane 4), and 2:1 ((b), lane 9), all three tested on a nanopore. As before, NEM is the target and PEG is the surrogate, each competing for binding sites on the PNA bound to the DNA backbone.

Figure 19 shows the evolution of the percentage of events with a duration less than 70 μs recorded over time in the population from Figure 18. Error bars quantify the uncertainty in the measured percentages. The fraction of events faster than 70 μs increases as the target:surrogate ratio increases.

DETAILED DESCRIPTION

Throughout this application, text relates to various embodiments of the apparatus, composition, system and method of the present invention. The various embodiments described are intended to provide multiple illustrative examples and should not be construed as descriptions of optional types. On the contrary, it should be noted that the descriptions of the various embodiments provided herein may overlap in scope. The embodiments discussed herein are merely illustrative and are not intended to limit the scope of the present invention.

Also throughout this disclosure, various publications, patents, and published patent specifications are cited by explicit citation. The disclosures of these publications, patents, and published patent specifications are hereby incorporated by reference into this disclosure in their entireties.

As used herein, the term "comprising" means that the systems, apparatuses, and methods include the recited components or steps, but do not exclude other components or steps. "Consisting essentially of" when used to define systems, apparatuses, and methods shall mean excluding other components or steps that have any material significance for the combination. "Consisting of shall mean excluding other components or steps. Embodiments defined by each of these transition terms are within the scope of the present invention.

All numerical expressions, including ranges, such as distances, sizes, temperatures, times, voltages, and concentrations, are approximate and vary in increments of 0.1 ((+) or (-)). It is understood that all numerical expressions are preceded by the term "about," although not always explicitly stated. It is also understood that, although not always explicitly stated, the components described herein are exemplary only and that equivalents of such components are known in the art.

As used herein, "a device comprising a nanopore separating an interior space" shall refer to a device having a pore comprising an opening within a structure that separates the interior space into two volumes or chambers. The device may also have more than one nanopore, with a common chamber between each pair of pores.

As used herein, the term "fusion molecule" refers to a molecule comprising at least two domains, one of which binds to a scaffold and the other binds to or reacts with a small molecule.

As used herein, the term "target small molecule" refers to a small molecule of interest that may or may not be present in a sample. In molecular biology and pharmacology, a small molecule is a low molecular weight (<1000 Dalton) organic compound that helps regulate biological processes or is a cleavage product (metabolite) of a larger molecule, either artificial (i.e., synthetic drugs) or natural product (e.g., protein). In the context of the present patent application, our definition of a small molecule is relatively broad, as we define it as any molecule that is too small to reliably generate a distinctive electrical signal when shuttled through a solid-state nanopore. In some embodiments, a small molecule can be, but is not limited to, any target molecule that is less than 10,000 Da in size. However, small molecules that are greater than 10,000 Da in size may also be difficult to detect in nanopores, such that the competition assays described herein can also be used to detect these small molecules.

As used herein, the term "surrogate molecule" or "surrogate small molecule" refers to a molecule that may be the same as or different from the target small molecule. The surrogate molecule competes with the target small molecule for the same binding site or reactive site on the fusion molecule. In some embodiments, the surrogate molecule is large enough to be detected in the nanopore. In some embodiments, the surrogate molecule is a small molecule that can bind to or be bound to a payload molecule. In this embodiment, the payload molecule facilitates detection of the surrogate small molecule in the nanopore. In some embodiments, the surrogate small molecule is adapted to or configured to bind to a payload molecule. In cases where the specification refers to a surrogate small molecule attached to a payload molecule, a small molecule without a payload may be used instead if it is capable of being detected in the nanopore when bound to the backbone/fusion molecule complex.

As used herein, the term "payload" or "payload molecule" refers to a molecule or compound that binds to a surrogate small molecule to enhance the selectivity and/or sensitivity of detection of a competing small molecule in a nanopore. In some embodiments, the payload molecule is bound to the surrogate small molecule at a 1:1 ratio.

As used herein, the term "skeleton" or "polymer backbone" refers to a negatively charged or positively charged polymer that is translocated through a nanopore when a voltage is applied. In some embodiments, the polymer backbone can be combined with or bound to a fusion molecule. In some aspects, the polymer backbone includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), DNA/RNA hybrid or polypeptide. The backbone can also be a chemically synthesized polymer, rather than a naturally occurring or biological molecule. In a preferred embodiment, the polymer backbone is dsDNA to allow for more predictable signals to be given when translocated through a nanopore, and to reduce the secondary structure present in ssDNA or RNA. In some embodiments, the polymer backbone includes a fusion molecule binding site that can be located at the backbone end or at both ends of the backbone. The backbone and fusion molecule can be connected by covalent bonds, non-covalent bonds, hydrogen bonds, ionic bonds, van der Waals forces, hydrophobic interactions, cation-pi interactions, planar stacking interactions or metallic bonds.

As used herein, the term "fusion" or "fusion molecule" refers to a macromolecule or compound having two domains: 1) a backbone-binding domain and 2) a small molecule-binding domain or reactive site. An example fusion molecule is a PNA (for binding to a dsDNA backbone) with an attached aptamer that is specific for the small molecule of interest. A second fusion example is a PNA binding domain fused to a chemical group for selective reactivity with a specific chemical group in the small molecule of interest. The small molecule binding domain or reactive chemical group has the ability to bind to the target small molecule of interest with high specificity and a known or measurable affinity, and also to bind to a surrogate small molecule with an attached payload.

As used herein, the term "binding" or "linking" refers to the formation of a chemical bond, such as a covalent bond, an ionic bond, or a metallic bond. Binding can include the stable association between two molecules by van der Waals forces, hydrophobic interactions, cation-pi interactions, and/or planar stacking interactions. Binding can include the formation of a bond between two (or more) chemically reactive groups such as ketones and nucleophiles using conventional chemical or biochemical coupling techniques (with or without catalysts). In some embodiments, the stable association between two molecules can be destroyed by competitive binding of another molecule that forms a stable interaction with the binding site.

As used herein, the term "binding site" or "binding domain" refers to an area on a molecule or compound that specifically binds to another molecule. In some embodiments, disclosed herein are fusion molecules comprising a binding domain specific for a polymer backbone and further comprising a binding domain specific for a target small molecule and a surrogate molecule. The stable association (e.g., docking) of the fusion molecule and other molecules at the binding domain can be formed by non-covalent intermolecular bonding such as ionic bonds, hydrogen bonds, and van der Waals forces between the binding domain and the target molecule or ligand, or by covalent bonding (i.e., by chemical or biochemical reactivity).

As used herein, the term "competition" or "competitive assay" refers to the competition between a surrogate molecule (e.g., a surrogate small molecule with an attached payload) and a target small molecule, the two molecular types attempting to bind to the target molecule binding domain on each scaffold/fusion molecule. Competition for binding to the target molecule binding domain can be competitive, non-competitive, or uncompetitive.

As used herein, the term "scaffold/fusion-target" or "scaffold/fusion-target complex" refers to a target small molecule that binds to a target molecule binding domain on a scaffold/fusion molecule complex.

As used herein, the term "scaffold/fusion-surrogate" or "scaffold/fusion-surrogate complex" refers to a surrogate molecule (e.g., a payload-bound surrogate small molecule) that binds to a target molecule binding domain on a scaffold/fusion molecule.

As used herein, the term "nanopore" refers to an opening (hole or channel) of sufficient size to allow a specifically sized polymer to pass through. Using an amplifier, a voltage is applied to drive the charged polymer through the nanopore, and the current through the pore detects whether the molecule has passed through the pore.

As used herein, the term "sensor" refers to a device that collects signals from a nanopore device. In many embodiments, the sensor comprises a pair of electrodes positioned on either side of a pore to measure ionic current across the pore as a molecule or other entity, particularly a polymer backbone, moves through the pore. In addition to electrodes, additional sensors, such as optical sensors, can detect optical signals in nanopore devices. Other sensors can be used to detect properties such as current blockade, electron tunneling current, charge-induced field effects, nanopore transit time, optical signals, light scattering, and plasmon resonance.

As used herein, the term "current measurement" refers to a series of measurements of the current flowing through the nanopore over time under an applied voltage. Current is expressed as a measure used to quantify events, and current normalized to voltage (conductance) is also used to quantify events.

As used herein, the term "open channel" refers to a baseline level of current through a nanopore channel within the noise range, where the current does not deviate from a threshold of values defined by the analysis software.

As used herein, the term "event" refers to a set of current impedance measurements that begins when the current measurement deviates from an open channel value by a defined threshold and ends when the current returns to within the threshold of the open channel value.

As used herein, the term "current impedance signature" refers to a collection of current measurements and/or patterns determined in a detected event. Multiple signatures may also be present in an event to enhance discrimination between molecule types.

As used herein, the term "target detection event" refers to a distinctive event or current impedance signature that signals the passage of a scaffold/fusion-target complex through a nanopore. As used herein, the term "surrogate detection event" refers to a distinctive event or current impedance signature that signals the passage of a scaffold/fusion-surrogate complex through a nanopore.

Surrogate target detection events can be due to increased size of the payload. It is the increased size of the payload molecule bound to the surrogate small molecule that enables differentiation between the scaffold/fusion-target complex and the scaffold/fusion-surrogate complex, where the differentiation is based on one or more differences in the current impedance characteristics.

Surrogate target detection events can be due to payload charge. It is the increased charge of the payload molecule bound to the surrogate small molecule that enables differentiation between the scaffold/fusion-target complex and the scaffold/fusion-surrogate complex, where the differentiation is based on one or more differences in the current impedance characteristics.

In addition to size and length, surrogate target detection events can be due to payloads having other characteristics that enable differentiation between a backbone/fusion-target complex and a backbone/fusion-surrogate complex, where differentiation is based on one or more differences in current impedance characteristics. Example characteristics include payload hydrophilicity, hydrophobicity, length, amino acid composition, base composition, or other chemical characteristics.

As used herein, the term "capture rate" refers to the number of events detected over time in a nanopore device. In some embodiments, the capture rate can specifically refer to the rate of capture and/or translocation of events associated with a particular complex. As described herein, the capture rate can be used to infer concentration compared to a control with a similar mass/charge ratio under similar nanopore conditions.

Competitive Analysis

Disclosed herein are methods and compositions for detecting target small molecules by using a competition analysis between a target and a surrogate and using a nanopore device for distinguishing target-bound and surrogate-bound macromolecules adapted for detection in a nanopore (e.g., by current impedance characteristics). In some embodiments, the methods, compositions, and devices disclosed herein are adapted to allow pure electronic counting of molecules bound to backbone DNA through a nanopore. The examples provided in the example "backbone/fusion of target binding" provide clear and stable signals that can be distinguished from "backbone/fusion of surrogate binding" each time a single backbone/fusion macromolecule passes through a nanopore. This allows for a fast and simple way to accurately and precisely detect and quantify target molecules from a mixed sample, thereby allowing the use of electronic detection (which does not require chemical or optical detection) to count 100s-1000s or larger target molecules individually within a few minutes and distinguish them from background non-target molecules. In addition, in view of the cheap hardware, low power requirements, small size, and tolerance to a wide range of nanopore geometries of the device, manufacturing and equipment costs are extremely low.

In some embodiments, provided herein are methods for conducting competitive binding assays to be analyzed in a nanopore device. In one embodiment, the competitive binding assay is performed using a saturation (equilibrium) assay, wherein the surrogate small molecule has a sufficient concentration to generate a saturation curve, from which the _Bmax value can be obtained. In some embodiments, a fixed amount of the scaffold-fusion is incubated with increasing concentrations of the surrogate small molecule and allowed to reach equilibrium. The scaffold is then passed through the pore and the amount of scaffold displaced by the small molecule is recorded. By comparing the amount of scaffold/fusion complex bound by the surrogate molecule against the concentration of the surrogate molecule, a logarithmic concentration-based response curve is generated, from which the maximum complex-binding ( _Bmax ) concentration of the surrogate small molecule can be determined. This _Bmax concentration can then be used in subsequent competition assays. The _KD for the binding interaction between the surrogate molecule and the fusion molecule can be calculated from a Scatchard plot, where the intercept on the X-axis is equal to _Bmax and the Y-axis is the surrogate-bound scaffold divided by the unbound scaffold. For this curve, the slope is equal to -1/ _KD . This _KD value (the concentration of the surrogate small molecule that produces 50% bound scaffold) is used when generating a standard curve as described below. Using the calculated _Bmax , a competition assay can be performed by diluting a test sample containing a small molecule of interest to a variety of concentrations, incubating each concentration with the backbone-fusion complex, and allowing the mixture to reach equilibrium. A surrogate molecule at a _Bmax concentration is then added to the mixture and incubated for a sufficient time for the reaction to reach equilibrium again (e.g., typically 5-10 minutes). The reaction is placed in a nanopore device, and a voltage is applied across the nanopore to induce translocation of the polymer backbone-fusion complex through the nanopore and observe the respective electronic signatures to determine whether the complex binds to the surrogate molecule or to the target molecule.

In some embodiments, the capture rate of the complex of alternative binding is recorded. This capture rate can then be mapped to a standard curve generated by measuring the capture rate (per second) (Y-axis) of the binding event relative to the concentration (X-axis) of the alternatively bound skeleton to estimate the concentration of the alternatively bound skeleton in the test sample. Since the number of the skeleton molecules of each incubation is known and each skeleton can only be combined with a small molecule, the number of small molecules can be determined. For example, if the rate of the skeleton of the alternative binding in the unknown sample matches the 50% binding rate on the standard curve, and 1 million skeletons/ml are used in the reaction, we infer that the small molecule concentration is 0.5 million/ml.

Assigning statistical significance to tests

In some embodiments, a collection of sensor measurements recorded over time is summarized and mathematical tools are applied to assign a numerical confidence value to the detection of a target small molecule suspected of being present in a sample (as described in detail in the previous section).

A quantitative method has recently been developed to distinguish molecular types from background (i.e., other molecular types) based on differences in the characteristics of the nanopore event population (Morin, T.J. et al., "Nanopore-based target sequence detection," submitted to PLoS On on December 31, 2015). This differentiation approach means that a specific molecular type can be detected in the presence of different types of other molecules and can be assigned statistical significance (e.g., detection of agent X with 99% confidence). We first summarize this method here to apply it to the examples provided below.

In general, there are two types of molecules in the chamber above the pore: Type 1 is the backbone/fusion-surrogate molecule, and Type 2 is the backbone/fusion-target molecule. Based on experimental data, we define event signature criteria based on the presence of a significant fraction of Type 2 events and a relatively small fraction of Type 1 events. The signature criteria can depend on δG, duration, the number and characteristics of levels within each event, and/or any other value or combination of values calculated from the event signal.

Note that event feature standards can be selected manually or by table look-up or in an automatic manner.For example, previous experiments can set up the performance of the positive and negative controls for a wide range of apertures and other conditions expected to exist in a given test, and when encountering suitable conditions (that is, for given alternative small molecules/payload types) for a given test, the selected standard determined from these controls can be used (in the form of table look-up).For example, based on the control run just before the sample, automatic standards can also be determined in real time.For the real-time detection of the target small molecule from the sample, as disclosed in the application, it is suitable and preferred approach to select automatic standards. Specifically, before test sample, the control producing type 1 event colony can be used for making the calculation automation of " type 2 event boundaries ", which sets up the standard for indicating " type 2-positive " events relative to " type 1-positive " events. Boundaries can be calculated (for example, the point is the event in the average offset relative duration graph) by any method around the point fitting curve in the 2D drawing. The curve fitting method can comprise least square method, linear or quadratic programming or any form of numerical optimization, and parameterizes the boundary by the coefficient of piecewise polynomial or spline.Calculating convex hull can provide boundary.More dimensional boundary fitting approach is also possible, for example, using 3 kinds of characteristics (3D boundary) that characterize event.The boundary obtained can be polygonal or smooth.The related art that is used to surround the subset of event (that is, as the mechanism of eliminating outliers when surrounding the point that is determined for automatic standard) with boundary (it comprises the event of certain percentage) is to calculate the z-quantile boundary of the boundary of the minimum area that is defined as the z score of total probability.For example, 95% quantile boundary is the boundary of the minimum area that comprises 95% (95% data) of total probability.Although probability density is unknown, this data and standard numerical technique can be used to estimate.

Once the criteria are selected (manually, by table lookup, or automatically), if the characteristic criteria are met for an event, the event is "labeled" as type 2. We define p as the probability that a capture event is type 2. In a control experiment without type 2 molecules, we know that p=0, and in an experiment testing type 2 molecules, we want to know whether p>0. We define the false positive probability q1=Pr(labeled|type 1 events). In a control experiment or experimental group with only backbone/fusion-surrogate molecules, q1 is determined with good accuracy from a reasonable number of capture events. In a detection experiment to determine whether a type 2 molecule is present in the bulk solution, the probability that a capture event is labeled is a function of p and can be approximately calculated as:

Q(p) = (number of events marked)/N

In this formula, N is the total number of events. A 99% confidence interval Q(p) ± Q _sd (p) can be calculated using Qsd(p) = 2.57*sqrt{Q(p)*(1-Q(p))/N}, where sqrt{} is the square root function. Over the course of the experiment, as the number of events N increases, the value of Q(p) converges and the uncertainty bound decreases. Plots of Q(p) ± Q _sd (p) as a function of recording time show how it evolves for each reagent type (Figures 10, 13, 17, and 19). In the control experiment without type 2 molecules, Q(0) = q1 was observed.

In the detection experiment, a Type 2 molecule is present with 99% confidence when the following criteria are true:

Q(p)-Q _sd (p)>q1 (1)

If the above criterion is true, we conclude that p > 0; if it is not true, we cannot say that p > 0. This framework is used in the examples provided below.

Estimation of concentration from measured capture rates

In some embodiments, a collection of sensor measurements recorded over time is aggregated and mathematical tools are applied to estimate the concentration of a target small molecule suspected to be present in a sample.

In some embodiments, the process can be repeated at different concentrations of one or more of the backbone, fusion, surrogate small molecule, and/or target small molecule suspected to be present in the sample (incubating the sample with the backbone/fusion reagent and performing the nanopore experiment). The data sets can then be combined to gather more information. In one embodiment, the total concentration of the target small molecule is estimated by applying mathematical tools to the aggregated data sets.

According to the literature (Wang, Hongyun et al., "Measuring and Modeling the Kinetics ofIndividual DNA-DNA Polymerase Complexes on a Nanopore." ACS Nano 7, no.5 (May28, 2013): 3876-86.doi:10.1021/nn401180j; Benner, Seico et al., "Sequence-Specific Detection of Individual DNA Polymerase Complexes in Real Time Using aNanopore."Nature Nanotechnology 2,no.11(October 28, 2007): 718-24 (doi: 10.1038/nnano.2007.344), a method that can apply biophysical models to nanopore data to quantify the binding and equilibrium dynamics between a target small molecule and its substrate, as well as the competition between the target and a surrogate small molecule. The study cited above by Wang et al. examined two mutually exclusive and competing binding states of a protein to its DNA substrate. The same modeling framework can be applied, but with two different molecules to generate two different binding configurations that can occur with each substrate molecule.

In our analysis, nanopores sample and measure single molecules from the bulk phase. In the presence of a target molecule, each backbone/fusion is either target-bound or alternatively bound, and the fraction of backbone/fusion-target complexes is proportional to the concentration of the target. At high target concentrations relative to alternative concentrations, target binding proceeds rapidly, and all backbone/fusion complexes are target-bound. At lower concentrations relative to alternative concentrations, alternative binding proceeds relatively rapidly, and all backbone/fusion complexes are alternatively bound. At intermediate concentrations, a non-zero percentage of backbone/fusion events is designated as target-bound (type 2), and this percentage remains constant over time when the reaction rapidly reaches equilibrium in the bulk phase chamber adjacent to the nanopore.

To estimate the total target small molecule concentration, the experiment can be repeated with a nanopore and a constant backbone/fusion concentration using two or more different surrogate molecule concentrations. Different surrogate molecule concentrations, from low (1pM) to high (100nM), can be titrated while maintaining the target molecule concentration using a portion of a common sample. By measuring the percentage and capture rate of the indicated target positive events, the modeling framework in Wang, Hongyun et al., ACS Nano 7, no.5 (May 28, 2013) allows estimation of the total target molecule concentration competing with surrogate molecules of known concentrations. The method can use a predetermined Kd value for the binding affinity between the target molecule binding domain on the target and fusion molecule or allow estimation of this Kd value. To estimate the total target small molecule concentration, a multi-nanopore array can also be implemented, wherein each nanopore measures different backbone/fusion-surrogate concentrations relative to the backbone/fusion-target to be estimated.

Composition

In some embodiments, provided herein are surrogate small molecules that bind to a payload molecule. In some embodiments, provided herein are surrogate small molecules that comprise a binding site for a payload molecule.

In some embodiments, the payload molecule can be a dendrimer, double-stranded DNA, single-stranded DNA, DNA aptamer, fluorophore, protein, polypeptide, nanorod, nanotube, fullerene, PEG molecule, liposome or cholesterol-DNA hybrid or chemically synthesized compound. In a preferred embodiment, the alternative small molecule and payload are directly or indirectly connected by covalent bonds, hydrogen bonds, ionic bonds, van der Waals forces, hydrophobic interactions, cation-pi interactions, planar stacking interactions or metal bonds, biotin-(streptavidin/neutravidin/monomeric avidin) avidin interactions. The payload increases the distinguishing characteristics of the alternative small molecule and is beneficial to detection, wherein the payload combined with the skeleton/fusion-small molecule has a current characteristic significantly different from the skeleton/fusion-small molecule without payload when passing through the nanopore. In some embodiments, the payload molecule can be combined with another molecule to affect the volume of the molecule, thereby further improving the sensitivity of distinguishing between the skeleton/fusion-small molecule without or with payload.

In some aspects, the surrogate small molecule comprises a chemical modification that causes or facilitates recognition and binding of the payload molecule. In some embodiments, the surrogate small molecule has sufficient characteristics, e.g., size, length, charge, etc., to make it suitable for detection in a nanopore by generating a recognizable current impedance characteristic when bound to the scaffold/fusion macromolecule.

In some embodiments, the small molecule of interest comprises a peptide, insulin, oxytocin, an amino acid, a protein or protein domain, a nucleotide, an oligomer, DNA, RNA, a hormone, a lipid, a cholesterol, a metabolite, a carbohydrate, a glycan, a peptidoglycan, a polysaccharide, a carbohydrate, an oligosaccharide, a polysaccharide, a phospholipid, a steroid, a chemically synthesized agonist and antagonist, a synthetic derivative (PNA, LNA, BNA), a polycyclic aromatic hydrocarbon (PAH), a carbon decomposition byproduct, a dioxin, a cyclohexamide, a vitamin, adenosine triphosphate and ATP analogs, a neurotransmitter, dopamine, L-dopa, serotonin, a metal, an electrolyte, an organometallic, an anesthetic and an anesthetic derivative, hyaluronic acid, a retinol. This list is not meant to be exclusive, as in some embodiments, the present invention provides a novel mechanism for detecting any small molecule of interest that can compete with a surrogate molecule for binding to a binding site on a fusion molecule.

In some embodiments, the polymer backbone includes a negatively or positively charged polymer that is translocated through the nanopore when a voltage is applied. In some embodiments, the polymer backbone includes a cleavable domain or a cleavable linker. In some embodiments, the polymer backbone is capable of binding to a fusion molecule comprising a cleavable linker or is bound to a fusion molecule comprising a cleavable linker and translocated through the pore when a voltage is applied. In some aspects, the polymer backbone includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), DNA/RNA hybrid or polypeptide. The backbone can also be a chemically synthesized polymer and is not a naturally occurring or biological molecule. In a preferred embodiment, the polymer backbone is dsDNA to allow for a more predictable signal when translocated through the nanopore and to reduce the secondary structure present in ssDNA or RNA. In some embodiments, the polymer backbone includes a fusion molecule binding site that can be located at the backbone end or at both ends of the backbone. The backbone and the fusion molecule can be connected by covalent bonds, hydrogen bonds, ionic bonds, van der Waals forces, hydrophobic interactions, cation-pi interactions, planar stacking interactions or metallic bonds. Alternatively, direct covalent tethering of the cleavable linker component to the backbone can connect the backbone and the fusion molecule. Alternatively, the fusion linker component can attach the cleavable linker to the backbone via direct covalent tethering. In preferred embodiments, the fusion molecule comprises a backbone-binding domain, which can be DNA, RNA, PNA, a polypeptide, a cholesterol/DNA hybrid, or a DNA/RNA hybrid.

In some embodiments, the molecules disclosed herein can be modified or have the ability to bind to a specified entity through a specific binding domain. Molecules, particularly proteins, that can specifically recognize binding motifs are known in the art. For example, protein domains such as helix-turn-helix, zinc fingers, leucine zippers, winged helices, winged helix-turn-helix, helix-loop-helix, and HMG boxes are known to bind to nucleotide sequences. Any of these molecules can be used as a payload molecule to bind to an amplicon or primer.

In some aspects, the binding domain can be a locked nucleic acid (LNA), a bridged nucleic acid (BNA), all types of protein nucleic acids (e.g., bisPNA, γ-PNA), a transcription activator-like effector nuclease (TALEN), a clustered regularly interspaced short palindromic repeats (CRISPR), or an aptamer (e.g., DNA, RNA, protein, or a combination thereof).

In some embodiments, the binding domain is one or more of a DNA binding protein (e.g., zinc finger protein), an antibody fragment (Fab), a chemically synthesized binder (e.g., PNA, LNA, TALENS, or CRISPR), or a chemical modification (i.e., reactive moiety) in a synthetic polymer backbone (e.g., thiolate, biotin, amine, carboxylate).

Nanopore devices

The provided nanoporous device includes at least one pore forming an opening in a structure that divides its interior space into two volumes, and a sensor configured to identify an object passing through the pore (e.g., by detecting a change in a parameter indicative of the object). Nanoporous devices used in the methods described herein are also disclosed in PCT Publication WO/2013/012881, which is incorporated by reference in its entirety.

sensor

As described above, in various aspects, the nanopore device further comprises one or more sensors for detecting target small molecules.

The sensor used in the device can be any sensor suitable for identifying the target polynucleotide amplicon that is combined with payload molecule or uncombined.For example, the sensor can be configured to identify the polymer backbone/fusion molecule combined with surrogate molecule or target micromolecule by measuring the electric current, voltage, pH value, optical signature or the dwell time relevant to polymer.In other aspects, the sensor can be configured to identify one or more single components of polymer backbone/fusion complex or one or more components (for example, surrogate molecule or target micromolecule) that are combined or connected with polymer backbone/fusion molecule complex.The sensor can be formed by any component that is configured to detect the change of measurable parameter, wherein this change indicates the entity that is combined with complex, the component of complex or preferably, the component that is combined or connected with complex.In one aspect, the sensor includes a pair of electrodes arranged in hole both sides to measure the cross-pore ion current when molecule or other entities move through hole.In some aspects, when the polymer backbone/fusion molecule segment through hole is combined with surrogate molecule or target molecule, the ion current across hole changes measurably. Such current changes can correspond to, for example, changes in the presence, absence, and/or size of a surrogate molecule (and bound entity) or small molecule of interest bound to the polymer backbone/fusion molecule complex in a predictable, measurable manner.

In a preferred embodiment, the sensor includes electrodes that apply a voltage and measure the current across the nanopore. Molecular translocation through the nanopore provides an electrical impedance (Z), which affects the current through the nanopore according to Ohm's law, V=IZ, where V is the applied voltage, I is the current through the nanopore, and Z is the impedance. Conversely, the conductance G=1/Z is monitored to detect and quantify nanopore events. When a molecule translocates through a nanopore in an electric field (e.g., under an applied voltage), the result is a current signature that can be associated with the molecule passing through the nanopore when the current signal is further analyzed.

When using residence time measurements from current signatures, the size of the component can be correlated to a specific component based on the length of time it takes to pass through the probing device.

In one embodiment, a sensor provided in a nanopore device measures an optical characteristic of a polymer, a component (or unit) of a polymer, or a component bound or attached to a polymer. An example of such a measurement includes identifying absorption bands characteristic of a particular unit by infrared (or ultraviolet) spectroscopy. In some embodiments, the sensor is an electrical sensor. In some embodiments, the sensor detects a fluorescent characteristic. A radiation source at the pore outlet can be used to detect this characteristic.

Example

The technology is further defined with reference to the following examples and experiments. It will be apparent to those skilled in the art that many modifications can be made without departing from the scope of the invention.

Example 1: Nanopore Detection of DNA

A solid-state nanopore is a nanoscale opening formed in a thin, solid membrane that separates two aqueous volumes. A voltage-clamp amplifier applies a voltage V across the membrane while measuring the ionic current flowing through the open pore. Unlike any other single-molecule sensor, the nanopore device can be packaged into a handheld form factor at a very low cost. When a single charged molecule, such as double-stranded DNA (dsDNA), is trapped by electrophoresis and driven through the pore, the measured current shift and the depth (δG = δI/V) and duration of the conductance shift are used to characterize the event (Figure 4(a)).

The value δG (also labeled ΔG) can be calculated as the average current excursion divided by the voltage. The value δG (also labeled ΔG) can also be calculated as the maximum current excursion divided by the voltage. The duration is calculated as the excursion width.

We placed 0.1 nM 3.2 kb dsDNA into a nanopore device with a 27 nm diameter nanopore. The solution in the nanopore device contained 1 M LiCl. We applied a 100 mV voltage across the nanopore to induce translocation of the dsDNA across the nanopore. This event was detected by a current sensor and analyzed as described below.

After recording many events during the experiment, the distribution of the events was analyzed to characterize the corresponding molecules. Figure 4(b) shows the event signature for 0.1 nM of 3.2 kb dsDNA passing through a 27 nm diameter nanopore at a voltage of V = 100 mV (1 M LiCl), resulting in 744 events recorded over 10 minutes. Two circled representative events are shown: the wider and shallower event corresponds to the DNA passing through unfolded; and the faster but deeper event corresponds to the DNA passing through folded. For dsDNA of ~1 kb and shorter, the DNA only passes through the pore in an unfolded state.

Example 2: Competition analysis with altered targets and surrogate molecules

To demonstrate our competition assay, we used a dsDNA backbone (i.e., polymer backbone) with a single bisPNA molecule (i.e., fusion molecule). The bisPNA was cysteine-tagged to react and bind with varying concentrations of small N-ethylmaleimide (NEM) (i.e., target small molecule), which competed with varying preselected amounts of 10 kDa maleimide polyethylene glycol (PEG-mal) (i.e., surrogate molecule). The target:surrogate ratios analyzed were 1:1 and 1:4.

Reagent preparation was as follows: a 25 μM solution of cysteine-labeled bis-peptide nucleic acid (bisPNA) was mixed with various concentrations of the small molecules N-ethylmaleimide (NEM, 125 g/mol) and/or 10 kDa maleimide polyethylene glycol (PEG-mal, 10,000 g/mol) at room temperature and allowed to react for 20 minutes. Coupling was performed in bisPNA binding buffer (0.01 M sodium phosphate, pH 7.4).

After a brief incubation, the reaction products were analyzed by reverse phase HPLC (Agilent 1100 series) to confirm the composition of the NEM-bisPNA and/or PEG-bisPNA conjugates. In RP-HPLC, the analyte is eluted from the non-polar stationary phase by increasing the concentration of the organic mobile phase over time and identified by its characteristic absorption spectrum. The peak absorbances of the corresponding reactants were 220 nm (10 kDa PEG-maleimide), 270 nm (PNA), and 303 nm (NEM). Because PNA gave the strongest signal of the reactants, it was used in all competition experiments to monitor the progression of the resulting reaction products of PNA-NEM and/or PNA-PEG.

The independent elution time and absorption spectrum of single reactant are first characterized by RP-HPLC to be used as the benchmark control (Fig. 5) of subsequent competitive reaction. PNA eluted at 6.12 minutes and showed strong absorbance at 270nm (Fig. 5 (a)). In addition, the peak eluted at 9.80 minutes is determined as the impurity in the PNA provided by the manufacturer, and does not participate in any following reaction. The PEG-maleimide molecule itself gives the small broad peak (Fig. 5 (b)) of 18.73min and independent NEM gives the very small peak (Fig. 5 (c)) of 5.71min. Due to the weak signal provided by competitive molecules PEG and NEM, the disappearance of the PNA absorption at 6.12 minutes and the appearance of the other new PNA peak with strong 270nm absorption are then used as the indication of successful PNA coupling.

When PNA was incubated with PEG-maleimide alone, PNA absorption was typically detected at an elution time of 6.12 min, indicating the disappearance of uncoupled PNA ( FIG. 6 , solid diamonds), and a new molecule with a strong absorption of 270 nm eluted at 17.86 min indicated the formation of a PNA-PEG conjugate ( FIG. 6 , solid squares). Similarly, when PNA was allowed to incubate with NEM alone, PNA reacted completely with the molecule and a new peak appeared at 9.5 minutes ( FIG. 6 , open circles). PEG and NEM were also allowed to react with PNA at an equimolar and 4:1 PEG-NEM ratio ( FIG. 6 , solid stars and solid crosses). As the NEM concentration increased relative to PEG, the intensity value of the peak at 17.86 min decreased between samples, indicating that NEM was able to successfully compete for the thiol-reactive groups of the cysteine-labeled PNA molecules.

NEM-PNA and/or PEG-PNA complexes were mixed with a 1074 bp double-stranded DNA (dsDNA) fragment in bisPNA binding buffer at 60°C for 2 hours to allow formation of the polymer backbone/fusion molecule complex (i.e., binding of the dsDNA to the bisPNA conjugate). The backbone-fusion molecule binding complex was confirmed by electrophoretic mobility shift analysis on a 5% polyacrylamide gel ( FIG. 7 ). Specifically, a 5% polyacrylamide gel was run to assess the binding of dsDNA by PNA molecules labeled (i.e., bound) with NEM (target) or 10 kDa PEG (surrogate). Bare 1074 bp dsDNA ( FIG. 7 , lane 1) was incubated with increasing amounts of NEM-bound PNA, ranging from a 10- to 100-fold molar excess of NEM-bound PNA relative to DNA ( FIG. 7 , lanes 2-5). A 50-fold molar excess of NEM-bound PNA was found to be sufficient to completely label the 1074 bp sequence ( FIG. 7 , lane 4) and was therefore used in subsequent nanopore analysis. PEG-labeled PNA similarly allowed binding of dsDNA at concentrations ranging from 10 to 50-fold molar excess ( FIG. 7 , lanes 7-9 ). The sample used for the nanopore analysis was one in which all the DNA present was bound to PEG-labeled PNA ( FIG. 7 , lane 9 , 50-fold molar excess).

Once validated, the polymer backbone/fusion complex bound to NEM or PEG-mal was diluted to the indicated concentrations in running buffer for nanopore analysis (1 M LiCl, 10 mM Tris, 1 mM EDTA, pH 8). All reagents in Example 1 were tested serially on the same 20-22 nm diameter pores in a 22 nm membrane. Only a buffer rinse was used between tests for each reagent type.

First, 0.5 nM DNA/PNA-NEM complex produced 346 events in 17 minutes. Most events were faster than 100 μs, with a median dwell time (IQR (interquartile range)) = 44 (20) μs, as shown in Figures 8 and 9. The capture rate was 0.33 sec ^-1 ( ^R2 = 0.9973). Subsequently, 1074 DNA+PNA+PEG were measured at 0.5 nM and produced 651 events in 20 minutes. Many events were longer than 100 μs, with a median dwell time (IQR) = 72 (275) μs, as shown in Figures 8 and 9. The capture rate was 0.68 sec ^-1 ( ^R2 = 0.9985).

By applying the framework established in the "Assigning Statistical Significance to Detection" section, we can assign statistical confidence to detecting the DNA/PNA-NEM complex as a type 2 molecule and DNA/PNA-PEG as a type 1 molecule. A suitable criterion is to label an event as type 2 if it is faster than 0.1 ms. DNA/PNA-PEG can be used as a negative control to calculate the false positive q1 = 0.585 (58.8%). The DNA/PNA-NEM (type 2) control yields Q(p) ± _Qsd (p) = 94.7977 ± 3.0752 with 99% confidence. From equation (1) of the mathematical framework, the result is Q(p) - _Qsd (p) = 0.917 > 0.585, which means that we can say that the DNA/PNA-NEM molecule is present with 99% confidence.

A plot of Q(p) ± _Qsd (p) as a function of recording time is shown for each reagent type (DNA/PNA-NEM and DNA/PNA-PEG) in Figure 10. It was observed that the DNA/PNA-NEM complex (backbone/fusion-target) was detected with 99% confidence within the first minute of recording.

We next varied PEG:NEM ratios of 80:20 and 50:50 to assess how competition for bisPNA binding sites shifted in nanopore measurements.

Binding of a solution of PNA molecules labeled with NEM (target) and 10 kDa PEG (surrogate) to dsDNA was examined on a 5% polyacrylamide gel (Figure 11). A solution of PNA, previously reacted with equimolar amounts of PEG and NEM (1:1), was mixed with a 1074 bp dsDNA fragment (i.e., the polymer backbone) in a 10- to 100-fold molar excess of PNA relative to DNA to bind the PNA molecules to the dsDNA (Figure 9, lanes 2-5). Nanopore analysis was performed on samples in which each DNA molecule was bound to a PNA-labeled molecule (Figure 11, lane 3). PNA was also mixed with a solution of PEG and NEM in which the PEG number exceeded the NEM at a 4:1 molar ratio. This solution of labeled PNA molecules was then allowed to invade DNA in a similar manner (lanes 7-9, 10- to 50-fold molar excess). Nanopore analysis was performed on samples in which each DNA molecule was bound to a PNA-labeled molecule (Figure 11, lane 9).

First, 0.2nM 1074DNA/PNA+PEG:NEM=80:20 generated 952 events in 19 minutes with a median dwell time (IQR) = 56 (44) μsec and a capture rate of 1.0 sec ^-1 ( ^R2 = 0.9984). The fraction of events faster than 0.1ms was Q(p) ± _Qsd (p) = 79.0966 +/- 3.3946 at 99% confidence. Subsequently, 0.2nM 1074DNA/PNA+PEG:NEM=50:50 generated 754 events in 15 minutes with a median dwell time (IQR) = 48 (20) μsec and a capture rate of 0.951 ^sec-1 ( ^R2 = 0.9978). As the relative amount of NEM relative to PEG increases, the fraction of events faster than 0.1 ms increases to Q(p) ± _Qsd (p) = 91.7772 +/- 2.577 with 99% confidence. Figure 12 shows overlay event plots for DNA/PNA-NEM and DNA/PNA-PEG controls, as well as DNA/PNA+PEG:NEM = 80:20 and DNA/PNA+PEG:NEM = 50:50. A DNA-only control is also shown. Figure 13 shows a plot of Q(p) ± _Qsd (p) as a function of recording time for each reagent type in Figure 12. The trend of increasing fractions with increasing relative amounts of NEM relative to PEG suggests that competition between NEM and PEG is detectable using nanopores. These measurements can be used to detect the presence of target small molecules, as shown here for NEM and with 99% confidence. These measurements can also be combined with biophysical models to estimate target small molecule concentrations.

Example 3: Competition analysis of target molecules with fixed substitutions and alterations

As in Example 2, a dsDNA backbone with a single bisPNA molecule was used in which a cysteine-labeled bisPNA was mixed with varying concentrations of small N-ethylmaleimide (NEM) as a model target small molecule and a constant amount of a model surrogate 10 kDa maleimide polyethylene glycol (PEG-mal). The surrogate and target small molecules competed for binding to the cysteine-labeled bisPNA. The concentration of the surrogate molecule was constant, in contrast to Example 2 in which both the target and the surrogate were varied. The relative ratios of target:surrogate utilized in this example were 1:2, 1:10, and 1:50. Complexes formed from each mixture were analyzed using a nanopore with a diameter of 20-22 nm and a 22 nm membrane.

Reagents were prepared as follows: a 25 μM solution of cysteine-labeled bisPNA was mixed with 250 μM 10 kDa PEG-mal and various concentrations of NEM in bisPNA binding buffer at room temperature for 20 minutes to allow PEG-mal or NEM to bind to the respective cysteine-labeled bisPNA.

After a brief incubation, the reaction products were analyzed by reverse phase HPLC (Agilent 1100 series) to confirm the composition of the NEM-bisPNA and/or PEG-bisPNA conjugates. The elution time and absorption spectrum of the individual reactants were first characterized by RP-HPLC to serve as a benchmark control for subsequent competitive reactions (Fig. 5). PNA eluted at 6.12 minutes and exhibited strong absorption at 270 nm (Fig. 5 (a)). The peak eluted at 9.80 minutes was determined to be an impurity in the PNA provided by the manufacturer and did not participate in any of the following reactions. The PEG-maleimide molecule itself gave a small broad peak of 18.73 min (Fig. 5 (b)) while the NEM alone gave a very small peak of 5.71 min (Fig. 5 (c)). Due to the weak signals given by the competing molecules PEG and NEM, the disappearance of the PNA absorption at 6.12 minutes and the appearance of another new PNA peak with strong absorption at 270 nm were then used as an indication of successful PNA coupling.

PNA was also incubated with a fixed amount of PEG while the target molecule NEM was titrated to demonstrate competition for thiol-reactive groups on cysteine using a different experimental design. First, 250 μM of PEG alone was incubated with 25 μM PNA, and a clear peak was detected at 17.86 min (Figure 14, solid squares). In separate reactions, NEM was then titrated from 5 μM (Figure 14, solid stars) to 125 μM (Figure 14, open triangles) in the presence of 250 μM PEG. As more NEM was added, a significant decrease in the 17.86-minute PNA-PEG peak was seen, while an increase in the 9.5-minute peak, indicative of a PNA-NEM conjugate, was detected. Overall, these results indicate that the target molecule NEM can successfully compete for thiol-reactive groups.

The NEM-PNA and/or PEG-PNA complexes were mixed with the 1074 bp dsDNA fragment in bisPNA binding buffer at 60°C for a period of 2 hours to allow binding of the cysteine-tagged bisPNA fusion molecules to the dsDNA polymer backbone.

The results of the incubation were observed by electrophoretic mobility shift analysis on a 5% polyacrylamide gel ( FIG. 15 ). Specifically, FIG. 15( a) shows a 5% polyacrylamide gel run to assess the binding of dsDNA to PNA molecules labeled with NEM (target). Bare 1074 bp dsDNA ((a), lane 1) was incubated with increasing amounts of NEM-labeled PNA labeled with PNA in a 10 to 100-fold molar excess relative to the DNA ((a), lanes 2-5). A 50-fold molar excess of NEM-labeled PNA was found to be sufficient to completely label the 1074 bp sequence and was therefore used in subsequent nanopore analysis ((a), lane 4). FIG. 12( b) shows a 10% polyacrylamide gel run to assess the binding of dsDNA to PNA molecules that had been coupled to 10 kDa PEG (alternative). 1074 bp DNA was similarly incubated with increasing concentrations of PNA-PEG, and samples in which each DNA molecule was bound to PNA were used in the nanopore analysis ((b), lane 4).

Once complex formation was confirmed, samples were diluted to the indicated concentrations in running buffer (1 M LiCl, 10 mM Tris, 1 mM EDTA, pH 8) and placed in the nanopore device for nanopore analysis ( FIG. 16 ). First, 0.2 nM of 1074 bp DNA-PNA-NEM generated 389 events in 15 minutes, (median, IQR) dwell time = (44, 16) μsec, and a capture rate of 0.45 sec ⁻¹ (R ² = 0.9972). Subsequently, 0.2 nM of 1074 bp DNA-PNA-PEG generated 251 events in 15 minutes, again with an increase in longer events, (median, IQR) dwell time = (60, 252) μsec, and a capture rate of 0.2742 sec ⁻¹ (R ² = 0.9972) for 25% trimmed data.

By applying the framework established in the "Assigning Statistical Significance to Detections" section, we assign statistical confidence to the detection of the DNA/PNA-NEM complex as a type 2 molecule and the DNA/PNA-PEG as a type 1 molecule. A suitable criterion is to label an event as type 2 if it is faster than 0.07 ms. The DNA/PNA-PEG can be used as a negative control to calculate the false positive q1 = 0.554 (55.4%). The DNA/PNA-NEM (type 2) control yields Q(p) ± _Qsd (p) = 95.3728 ± 2.7436 with 99% confidence. From equation (1) of the mathematical framework, the result is Q(p) - _Qsd (p) = 0.926 > 0.554, which means that we can say that the DNA/PNA-NEM molecule is present with 99% confidence.

The Q(p)± _Qsd (p) curves as a function of recording time are shown for each reagent type (DNA/PNA-NEM and DNA/PNA-PEG) in Figure 17. It was observed that the DNA/PNA-NEM complex was detected with 99% confidence within the first minute of recording.

We next varied PEG:NEM ratios of 50:1, 10:1, and 2:1 to assess how competition for bisPNA binding sites was observed in the nanopore device, and particularly when PEG substitution was held constant.

A 5% polyacrylamide gel ( FIG. 18 ) was run to evaluate the binding of solutions of PNA molecules labeled with NEM (target) and 10 kDa PEG (surrogate) to dsDNA. FIG. 18( a ) shows a 10% polyacrylamide gel run to evaluate the DNA binding ability of PNA that had been reacted with a solution containing PEG and NEM at a 50:1 molar ratio. 1074 bp DNA was incubated with increasing amounts of the fully reacted PNA at concentrations ranging from 10 to 100-fold molar excess ( FIG. 18( a ), lanes 2-5). Nanopore analysis was performed on samples that demonstrated complete DNA binding by both PNA-PEG and PNA-NEM species ( FIG. 18( a ), lane 4). FIG. 18( b ) shows a 10% polyacrylamide gel run to evaluate the DNA binding ability of PNA bound to PEG and increasing amounts of NEM (10:1 and 2:1 PEG-NEM molar ratios, respectively). 1074 bp DNA was incubated with increasing amounts of bound PNA at concentrations ranging from 10 to 100-fold molar excess (lanes 2-5 = 10:1 ratio, lanes 7-10 = 2:1 ratio). Nanopore analysis was performed on samples that exhibited complete DNA binding by both PNA-PEG and PNA-NEM species (Figure 18(b), lanes 4 and 9).

First, on the same pore, using a population indistinguishable from the PEG-only analysis, 1074 bp DNA-PNA-10xPEG and 0.2x NEM (50:1 surrogate:target) produced 337 events in 15 minutes with a (median, IQR) dwell time = (68,210) μsec. The fraction of events faster than 0.07 ms was Q(p) ± Q _sd (p) = 51.3353 +/- 7.0132 at 99% confidence, which is not high enough to infer detection of the presence of DNA/PNA-NEM. That is, since equation (1) is not satisfied, we cannot say that DNA/PNA-NEM is present in this test at a 50:1 surrogate:target ratio. Subsequently, 1074 bp DNA-PNA-10xPEG and 1x NEM produced 472 events in 30 minutes with a (median, IQR) dwell time = (60,76) μsec. As the relative amount of NEM relative to PEG increases, the fraction of events faster than 0.07 ms increases to Q(p) ± Q _sd (p) = 62.0763 +/- 5.7526 at 99% confidence. From the mathematical framework of equation (1), the result is Q(p) - Q _sd (p) = 0.563 > 0.554, which means that we can say that the DNA/PNA-NEM molecule is present with 99% confidence. Finally, 1074 bp DNA-PNA-10x PEG and 5x NEM produced 687 events in 30 minutes with a (median, IQR) dwell time = (52, 48) μsec. As the relative amount of NEM relative to PEG increases, the fraction of events faster than 0.07 ms further increases to Q(p) ± Q _sd (p) = 70.0146 +/- 4.5029 at 99% confidence. Again, from the mathematical framework of equation (1), the result is Q(p) _-Qsd (p)=0.655>0.554, which means that we can say that the DNA/PNA-NEM molecule is present with 99% confidence. Figure 19 shows the Q(p)± _Qsd (p) curves as a function of recording time for each reagent type.

As in Example 2, the trend of increasing scores with increasing amounts of NEM relative to PEG indicates that competition between NEM and PEG is detectable using nanopores. These measurements can be used to detect the presence of target small molecules, as shown here for NEM with 99% confidence. These measurements can also be combined with biophysical models to estimate target small molecule concentrations.

At 99% confidence, Q(end) = 63.5015 +/- 6.7551.

Claims (56)

1. A method for detecting the presence or absence of a target molecule suspected of being present in a sample, comprising: A device comprising a layer, wherein the layer divides the internal space of the device into a first volume and a second volume, wherein the layer comprises nanopores connecting the first volume and the second volume, and wherein the device comprises a sensor configured to identify an object passing through the nanopores; The invention provides alternative molecules, fusion molecules, and polymer backbones, wherein the fusion molecule includes a polymer backbone binding domain adapted to bind the polymer backbone to form a backbone/fusion molecule complex, and the fusion molecule includes a target molecule binding domain adapted to bind the alternative molecule or the target molecule. Competitive analysis is performed by combining the substitute molecule and the fusion molecule with the sample, wherein if the target molecule is present in the sample, the target molecule competes with the substitute molecule to bind to the target molecule binding domain; The sample is loaded into the first volume; A voltage is applied across the nanopores, wherein the first volume comprises the polymer backbone, the fusion molecule, the substitute molecule, and the sample suspected of containing the target molecule, wherein the polymer backbone hybridizes with the fusion molecule, and wherein the fusion molecule hybridizes with either the substitute molecule or the target molecule. 2. The method of claim 1, wherein the voltage induces the backbone/fusion molecular complex bound to the target molecule or the alternative molecule to translocate from the first volume through the nanopores to generate a change in electrical signal detected by the sensor. 3. The method of claim 2 further includes recording changes in the electrical signal as a function of time. 4. The method of claim 3 further comprises analyzing changes in the electrical signal as a function of time to determine the presence or absence of the target molecule in the sample. 5. The method of any one of claims 1-4, wherein the competition analysis is performed after the sample is loaded into the first volume. 6. The method of any one of claims 1-4, wherein the competition analysis is performed before the sample is loaded into the first volume. 7. The method of any one of claims 1-4, wherein the alternative molecule comprises maleimide polyethylene glycol. 8. The method of any one of claims 1-4, wherein the alternative molecule comprises a chemically reactive group selected from the group consisting of ketones, aldehydes, isocyanates, amines, carboxylic acids, halides, esters, maleimides, thiols, bicyclic carbonimides, pyridyl groups, pyridine disulfides, and acetyl groups. 9. The method of any one of claims 1-4, wherein the alternative molecule comprises a strong or weak nucleophile or an electrophile. 10. The method of any one of claims 1-4, wherein the alternative molecule comprises a peptide, a dendritic molecule, a nucleic acid, a nano or microbead or particle, a quantum dot, a protein, a polynucleotide, a liposome, an antibody, or an antibody fragment. 11. The method of any one of claims 1-4, wherein the alternative molecule comprises a payload binding site adapted to bind the payload molecule. 12. The method of claim 11, wherein the effective payload molecule is selected from: dendritic molecules, double-stranded DNA, single-stranded DNA, DNA aptamers, fluorophores, proteins, antibodies, peptides, nanobeads, nanorods, nanotubes, nanoparticles, fullerenes, PEG molecules, liposomes, or cholesterol-DNA hybrids. 13. The method of claim 11, wherein the effective load molecule comprises a charge. 14. The method of claim 13, wherein the charged effective load molecule is selected from: peptides, amino acids, charged nanoparticles, synthetic molecules, nucleotides, polynucleotides, metals, or ions. 15. The method of claim 11, wherein the substitute molecule binds to the payload molecule through an interaction selected from: covalent bonds, hydrogen bonds, ionic bonds, van der Waals forces, hydrophobic interactions, cation-pi interactions, planar stacking interactions, metallic bonds, and biotin-avidin interactions. 16. The method of any one of claims 1-4, wherein the target molecule comprises a small molecule with a size less than 30,000 Da, 20,000 Da, 10,000 Da, 5,000 Da, 2,000 Da, 1,000 Da, 500 Da, 200 Da, 100 Da, 50 Da, 20 Da, or 10 Da. 17. The method of any one of claims 1-4, wherein the target molecule comprises peptides, insulin, oxytocin, amino acids, proteins or protein domains, nucleotides, oligomers, DNA, RNA, PNA, LNA, BNA, hormones, lipids, cholesterol, metabolites, sugars, polysaccharides, peptidoglycans, polysaccharides, phospholipids, steroids, chemically synthesized agonists and antagonists, synthetic derivatives of polynucleotides, polycyclic aromatic hydrocarbons, carbon decomposition byproducts, dioxins, cyclohexylamide, vitamins, adenosine triphosphate and ATP analogs, neurotransmitters, dopamine, L-DOPA, serotonin, metals, electrolytes, organometallic substances, anesthetics and anesthetic derivatives, hyaluronic acid, or retinol. 18. The method of any one of claims 1-4, wherein the fusion molecule comprises a peptide nucleic acid. 19. The method of any one of claims 1-4, wherein the fusion molecule comprises bridging nucleic acid, locked nucleic acid, biotin, streptavidin, streptavidin derivative, zinc finger protein, ZFP binding domain, CRISPR domain, TALEN, DNA, PNA, or RNA oligomer. 20. The method of claim 19, wherein the fusion molecule comprises a cysteine-labeled bispeptide nucleic acid. 21. The method of any one of claims 1-4, wherein the polymer backbone comprises a negatively or positively charged polymer adapted to translocate through the nanopores from the first volume to the second volume when a potential is applied to the nanopores. 22. The method of any one of claims 1-4, wherein the polymer backbone comprises molecules selected from the group consisting of deoxyribonucleic acid, ribonucleic acid, peptide nucleic acid, DNA/RNA hybrids, and polypeptides. 23. The method of any one of claims 1-4, wherein the sensor is configured to identify an object passing through a single nanopore. 24. The method of any one of claims 1-4, wherein the sensor is an electrical sensor. 25. The method of claim 24, wherein the sensor detects a current through the nanopores when a voltage is applied across the nanopores. 26. The method of any one of claims 3-4, wherein the analysis of the change in the electrical signal as a function of time comprises separating events resulting from the translocation of the skeleton/fusion complex bound to the alternative molecule through the nanopores and events resulting from the translocation of the skeleton/fusion complex bound to the target molecule through the nanopores. 27. The method of any one of claims 1-4, wherein the method provides a detection confidence level of greater than 90%, 95%, 98%, or 99% for the target molecule. 28. The method of any one of claims 1-4, wherein the sample is not purified before being loaded into the first volume. 29. The method of any one of claims 1-4, wherein the nanopore diameter is at least 5 nm, 10 nm, 20 nm, 30 nm, 40 nm or 50 nm. 30. The method of claim 11, wherein the device comprises at least two tandem nanopores, and wherein the amplicon bound to the payload molecule is simultaneously located within the at least two tandem nanopores during translocation. 31. A reagent kit comprising... A device comprising nanopores, wherein the device includes a layer dividing the internal space of the device into a first volume and a second volume, wherein the layer includes nanopores passing through the layer connecting the first volume and the second volume, and wherein the device includes a sensor configured to identify objects passing through the nanopores; The fusion molecule comprises a substitute molecule, a fusion molecule, and a polymer backbone, wherein the fusion molecule includes a polymer backbone binding domain adapted to bind the polymer backbone to form a backbone/fusion molecule complex, and the fusion molecule includes a target molecule binding domain adapted to bind the substitute molecule or the target molecule; and This is an instruction for detecting the presence or absence of the target molecule by observing the results of a competitive analysis in the device. 32. The kit of claim 31, wherein the alternative molecule comprises maleimide polyethylene glycol. 33. The kit of claim 31, wherein the alternative molecule comprises a chemically reactive group selected from the group consisting of ketones, aldehydes, isocyanates, amines, carboxylic acids, halides, esters, maleimides, thiols, bicyclic carbonimides, pyridyl groups, pyridine disulfides, and acetyl groups. 34. The kit of claim 31, wherein the alternative molecule comprises a weak or strong nucleophile or electrophile. 35. The kit of claim 31, wherein the alternative molecule comprises peptides, dendritic molecules, nucleic acids, nano or microbeads or particles, quantum dots, proteins, polynucleotides, liposomes or antibodies. 36. The kit of claim 31, wherein the alternative molecule comprises a payload binding site adapted to bind the payload molecule. 37. The kit of claim 36, wherein the payload molecule is selected from: dendritic molecules, double-stranded DNA, single-stranded DNA, DNA aptamers, fluorophores, proteins, antibodies, peptides, nanobeads, nanorods, nanotubes, nanoparticles, fullerenes, PEG molecules, liposomes, or cholesterol-DNA hybrids. 38. The kit of claim 36, wherein the payload molecule comprises a charge. 39. The kit of claim 38, wherein the charged effective charge molecule is selected from: peptides, amino acids, charged nanoparticles, synthetic molecules, nucleotides, polynucleotides, metals or ions. 40. The kit of claim 36, wherein the alternative molecule binds to the payload molecule through an interaction selected from: covalent bonds, hydrogen bonds, ionic bonds, van der Waals forces, hydrophobic interactions, cation-pi interactions, planar stacking interactions, metallic bonds, and biotin-antibiotin interactions. 41. The kit of claim 31, wherein the target molecule comprises a small molecule with a size less than 30,000 Da, 20,000 Da, 10,000 Da, 5,000 Da, 2,000 Da, 1,000 Da, 500 Da, 200 Da, 100 Da, 50 Da, 20 Da, or 10 Da. 42. The kit of claim 31, wherein the target molecule comprises a molecule selected from the group consisting of: N-ethylmaleimide, peptides, insulin, oxytocin, amino acids, proteins or protein domains, nucleotides, oligomers, DNA, RNA, PNA, LNA, BNA, hormones, lipids, cholesterol, metabolites, sugars, polysaccharides, peptidoglycans, polysaccharides, phospholipids, steroids, chemically synthesized agonists and antagonists, synthetic derivatives, polycyclic aromatic hydrocarbons, carbon decomposition byproducts, dioxins, cyclohexylamide, vitamins, adenosine triphosphate and ATP analogs, neurotransmitters, dopamine, L-DOPA, serotonin, metals, electrolytes, organometallic substances, anesthetics and anesthetic derivatives, hyaluronic acid or retinol. 43. The kit of claim 31, wherein the fusion molecule comprises a peptide nucleic acid. 44. The kit of claim 43, wherein the fusion molecule comprises a cysteine-labeled bispeptide nucleic acid. 45. The kit of claim 31, wherein the fusion molecule comprises a bridging nucleic acid, a locked nucleic acid, biotin, streptavidin or a derivative thereof, a zinc finger protein or ZFP binding domain, a CRISPR domain, TALEN, a DNA or RNA oligomer, which is fused to a domain that binds to the target small molecule. 46. The kit of claim 31, wherein the polymer backbone comprises a negatively or positively charged polymer adapted to translocate through the nanopores from the first volume to the second volume when a potential is applied to the nanopores. 47. The kit of claim 31, wherein the polymer backbone comprises molecules selected from the group consisting of deoxyribonucleic acid, ribonucleic acid, peptide nucleic acid, DNA/RNA hybrids, and polypeptides. 48. The kit of claim 31, wherein the sensor is configured to identify an object passing through a single nanopore. 49. The kit of claim 31, wherein the sensor is an electrical sensor. 50. The kit of claim 49, wherein the sensor detects the current through the nanopores when a voltage is applied across the nanopores. 51. A method for quantifying the amount of a target small molecule present in a sample, comprising: A device comprising a layer, wherein the layer divides the internal space of the device into a first volume and a second volume, wherein the layer comprises nanopores connecting the first volume and the second volume, and wherein the device comprises a sensor configured to identify an object passing through the nanopores; The invention provides alternative molecules, fusion molecules, and polymer backbones, wherein the fusion molecule includes a polymer backbone binding domain adapted to bind the polymer backbone to form a backbone/fusion molecule complex, and the fusion molecule includes a target molecule binding domain adapted to bind the alternative molecule or the target molecule. The sample is subjected to competitive analysis by combining the substitute molecule and the fusion molecule, wherein if the target molecule is present in the sample, the target molecule competes with the substitute molecule for binding to the target molecule binding domain; The sample is loaded into the first volume; A voltage is applied across the nanopores, wherein the first volume comprises the polymer backbone, the fusion molecule, the substitute molecule, and the sample suspected of containing the target molecule, wherein the polymer backbone hybridizes with the fusion molecule, and wherein the fusion molecule hybridizes with the substitute molecule or the target molecule; The capture rate of the backbone/fusion molecule bound to the target small molecule in the nanopores is compared with the capture rate of the backbone/fusion molecule bound to the alternative molecule in the nanopores to quantify the amount of the target small molecule in the sample. 52. The method of claim 51, wherein the voltage induces a translocation of the backbone/fusion molecular complex bound to the target molecule or the alternative molecule from the first volume through the nanopores to generate a change in electrical signal detected by the sensor. 53. The method of claim 52 further includes recording changes in the electrical signal as a function of time. 54. The method of claim 53 further comprises analyzing changes in the electrical signal as a function of time to determine the presence or absence of the target small molecule in the sample. 55. The method of claim 51, wherein the competition analysis is performed after the sample is loaded into the first volume. 56. The method of claim 51, wherein the competition analysis is performed before the sample is loaded into the first volume.