HK40061244A

HK40061244A - Isothermal amplification with electrical detection

Info

Publication number: HK40061244A
Application number: HK62022050518.9A
Authority: HK
Inventors: 方日勳; 约瑟夫‧卡尔‧盖特里; 布伦纳‧赫恩‧洛德; 蒋玉民; 罗纳德‧菲利普‧基亚雷洛
Original assignee: 阿尔韦奥科技公司
Priority date: 2018-12-20
Filing date: 2019-12-18
Publication date: 2022-05-27

Description

Isothermal amplification with electrical detection

RELATED APPLICATIONS

Priority of U.S. provisional patent application 62/783117 entitled "international AMPLIFICATIONs WITH ELECTRICAL DETECTION" filed on 12, 20, 2018, this application claims priority, which is hereby expressly incorporated herein in its entirety by reference.

Sequence listing

This application is filed with a sequence listing in electronic format. A sequence listing is provided for a file created at 12, 7 and 2019 under the name ALVEO021WOSEQLISTING, which is about 5Kb in size. The information in the sequence listing in electronic format is incorporated by reference herein in its entirety.

Technical Field

Some embodiments of the methods provided herein relate to amplifying and detecting target nucleic acids. Some such embodiments include performing Recombinase Polymerase Amplification (RPA) and optionally a second isothermal amplification reaction, such as loop-mediated isothermal amplification (LAMP). Some embodiments further comprise detecting the presence of amplification products generated by RPA with or without a second isothermal amplification (e.g., LAMP) by measuring or analyzing modulation of an electrical signal (e.g., impedance) that is desired to be compared to a control.

Background

Pathogens in a sample can be identified by detecting specific genomic material (DNA or RNA). In addition to pathogen detection, many other biomarkers can be used for testing, including molecules that provide early detection of cancer, important prenatal information, or a better understanding of the patient's microbiota. In conventional nucleic acid testing ("NAT"), genomic material in a sample can first be replicated exponentially using a molecular amplification procedure known as polymerase chain reaction ("PCR") until the amount of DNA present is sufficient to be measurable. In the case of RNA (the genomic material of many viruses), an additional step may be included to first transcribe the RNA into DNA prior to amplification by PCR.

Disclosure of Invention

Some embodiments include a method of amplifying and detecting a target nucleic acid, the method comprising: providing, preferably in a single container, a Recombinase Polymerase Amplification (RPA) reagent solution comprising a target nucleic acid, primers each complementary to a region of the target nucleic acid, a buffer, dntps, a recombinase, a single-stranded DNA binding protein (SSB), and a strand-displacing DNA polymerase; combining, preferably in the single container, the RPA reagent solution with a second reagent solution configured for a second isothermal amplification reaction (e.g., LAMP) that does not use a recombinase to produce an amplification reaction solution; performing RPA in the amplification reaction solution, and optionally the second isothermal amplification, preferably in the single vessel, to produce amplified target nucleic acid; optionally, helical rpa (helical rpa) is performed using pairs of primers, the forward and reverse primer sequences being reverse complementary to each other at their 5 'ends and their 3' end sequences being complementary to the target sequence; and detecting the presence of amplified target nucleic acid by measuring modulation of an electrical signal (e.g., impedance) in the amplification reaction solution when the amplification reaction is subjected to an electric field, as compared to a control.

In some embodiments, the detection of the amplified target nucleic acid is performed in a device comprising a test well comprising an excitation electrode and a sensor electrode, and wherein the detecting further comprises: applying an excitation signal from a reader device to an excitation electrode; sensing a signal from the test well using the excitation electrode, wherein the signal is representative of an impedance of the amplification reaction solution; and transmitting the signal to a reader device, wherein the reader device analyzes the signal.

In some embodiments, the recombinase enzyme comprises UvsX or RecA. In some embodiments, the SSB comprises gp32 or e. In some embodiments, the strand displacement DNA polymerase includes Bst DNA polymerase large fragment, Bst 2.0 polymerase, Bst 3.0 polymerase, Gsp polymerase, Sau polymerase, Bsu DNA polymerase large fragment, Deep VentR DNA polymerase, Deep VentR (exo-) DNA polymerase, Klenow fragment (3'→ 5' exo-), DNA polymerase I large (Klenow) fragment, phi29 DNA polymerase, VentR DNA polymerase, or VentR (exo-) DNA polymerase, or any combination thereof.

In some embodiments, the RPA reagent solution further comprises a reagent selected from the group consisting of: tris-acetate, polyethylene glycol, trehalose, potassium acetate, creatine kinase, phosphocreatine, adenosine triphosphate, or a recombinase-loaded protein (e.g., uvsY), or wherein the RPA reagent solution or the amplification reaction solution does not contain a primer with a detectable label or dye. In some embodiments, the amplification reaction solution comprises DTT at a concentration of 0.5mM to 1mM, 0.25mM to 0.5mM, about 0.25mM, 0mM to 0.25mM, or 0 mM. In some embodiments, the amplification reaction solution comprises DTT at a concentration of 1mM-10mM, about 5mM, or 5mM, and calcium chloride at a concentration of 5mM-15mM, calcium chloride at a concentration of about 0.9mM, calcium chloride at a concentration of 0.9mM, Ca at a concentration of 5mM-15mM ²⁺Ca of about 0.9mM²⁺Or 0.9mM Ca²⁺. In some embodiments, the amplification reaction solution comprises a concentration of 1mM-20mM, 1mM-5mM, 5mM-10mM, 7mM-9mM, 8mM, about 8mM, 10mM-20mM, about 13mM, or 13mMMagnesium or magnesium ions. In some embodiments, the amplification reaction solution comprises a dNTP mixture at a concentration of 1mM-10mM, 1mM-3mM, about 1.8mM, 5mM-6mM, about 5.6mM, or 5.6 mM. In some embodiments, the amplification reaction solution further comprises a blocking region oligonucleotide comprising a nucleic acid sequence that is reverse complementary to a portion of the nucleic acid sequence of one or more of the primers.

In some embodiments, the RPA comprises a leading strand RPA (lsrpa), a synchronous leading strand, and a following strand synthetic or nested RPA. In some embodiments, the second isothermal amplification comprises self-sustained sequence replication reaction (3SR), 90-I, BAD Amp, cross-primer amplification (CPA), isothermal index amplification reaction (EXPAR), isothermal chimeric primer-primed nucleic acid amplification (ICAN), Isothermal Multiple Displacement Amplification (IMDA), ligation-mediated SDA, multiple displacement amplification, polymerase helix reaction (PSR), restriction cascade index amplification (RCEA), smart amplification program (SMAP2), Single Primer Isothermal Amplification (SPIA), transcription-based amplification system (TAS), transcription-mediated amplification (TMA), Ligase Chain Reaction (LCR) or multiple cross-displacement amplification (MCDA), rolling circle Replication (RCA), nickase amplification reaction (NEAR), or nucleic acid sequence-based amplification (NASBA).

In some embodiments, the second isothermal amplification comprises loop-mediated isothermal amplification (LAMP). In some embodiments, the amplification reaction solution comprises LAMP-compatible primer oligonucleotides. In some embodiments, the amplification reaction solution comprises a LAMP-compatible FIP, BIP, LF, or LB primer oligonucleotide, and a RPA-compatible primer. In some embodiments, the amplification reaction solution comprises a LAMP-compatible FIP, BIP, LF, LB, F3, or B3 primer oligonucleotide.

In some embodiments, the RPA and the second isothermal amplification (e.g., LAMP) are performed at the same or substantially the same temperature. In some embodiments, the RPA or second isothermal amplification (e.g., LAMP) is performed at 25 ℃, 30 ℃, 37 ℃, 40 ℃, 45 ℃, 50 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, or at a temperature within a range defined by any two of the aforementioned temperatures. In some embodiments, RPA is performed at a lower or higher temperature than the second isothermal amplification (e.g., LAMP). In some embodiments, the RPA is performed at 37 ℃, about 37 ℃, 40 ℃, or about 40 ℃, and the second isothermal amplification (e.g., LAMP) is performed at 60 ℃, about 60 ℃, 65 ℃, or about 65 ℃.

In some embodiments, the primer does not comprise a label, marker, or dye. In some embodiments, amplification and detection are performed in the absence of a detection reagent (e.g., a dye, a clouding agent, a fluorophore, a double-stranded nucleic acid intercalator, a sequencing index, or a nanoparticle).

Some embodiments include a method of amplifying and detecting a target nucleic acid, the method comprising: providing, preferably in a single container, a Recombinase Polymerase Amplification (RPA) reagent solution comprising a target nucleic acid, primers each complementary to a region of the target nucleic acid, a buffer, dntps, a recombinase, a single-stranded DNA binding protein (SSB), and a strand-displacing DNA polymerase; performing RPA in an RPA reagent solution to produce amplified target nucleic acids; combining, preferably in the single vessel, the amplified target nucleic acid with a second reagent solution configured for a second isothermal amplification reaction (e.g., LAMP) that does not use a recombinase to produce a second amplification reaction solution; performing a second isothermal amplification of the second amplification reaction solution to produce further amplified target nucleic acids; and detecting the presence of amplified target nucleic acid by measuring modulation of an electrical signal (e.g., impedance) in the amplification reaction solution when subjecting the amplification reaction to an electric field, optionally compared to a control.

In some embodiments, combining the amplified target nucleic acid with a second reagent solution configured for a second isothermal amplification reaction comprises: adding a second reagent solution to the RPA reagent solution after performing RPA to produce the amplified target nucleic acid. In some embodiments, combining the amplified target nucleic acid with a second reagent solution configured for a second isothermal amplification reaction comprises: adding the RPA reagent solution or a portion of the RPA reagent solution to the second reagent solution after performing RPA to produce the amplified target nucleic acid.

In some embodiments, the recombinase enzyme comprises UvsX or RecA. In some embodiments, the SSB comprises gp32 or an e. In some embodiments, the strand displacement DNA polymerase comprises a Bst DNA polymerase large fragment, Bst 2.0 polymerase, Bst 3.0 polymerase, Gsp polymerase, Sau polymerase, a Bst DNA polymerase large fragment, Deep VentR DNA polymerase, Deep VentR (exo-) DNA polymerase, Klenow fragment (3'→ 5' exo-), DNA polymerase I large (Klenow) fragment, phi29 DNA polymerase, VentR DNA polymerase, or VentR (exo-) DNA polymerase. In some embodiments, the RPA reagent solution further comprises a reagent selected from the group consisting of: tris-acetate, polyethylene glycol, trehalose, potassium acetate, creatine kinase, phosphocreatine, recombinase-loaded proteins (e.g., uvsY), or wherein the RPA reagent solution or the amplification reaction solution does not contain a primer with a detectable label, marker, or dye.

In some embodiments, the amplification reaction solution comprises DTT at a concentration of 0.5mM to 1mM, 0.25mM to 0.5mM, about 0.25mM, 0mM to 0.25mM, or 0 mM. In some embodiments, the amplification reaction solution comprises DTT at a concentration of 1mM-10mM, about 5mM, or 5mM, and calcium chloride at a concentration of 5mM-15mM, calcium chloride at a concentration of about 0.9mM, calcium chloride at a concentration of 0.9mM, Ca at a concentration of 5mM-15mM²⁺Ca of about 0.9mM²⁺Or 0.9mM Ca²⁺. In some embodiments, the amplification reaction solution comprises magnesium or magnesium ions at a concentration of 1mM-20mM, 1mM-5mM, 5mM-10mM, 7mM-9mM, 8mM, about 8mM, 10mM-20mM, about 13mM, or 13 mM. In some embodiments, the amplification reaction solution comprises a dNTP mixture at a concentration of 1mM-10mM, 1mM-3mM, about 1.8mM, 5mM-6mM, about 5.6mM, or 5.6 mM. In some embodiments, the amplification reaction solution further comprises a blocking region oligonucleotide comprising a nucleic acid sequence that is reverse complementary to a portion of the nucleic acid sequence of one or more of the primers. In some embodiments of the methods described herein, the amplification reaction solution comprises TCEP. In some embodiments, the TCEP in the amplification reaction solution is 5mM, about 5mM, 4mM-5mM, 5mM-6mM, 4mM-6mM, 3mM-7mM, 2mM-8mM, 1mM-9mM, or 1mM-10 mM. In some embodiments of the methods described herein, the amplification reaction solution comprises a reducing agent (e.g., DTT or TCEP). At one end In some embodiments, the reducing agent in the amplification reaction solution is 5mM, about 5mM, 4mM-5mM, 5mM-6mM, 4mM-6mM, 3mM-7mM, 2mM-8mM, 1mM-9mM, or 1mM-10 mM.

Some embodiments include a method of amplifying and detecting a target nucleic acid, the method comprising: performing Recombinase Polymerase Amplification (RPA) and loop-mediated isothermal amplification (LAMP) on a target nucleic acid in a single vessel to produce amplified target nucleic acid, preferably without separating or purifying the amplified target nucleic acid between the RPA amplification and the LAMP amplification, e.g., in a single vessel; and detecting the presence of amplified target nucleic acid by measuring modulation of an electrical signal (e.g., impedance) in the amplification reaction solution when subjecting the amplification reaction to an electric field, optionally compared to a control.

In some embodiments, RPA and LAMP amplification are performed at the same or substantially the same temperature. In some embodiments, the RPA or LAMP amplification is performed at 25 ℃, 30 ℃, 37 ℃, 40 ℃, 45 ℃, 50 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, or at a temperature within a range defined by any two of the above temperatures. In some embodiments, RPA is performed at a lower or higher temperature than LAMP amplification. In some embodiments, the RPA is performed at 37 ℃, about 37 ℃, 40 ℃, or about 40 ℃, and the LAMP amplification is performed at 60 ℃, about 60 ℃, 65 ℃, or about 65 ℃.

In some embodiments, the primers in the RPA or LAMP amplification do not comprise a label, marker, or dye. In some embodiments, RPA and LAMP amplification and detection are performed in the absence of detection reagents (e.g., dyes, clouding agents, fluorophores, double-stranded nucleic acid intercalators, sequencing indexes, or nanoparticles).

In some embodiments, the detection of the amplified target nucleic acid is performed in a device comprising a test well comprising an excitation electrode and a sensor electrode, and wherein the detecting further comprises: applying an excitation signal from a reader device to an excitation electrode; sensing a signal from the test well using the excitation electrode, wherein the signal is representative of an impedance of the amplification reaction solution; and in some embodiments, the signal is transmitted to a reader device, wherein the reader device analyzes the signal.

Drawings

Fig. 1A-1D depict exemplary cassettes for detection of a target.

Figure 2 depicts another exemplary cassette for detection of a target.

Fig. 3A and 3B depict another exemplary cartridge for detection of a target.

Fig. 4A-4G depict various examples of electrodes that may be used in the test wells of the cartridges of fig. 1A-3B, 51A-53B, or in the test wells or channels of another suitable target detection cartridge described herein.

Fig. 5A depicts a first or excitation electrode and a second or signal electrode, which may be spaced apart from each other within the test well of the cartridges of fig. 1A-3B, 51A-53B, or within the test well or channel of another suitable target detection cartridge described herein.

FIG. 5B depicts an exemplary signal that may be extracted from the signal electrode of FIG. 5A.

Fig. 5C depicts resistive and reactive components extracted from the signal shown in fig. 5B generated based on an example positive test.

Fig. 5D depicts the resistive and reactive components extracted from the signals shown in fig. 5B from the test of the example of the positive control and the test of the example of the negative control.

Fig. 5E depicts resistive and reactive components extracted from a signal, as shown in fig. 5B, generated based on a positive test of another example.

Fig. 6 depicts a schematic block diagram of an example reader device that may be used with the cartridges described herein.

FIG. 7A depicts a flowchart of an example process for operating a reader device during testing as described herein.

Fig. 7B depicts a flowchart of an example process for analyzing test data to detect a target as described herein.

FIG. 8 depicts a protocol for an amplified immunoassay (amplification immunoassay).

FIG. 9 depicts a bead-based amplified immunoassay protocol.

FIG. 10 depicts a magnetic bead based amplification immunoassay protocol.

FIG. 11 depicts a first electrode (or actuation electrode) and a second electrode (or signal electrode) that may be spaced apart from each other along a channel.

Fig. 12 is a graph showing the change in the impedance of a signal depending on the excitation frequency and after the LAMP reaction occurs in the channel (where the left-side inequality may define the frequency region).

Fig. 13 is a graph showing that the impedance is capacitor-like and out of phase (nearly 90 °) with the excitation voltage in the two extreme regions.

Fig. 14 is a graph depicting the measured impedance of the sample chip versus the excitation frequency.

FIG. 15 is a graph depicting the response of a synchronized detector plotted against a dimensionless conductivity.

FIG. 16 is a graph depicting the results of a model indicating the consistency of detector output for a given step with detector output and frequency for a wide range of conductivities.

Fig. 17A and 17B depict embodiments of detection systems that can be used to detect the presence or absence of particular nucleic acids and/or particular nucleotides in a sample. Fig. 17A is a top view and fig. 17B is a cross-sectional side view of the system.

FIG. 18 is a process flow diagram illustrating an embodiment of an apparatus for detecting a target.

FIG. 19 is a process flow diagram illustrating an embodiment of an apparatus for detecting a target.

Fig. 20 depicts an example fluid cartridge.

Fig. 21 is a plan view of the example fluidic cartridge of fig. 20.

Fig. 22 depicts an example configuration of an electrode.

FIG. 23 depicts an exemplary channel.

FIG. 24 is a graph depicting sensor voltage before amplification (-control) and after amplification (+ control) as a function of time.

FIG. 25 is a graph depicting pre-amplification (-control) and post-amplification (+ control) sensor voltage versus time for 0% whole blood.

FIG. 26 is a graph depicting pre-amplification (-control) and post-amplification (+ control) sensor voltage versus time for 1% whole blood.

FIG. 27 is a graph depicting sensor voltage before amplification (-control) and after amplification (+ control) for 5% whole blood over time.

FIG. 28 is a graph depicting sensor voltage before amplification (-control) and after amplification (+ control) as a function of time for 0% whole blood for an unfiltered sample.

FIG. 29 is a graph depicting sensor voltage before amplification (-control) and after amplification (+ control) for 0% whole blood for filtered samples as a function of time.

Figure 30 depicts a graph of time as a function of target loading with error bars showing standard deviation.

FIG. 31 depicts conductivity maps of various samples from pre-amplification vial (-control) and post-amplification vial (+ control).

Figure 32 depicts a magnetic bead-based amplified immunoassay protocol for detection of HBsAg.

FIG. 33 depicts a diagram illustrating detection of HBsAg.

FIG. 34 depicts a diagram illustrating the detection of HBsAg with low ion buffer (T10).

Fig. 35 depicts a graph illustrating impedance characteristics of a fluidic cartridge.

Figure 36A depicts a plot of the out of phase signal of LAMP performed on cassettes at 65 ℃.

Figure 36B depicts a plot of the in-phase signal of LAMP performed on the cassette at 65 ℃.

Figure 36C depicts a plot of the out of phase signal of LAMP performed on the cassette at 67 ℃.

Figure 36D depicts a plot of the in-phase signal of LAMP performed on the cassette at 67 ℃.

Figure 36E depicts a plot of the out of phase signal of LAMP performed on the cassette at 67 ℃.

Figure 36F depicts a plot of the in-phase signal of LAMP performed on the cassette at 67 ℃.

FIG. 37 depicts an example of RecA/primer loading.

FIG. 38A is a schematic diagram depicting an example of steps for leading strand recombinase polymerase amplification (lsRPA).

FIG. 38B is a schematic diagram depicting an example of the steps of leading strand recombinase polymerase amplification (lsRPA).

FIG. 39A is a schematic diagram depicting an example of steps for polymerase amplification of the leading and trailing strands.

FIG. 39B is a schematic diagram depicting an example of steps for polymerase amplification of the leading and trailing strands.

FIG. 39C is a schematic diagram depicting an example of steps for polymerase amplification of the leading and trailing strands.

FIG. 39D is a schematic diagram depicting an example of steps for polymerase amplification of the leading and trailing strands with the strand recombinase.

Figure 40 depicts an example of nested primers selected for nested RPAs.

FIG. 41 is a diagram of an example of a method described herein.

FIG. 42 is a diagram of an example of a method described herein.

FIG. 43 is a diagram of an example of a method described herein.

FIG. 44 is a chart depicting the results of a cartridge test.

Fig. 45A is a graph illustrating an amplification curve according to some embodiments.

Fig. 45B is a graph illustrating a melting curve according to some embodiments.

Fig. 45C is a graph illustrating an amplification curve according to some embodiments.

Fig. 45D is a graph illustrating a melting curve according to some embodiments.

Fig. 45E is a table with Ct data according to some embodiments.

Fig. 46A is an amplification graph of RPA according to some embodiments.

Fig. 46B is an amplification graph of RPA according to some embodiments.

Fig. 47A is an amplification graph of an RPA stage according to some embodiments.

Fig. 47B is an amplification graph of the RPA stage according to some embodiments.

Fig. 48A is an amplification chart of the LAMP stage according to some embodiments.

Fig. 48B is a melting curve graph of the LAMP stage according to some embodiments.

Fig. 48C is an amplification chart of the LAMP stage according to some embodiments.

Fig. 48D is a melting curve graph of the LAMP stage according to some embodiments.

Fig. 48E is an amplification chart of the LAMP stage according to some embodiments.

Fig. 48F is a melting curve graph of the LAMP stage according to some embodiments.

Fig. 49A is an amplification chart according to some embodiments.

Fig. 49B is an amplification chart according to some embodiments.

Fig. 49C is an amplification chart according to some embodiments.

Fig. 49D is an amplification chart according to some embodiments.

Fig. 50A is an amplification chart according to some embodiments.

Fig. 50B is an amplification chart according to some embodiments.

Fig. 50C is an amplification chart according to some embodiments.

Fig. 51A-51C depict an exemplary handheld system for detecting a target.

Fig. 52A-52F depict exemplary cartridges for detecting a target that may be used in the handheld systems of fig. 51A-51C.

Fig. 53A-53E depict a mechanical fluid transfer arrangement for the example cartridge of fig. 52A-52F.

FIG. 54 depicts a flowchart of an example program for operating a reader device during testing as described herein.

55A-55D depict example user interfaces of user devices implementing example test programs in communication with a reader device as described herein.

Fig. 56A and 56B depict an exemplary handheld system for detecting a target.

Fig. 57A-57K depict exemplary cartridges for detecting a target that can be used in the handheld systems of fig. 56A and 56B.

Fig. 58A-58D depict mechanical fluid transfer devices of the example cartridges of fig. 57A-57J.

Fig. 59 depicts a schematic block diagram of an example reader device that may be used with the cartridges described herein.

Fig. 60A-60I depict exemplary cartridges for detecting a target that can be used in conjunction with the handheld systems disclosed herein.

Detailed Description

Aspects disclosed herein concern the use of amplification and non-contact electrical sensing for detecting the presence of a target in a sample. Such diagnostic platforms can replace the complex optical systems and expensive fluorescent labels used for optical detection and the electrodes and electroactive agents used in existing electrochemical and FET technologies with common electronics. In some aspects, amplification may be isothermal (e.g., with RPA with or without LAMP). The platform described herein is inexpensive, rugged, portable, and consumes less power than conventional diagnostic systems. In some aspects, the diagnostic platform is small enough to fit in the palm of a consumer's hand and can be performed on-site, for example, at a doctor's office, at home, at a location remote from a medical facility.

Certain embodiments provided herein include aspects disclosed in: U.S.62/782610 entitled "METHODS AND COMPOSITIONS TO REDUCE NONSPECIFIC AMPLIFICATION IN ISOTHERMAL AMPLIFICATION REACTIONS" filed on 20/12 months IN 2018; U.S.62/783104 entitled "HANDHELD IMPEDANCE-BASED DIAGNOSTIC TEST SYSTEM FOR DETECTING ANALYTES" filed 12, 20 and 2018; AND U.S.62/783051 entitled "METHODS AND COMPOSITIONS FOR DETECTION OF AMPLIFICATION OF PRODUCTS", filed on 20.12.2018, the entire contents OF which are each expressly incorporated by reference in their entirety. Certain embodiments provided herein also include aspects disclosed in u.s.2016/0097740, u.s.2016/0097741, u.s.2016/0097739, u.s.2016/0097742, u.s.2016/0130639, and u.s.2019/0232282, each of which is expressly incorporated by reference in its entirety.

Many commercially available nucleic acid detection platforms utilize conventional PCR, requiring temperature cycling, fluorescent labeling, and optical detection instrumentation. These factors result in expensive laboratory-based instruments that utilize delicate vibration-sensitive detectors, expensive fluorescent markers, and have large footprints. The device requires operation and frequent calibration by trained personnel.

These large, bulky platforms make the conventional use of traditional NATs difficult to use in the clinic, let alone at home. NAT remains an expensive and slow strategy closely associated with centralized laboratory facilities. In contrast, the techniques of this disclosure avoid these challenges.

A barrier to point-of-care ("POC") testing is the potential inhibition of amplification by interferents often encountered in crude, unprocessed clinical samples (e.g., whole blood or mucus). Mitigation of amplification inhibitors may challenge direct detection of target nucleic acids from clinically relevant biological samples.

Conventional detection strategies typically rely on fluorescence detection techniques. Such techniques can be complex, more expensive, and require sophisticated optical systems. However, the present disclosure generally relies on electrical detection systems. Such electrical detection systems may utilize microelectronics that consumes relatively low power and can be manufactured at reduced cost due to high volume manufacturing. Thus, electrical detection of genomic material can shift the development of the computer industry to biological assay sensing.

Existing electronic methods for monitoring amplification may require binding of electrochemically active labels to the surface or selective binding of amplified substances. However, when used in real-world clinical applications, these techniques often suffer from slow response times, biofouling of the binding surfaces or electrodes resulting in poor signal-to-noise ratios, and limitations on the lifetime and reliability of the device. While potentially enabling high sensitivity, the use of electrochemical or field effect transistor "FET" detection adds a level of complexity to the detection. This may result in a more expensive and less robust policy than is typically required by POC and other consumer applications. Therefore, an additional diagnostic device is clearly needed.

The platforms disclosed herein rely on the measurement of conductivity changes that occur during nucleic acid amplification. In summary, the number and mobility of charged molecules is altered during biochemical synthesis of DNA from nucleoside triphosphates. This in turn causes the solution conductivity to change as amplification progresses. Contactless conductivity detection using frequency dependent capacitive coupling ("fC)⁴D ") to sense such changes in solution conductivity.

In some embodiments, fC⁴D measuring the electrical properties of the solution using a pair of electrodes in close proximity to, but not in contact with, the fluid located in the amplification chamber. The ability to measure solution properties in this manner without direct contact avoids the challenges of surface contamination common to other electrical measurement methods.

In some embodiments, fC is utilized⁴D applies a high frequency alternating current ("AC") signal to the excitation electrode. The signal is capacitively coupled through the solution where it is detected at the signal electrode. By comparing the excitation signal to the signal at the signal electrode, the conductivity of the solution can be determined.

Based on fC, the method is known through high-resolution finite element model and empirical research⁴The specific tolerances of the technique of D may enable optimal detection sensitivity and dynamic sensing range for a particular implementation of the platform. Such calculated and empirically determined parameters of microfluidic dimensions, capacitive coupling characteristics and applied frequency enable the determination of effective parameters for detecting changes in solution conductivity. In some embodiments, the parameters corresponding to the best detection may be interdependent variables. The measured impedance is a function of solution resistance, capacitance, and applied frequency according to the following equation:

Z＝R-(1/pi*f*C)*j

As the thickness of the electrode passivation layer increases, the parasitic capacitance due to this layer increases. Thus, the capacitance relative to the passivation layer can be selected to pass fC⁴D measures the optimal AC frequency for the solution conductivity.

Overview of exemplary cartridges, readers, and Signal processing

In some aspects, a system for detecting a target in a sample includes a removable fluidic cartridge connectable to a companion reader device. The user may apply the sample to the cartridge and then insert it into the reader device. The reader device is configured to perform a test procedure using the cartridge and analyze the test data to determine the presence, absence, or amount of the target in the sample. For example, the cartridge may be provided with the desired reagents, proteins, or other chemicals for the amplification procedure by which the target originally present in the sample is amplified. In particular, some cartridges may be provided with desired chemicals for nucleic acid testing, as described herein, wherein genomic material in a sample is exponentially copied using a molecular amplification procedure. The cartridge may also include a test well for containing the amplification procedure, where a test well refers to a well, chamber, channel, or other geometry configured for containing (or substantially containing) components of the amplification procedure and the test fluid. The reader device may maintain a desired temperature or other test environment parameter of the cartridge to facilitate the amplification procedure, and may electronically monitor the test wells of the cartridge throughout some or all of the amplification procedures. Thus, the reader device can collect signal data representative of the impedance of the test well over time during the amplification procedure, and can analyze the impedance as described herein to determine the presence, absence, or amount of the target in the sample. As an example, the amplification procedure may be in the range of 5 minutes to 60 minutes, some examples may be in the range of 10 minutes to 30 minutes. Preferably, in some embodiments, the amplification product is detected when suspended in the fluid within the well or when moved in the fluid within the well such that the amplification product is not attached to or spaced apart from the well or immobilized or bound to a probe (the probe is bound to the well or a particle within the well). In other embodiments, amplification products are detected when they are attached to, or separated from (e.g., immobilized or bound to, a probe that is bound to) a well or a particle within a well.

Such a system may advantageously provide target detection that may be performed in a clinical setting or even at the home of the user, rather than requiring the sample to be sent to a laboratory for amplification and analysis. In a clinical setting, this can avoid the delay of conventional nucleic acid testing, thereby enabling a clinician to make a determination of a diagnosis within a typical schedule of a patient's office visit. In this regard, the disclosed system enables a clinician to plan a treatment for a patient during their first office visit, rather than requiring the clinician to wait hours or even days to retrieve test results from the laboratory. For example, when a patient visits a clinic for a visit, a nurse or other healthcare practitioner can collect a sample from the patient and initiate testing using the described system. The system may provide test results when a patient consults their physician or clinician to determine a treatment plan. The disclosed system may avoid delays associated with laboratory testing that may negatively impact patient treatment and outcome, particularly when used to diagnose rapidly progressing lesions.

As another benefit, the disclosed system may be used outside of a clinical setting (e.g., on-site, in a rural setting where access to an established medical clinic is not readily available) to detect a health condition such as an infectious disease (e.g., ebola virus) to enable appropriate personnel to take immediate action to prevent or mitigate the spread of the infectious disease. Similarly, the disclosed system may be used on-site or in locations with suspected hazardous contaminants (e.g., anthrax) to quickly determine whether a sample contains a hazardous contaminant, thereby enabling appropriate personnel to immediately take action to prevent or mitigate exposure of the person to the contaminant. In addition, the disclosed system can be used to detect contaminants in blood or plasma supplies or contaminants in the food industry. It will be appreciated that the disclosed system may provide similar benefits in other scenarios, where real-time detection of the target enables more efficient action than delayed detection via sending the sample to an off-site laboratory.

Another benefit of such systems is their use with low cost disposable single use cartridges and reusable reader devices that can be used multiple times with different cartridges and/or for testing of different targets.

Fig. 1A-1D depict an exemplary cartridge 100 configured for detection of a target. As described herein, the target can be a viral target, a bacterial target, an antigenic target, a parasitic target, a microRNA target, or an agricultural analyte. Some embodiments of the cartridge 100 may be configured for testing a single target, while some embodiments of the cartridge 100 may be configured for testing multiple targets.

Fig. 1A depicts the pod 100 having a lid 105, the lid 105 being disposed on a base 125 thereof. In use, the lid 105 may operate to enclose the provided sample within the cartridge 100, thereby preventing the test operator from being exposed to the sample and preventing any liquid from escaping into the electronics of the associated reader device. The cover 105 may be permanently secured to the base 125, or in some embodiments, the cover 105 may be removable. The cover 105 may be formed of a suitable material (e.g., plastic) and may be opaque as described, or may be translucent or transparent in other examples.

The lid 105 includes an aperture 115 located above the sample introduction region 120 of the base 125. As used herein, "above" means that the aperture 115 is located at an upper portion of the sample introduction region 120 when the cartridge 100 is viewed from a top-down perspective, normal to the planar surface of the lid 105 that includes the aperture 115. The lid 105 also includes a cap 110, the cap 110 being configured to fluidly close the aperture 115 before and after providing the sample through the aperture 115. The cap 110 includes a cylindrical protrusion 111, a release tab 113, and a hinge 112; when the cap 110 is closed with the aperture 115, the cylindrical protrusion 111 blocks the aperture 115; the release tab 113 is configured to assist a user in pulling the cap 110 out of the aperture 115 when the cap 110 is closed with the aperture 115; and the hinge 112 is configured to enable the cap 110 to be removed from the aperture 115 and out of the sample supply flow path (path) while maintaining the cap 110 secured to the cover 105. It will be appreciated that other variations in the shape of the cap 110 may be similarly used to achieve closure of the aperture 115, and in some embodiments, the hinge 112 and/or release tab 113 may be modified or omitted. In the illustrated embodiment, the cover 105 and the cap 110 are integrally formed as a single piece of material, however in other embodiments, the cap 110 may be a separate structure from the cover 105.

In use, a user opens the cap 110 and applies a sample, potentially containing a target, to the sample introduction region 120 of the base 125 through the aperture 115 in the cap. For example, a user may prick a finger and apply a whole blood sample to the sample introduction region 120 (e.g., via a capillary tube). The cartridge 100 may be configured to accept one or more liquid, semi-solid, and solid samples. After applying the sample, the user may close the cap 110 to close the aperture 115. Advantageously, closing the fluid flow channel inlet of the base 125 allows the sample (and other liquids) to move through the fluid flow channel of the base 125 to the test wells. For example, as described herein, a user may insert the closed cartridge 100 containing the sample into a reader device, and the reader device may activate an optional pneumatic interface for moving the sample to the test wells. The fluid flow channels and test wells are described in more detail with respect to fig. 1B and 1C, and an exemplary reader device is described with respect to fig. 6.

The cover 105 also includes a recess 130 for exposing an electrode interface 135 of the base 125, described in more detail below. In some embodiments, the cover 105 may include a removable tab or sheath for protecting the electrode interface 135 prior to use.

FIG. 1B depicts the cartridge 100 of FIG. 1A with the cover removed to expose features of the base 125. The base 125 may be formed of a fluid impermeable material, such as an injection molded or milled acrylic material or a plastic material. The base 125 includes a sample introduction region 120, a blister pack 140, a pneumatic interface 160, a test region 170A including a test well 175, and a fluid flow channel 150 configured for mixing an applied sample with a liquid contained in the blister pack 140 and for carrying the mixed liquid to the test well 175. It will be appreciated that the particular geometry or relative arrangement of these features may vary in other embodiments.

The blister pack 140 comprises a film (e.g., thermoformed plastic) forming an enclosed chamber containing a liquid for mixing with the applied sample. These liquids may include amplification reagents, buffer solutions, water, or other desired liquid components for the testing procedure. The particular selection and chemistry of these liquids can be tailored to the particular target or targets for which the cartridge 100 is designed for testing. Some embodiments of blister pack 140 may additionally include non-liquid compounds dissolved or suspended in the contained liquid. The blister pack 140 may be secured to the base 125, for example, within a fluid-tight chamber having a pneumatic fluid channel 161 leading into the chamber and an aperture 141 leading out of the chamber into the fluid channel 150. For example, a ring of pressure sensitive adhesive disposed along the outer edge of one or both surfaces of blister package 140 may be used to secure blister package 140 in place.

In use, a user or reader device may mechanically actuate a spike (e.g., a needle or other object having a sharp point) to pierce the blister package 140 and release its liquid contents through the aperture 141 and into the first section 151 of the fluid flow channel 150. A spike may be incorporated into the cartridge 100, for example in a chamber containing a blister pack 140, the blister pack 140 having a chamber in fluid communication with the first section 151 of the fluid flow channel. As used herein, fluid communication refers to the ability to transfer fluids (e.g., liquids, gases). In another embodiment, a user or reader device may press a lower surface of blister package 140 (which, although not illustrated, is opposite the surface visible in fig. 1B) to push it up into the prongs and pierce blister package 140. In other embodiments, the spike may be omitted and the blister package 140 may be compressed by a user or reader device until the pressure of its liquid contents causes the blister package 140 to rupture. Although described as a rupturable blister package, other embodiments may implement a mechanically openable chamber configured to similarly release the contained liquid into the first section 151 of the fluid channel 150.

As described above, after applying the sample, the user closes the aperture 115 of the cap, thereby closing the fluid flow channel 150 within the cartridge 100. The pneumatic interface 160 is configured to provide a fluid or medium (e.g., air) through the blister pack chamber into the enclosed fluid flow channel 150 to facilitate fluid flow in a desired direction along the fluid flow channel 150 to the test wells 175. The pneumatic interface 160 may be an aperture that opens into and is in fluid communication with a pneumatic fluid channel 161, which in turn opens into and is in fluid communication with the blister pack 140 or a chamber containing the blister pack 140. In some embodiments, the pneumatic interface 160 may be a compressible one-way valve that pushes ambient air into the pneumatic fluid flow channel 161 when compressed and absorbs ambient air from its environment when depressurized. In such an embodiment, repeated compression of the pneumatic interface 160 may push the fluid in the cartridge along the fluid flow path.

Fluid flow channel 150 includes sections 151, 152, 153, 154, 155, and 156, as well as sample introduction region 120, test well 175, test well inlet flow channel 176, and test well outlet flow channel 177. The first section 151 of the fluid flow channel 150 leads from the blister pack 140 to the sample introduction region 120. A second section 152 of the fluid flow channel 150 leads from the sample introduction region 120 to a mixing chamber 153. The mixing chamber 153 is a third section of the fluid flow channel 150 and widens with respect to the second section 152 and the fourth section 154. The fourth section 154 of the fluid flow passage 150 leads from the mixing chamber 153 to the fifth section 155 of the fluid flow passage. A fifth section 155 of the fluid flow channel 150 is formed in the testing region 170A. The fifth section 155 of the fluid flow channel 150 opens into the first test well inlet flow channel 176 and the sixth section 156 of the fluid flow channel 150. The sixth sections 156 of the fluid flow channels 150 each form a continuation of the fluid flow channel 150 between adjacent test well inlets up to the last test well inlet 176. Test well inlet flow channel 176 fluidly connects test well 175 to fluid flow channel 150 and may be closed by valve 174 (e.g., to prevent cross amplification between test wells). Test well outlet flow channel 177 leads from test well 175 to outlet aperture 178, outlet aperture 178 allowing gas to escape from test well 175 and exit cartridge 100.

In some embodiments, uniform or homogeneous mixing of the liquid from the blister pack 140 with the applied sample may produce more accurate test results. In this regard, the mixing chamber 153 is configured to promote uniform mixing of the liquid from the blister pack 140 with the applied sample, for example, by including a cross-sectional shape and/or curved regions that promote turbulent flow rather than laminar flow of the liquid within the mixing chamber 153. Turbulence is a flow state in fluid dynamics that is characterized by chaotic changes in the pressure and flow rate of a fluid. Turbulent flow is in contrast to laminar flow, which occurs when a fluid flows in parallel layers without disruption between the layers.

The sections 151, 152, 153, 154 of the fluid flow channel 150 may be completely enclosed within the material of the base 125, or may have three surfaces formed by the material of the base 125, and the cover 105 forms an upper surface that closes off these channels. The sections 155, 156 of the fluid flow channel 150, as well as the test well inlet flow channel 176 and the test well outlet flow channel 177, may be completely enclosed within the material of the base 125, may have three surfaces formed by the material of the base 125, and the cover 105 forms an upper surface that encloses these features, or may have two surfaces formed by the material of the base 125, and the circuit board 179 forms a lower surface of these features and the cover 105 forms an upper surface of these features.

Fig. 1C illustrates a flow direction along the fluid flow channel 150 with circled numbers shown as labels at certain points along the fluid flow channel. Circled numbers are discussed below as example steps of progression of fluid 180 as it travels through fluid flow channels 150 within cartridge 100, each step including a directional arrow showing the direction of fluid travel at that step.

Prior to step (1), the user applies the sample at the sample introduction area 120. For clarity and brevity of FIG. 1C, components labeled with reference numbers in FIG. 1B are not labeled in FIG. 1C. Also prior to step (1), the blister pack 140 is ruptured to allow its liquid contents to be released from its previously closed chamber.

In step (1), air or other fluid flowing from the pneumatic interface 160 travels along the pneumatic fluid flow path 161 in the direction shown to the ruptured blister pack 140.

In step (2), the liquid released from the ruptured blister pack 140 (referred to herein as the "reaction premix") travels through the aperture 141 in the direction shown and into the first section 151 of the fluid flow channel 150. The reaction premix continues to flow along the first section 151 until step (3) as it enters the sample introduction region 120 and begins to carry the sample on its own further along the fluid flow path.

In step (4), the reaction premix and sample exit the sample introduction region 120 and flow along the second section 152 of the fluid flow channel 150 in the direction shown. The volume of the reaction premix may be preselected to completely flush or substantially completely flush the applied sample from the sample introduction region 120 and/or to at least fill the test well 175 and its corresponding inlet flow channel 176.

In step (5), the reaction premix and sample flow into the inlet of the wider third section 153 of the fluid flow channel 150 in the direction shown, and in step (6), the reaction premix and sample are mixed into a uniform solution, wherein the sample is uniformly distributed throughout the reaction premix. As described above, the third section 153 includes a planar mixing chamber and a curved section configured to facilitate mixing of the reaction premix and the sample. In some embodiments, the fluid velocity defined by the pneumatic interface 160 may be selected to further promote this mixing.

In step (7), the mixed reaction premix and sample (referred to as the "test fluid") exits the mixing chamber 153 and enters the fourth section 154 of the fluid flow channel 150 (which leads to the test zone 170A).

In step (8), the test fluid travels along the fifth section 155 of the fluid flow channel 150 in the direction shown, through the test region 170A to the test aperture 175.

In step (9), the test fluid reaches the first test well inlet flow channel 176 and its flow is directed along three possible flow channels shown trifurcated from the arrow of the fluid flow channel of step (9).

The flow path of step (10) shows the test fluid flowing further along section 156 of fluid flow path 150 to the subsequent test well inlet flow path 176. Optionally, valve 174 at test port inlet flow passage 176 may be closed, preventing the flow of test fluid to step (10).

The flow path of step (11) illustrates the optional flow of the gaseous portion of the test fluid through valve 174. In some embodiments, valve 174 may include a liquid impermeable, gas permeable filter to allow any gas present in the test fluid to vent through valve 174 before entering test well 175. In some embodiments, valve 174 may not be configured to vent gas.

The flow path of step (12) shows the direction of flow of the test fluid into test hole 175. In some embodiments, valve 174 may be closed to close test well 175 when a predetermined trigger occurs. Triggering may occur after a predetermined volume of liquid corresponding to at least the volume of the test well 175 (and the additional inlet and outlet flow channels 176, 177) has flowed along the flow channel of step (12). Another example of valve closure triggering may occur after a predetermined amount of time has elapsed, which corresponds to the time that the volume of liquid is expected to flow along the flow path of step (12). In another embodiment, the trigger may be the deactivation of the pneumatic interface 160, at which point the fluid may begin to flow in a reverse direction along the flow path as shown, resulting in cross-contamination of the amplification process occurring in the different test wells. In some embodiments, the depicted position of valve 174 may instead be a gas outlet aperture, optionally covered with a liquid impermeable, gas permeable filter, and the depicted valve may be disposed along test well inlet flow channel 176 or along fluid flow channel section 156.

The flow channels of step (13) illustrate the direction of the test fluid or its gaseous components exiting the test aperture 175 through the outlet flow channels 177. The outlet flow channels 177 may be channels leading from the test holes 175 to the outside, and the test fluid may be pushed into the outlet flow channels 177 by the pressure provided by the pneumatic interface 160. In some embodiments, a liquid impermeable, gas permeable filter can be provided at the interface of the test well 175 and the outlet flow passage 177, such that only the gaseous component of the test fluid flows through the outlet flow passage 177.

At step (14), gas from the test fluid is exhausted from the cartridge 100 through the outlet aperture 178. The exit aperture 178 may be covered by a liquid impermeable, gas permeable filter to allow gas to escape the cartridge 100 and prevent liquid from escaping the cartridge 100. Advantageously, allowing and facilitating the venting of gas from the test fluid minimizes the amount of gas remaining in the test wells, maximizing the amount of liquid in the test wells. Minimizing the likelihood of bubble formation in the flow channel between the electrodes advantageously results in a more reliable signal and more accurate test results, as described below.

Returning to fig. 1B, test area 170A includes section 155 and section 156 of fluid flow channel 150, test well 175, test well inlet flow channel 176, test well outlet flow channel 177, aperture/valve 176, aperture/valve 178, and circuit board 179. Circuit board 179 includes electrodes 171A and 171B of the test well, conductors 172 for carrying electrical current or other electrical signals, and electrode interface 135. The electrode interface 135 includes a contact plate 173, one half of the contact plate 173 being configured for connecting the excitation electrodes of the test wells with a voltage or current source of the reader device, and the other half of the contact plate 173 being configured for electrically connecting the signal electrodes of the test wells with signal reading conductors of the test device. For clarity of fig. 1B, only some of the repeating features of test area 170A are labeled with reference numbers.

The circuit board 179 may be a printed circuit board, such as a screen printed circuit board or a screen printed circuit board having multiple layers. The circuit board 179 may be printed onto a flexible plastic substrate or a semiconductor substrate. Circuit board 179 may be formed at least in part from a material separate from base 125 and secured to the underside of base 125, while overlying region 126 of base 125 includes section 155 and section 156 of fluid flow channel 150, test wells 175, test well inlets 176, test well outlets 178, and apertures/valves 176, 178. For example, circuit board 179 can be an acrylic multilayer printed circuit board that is adhered, secured, or laminated to overlying region 126. The electrode interface 135 may extend beyond the edge of the overlying region 126. Test hole 175 may be formed as an opening in the material overlying region 126, such that electrodes 171A, 171B of circuit board 179 are exposed in hole 175. In this regard, the electrodes 171A, 171B may be in direct contact with the fluid flowing into the bore 175. The circuit board 179 may be greased by having resin on its upper surface to create a smooth flat surface at the bottom of the test well.

Solid dry components of the test procedure, such as primers and proteins, may be provided to test wells 175. The particular selection and chemistry of these dry ingredients can be tailored to the particular target or targets for which cartridge 100 is designed to test. The same or different dry ingredients may be provided to test wells 175. These dry ingredients may be hydrated with a liquid that flows into the test wells (e.g., liquid from blister pack 140 that mixes with the applied sample) so that they are activated for the testing procedure. Advantageously, providing the liquid component in blister pack 140 and the dry solid component in test wells 175 separately enables the cartridge 100 to be stored prior to use to contain the components required for the amplification procedure, while also delaying initiation of amplification until after application of the sample.

Test holes 175 are depicted as circular holes arranged in two rows at staggered distances from electrode interface 135. Test hole 175 may be generally cylindrical (e.g., formed as a circular opening in the material of overlying region 126) and bounded by planar surfaces of its upper side (e.g., cover 105 or a portion of overlying region 126) and lower side (e.g., circuit board 179). Each test well 175 contains two electrodes 171A and 171B, one of which is an excitation electrode configured to apply a current to the sample in the test well 175, and the other of which is a signal electrode configured to detect a current flowing from the excitation electrode through the liquid sample. In some embodiments, a thermistor may be provided to one or more test wells in place of an electrode to provide monitoring of the temperature of the fluid within cartridge 100.

Each test well may be monitored independently of the other test wells so that each test well may constitute a different test. Depicted electrodes 171A and 171B in each test well are linear electrodes placed parallel to each other. The described arrangement of test wells 175 provides a compact test area 170A with access to each test well 175 from the fluid flow channels 150. Some embodiments may include only a single test well, and various embodiments may include more than two test wells arranged in other configurations. Furthermore, the shape of the test wells may vary in other embodiments, and the electrode shape may be any of the electrodes shown in fig. 4A-4G.

In some embodiments, bubbles within test well 175 (particularly if the bubbles are located along the current flow path between electrode 171A and electrode 171B) can generate noise in the signal picked up by the signal electrodes. This noise may reduce the accuracy of test results determined based on signals from the signal electrodes. The desired high quality signal can be obtained when only liquid is present along the current flow path, or when there are minimal bubbles along the current flow path. As described above, any air that is initially present in the fluid flowing along the fluid flow passage 150 may be pushed out through the exit apertures 178. In addition, electrodes 171A, 171B, and/or test wells 175 can be shaped to mitigate or prevent the buildup of liquid samples, where air or gas bubbles form in the liquid sample and collect along the electrodes.

For example, electrode 171A and electrode 171B are located at the bottom of test well 175. This may allow any air or gas to rise to the top of the fluid in the test well and away from the flow channel between the electrodes. As used herein, the bottom of a test well refers to the portion of the test well in which heavier liquids settle due to gravity, and the top of the test well refers to the portion of the test well in which lighter gases rise above the heavier liquids. In addition, electrodes 171A and 171B are positioned away from the periphery or edge of test well 175 where bubble nucleation typically occurs.

In addition, electrodes 171A and 171B may be formed from a thin, flat layer of material having a minimum height relative to the lower circuit board layer that forms the bottom of the test well. In some embodiments, electrodes 171A and 171B may be formed using electrodeposition and patterning to form a thin layer of metal film (e.g., about 300nm in height). This minimum height may help prevent or mitigate entrapment of gas bubbles along the interface between the electrode and the underlying layer. In some embodiments, a layer of conductive material may be deposited on top of each electrode to create a smoother transition between the edges of the electrodes and the bottom of the test well. For example, a thin polyimide layer (e.g., about 5 microns in height) may be deposited on top of the electrodes, or the circuit board may be butter coated. Additionally or alternatively, the electrode may be located in a trench in the underlying layer, the trench having a depth approximately equal to the height of the electrode. These and other suitable methods may result in an electrode that is substantially flat or flush with the bottom surface of the hole.

Advantageously, the above-described features may help to keep electrodes 171A and 171B surrounded by liquid and prevent or reduce bubbles from being located along the current flow path between electrodes 171A and 171B.

Fig. 1D is a line drawing depicting a top plan view of the testing region 170B of the cartridge 100. As with fig. 1B, certain repeated features are labeled with reference numbers in only one location for the sake of brevity and clarity of the drawing of fig. 1D.

Test zone 170B is an alternate embodiment of test zone 170A, the difference between the two embodiments being a different electrode configuration within test well 175. In an embodiment of test zone 170B, test wells are provided with ring electrode 171C and ring electrode 171D. Any of linear electrodes 171A and 171B of test area 170A may be either an excitation electrode or a signal electrode. In an embodiment of test region 170B, inner electrode 171D is an excitation electrode and outer electrode 171C is a signal electrode.

The inner electrode 171D may be a disk-shaped or circular electrode connected to a current supply conductor 172B, which in turn is connected to a current supply plate 173 of the electrode interface 135, which transmits a current (e.g., an AC current at a specified frequency) from the reader device to the inner electrode 171D. Inner electrode 171D may be positioned in the center of test well 175. The outer electrode 171C is a semicircular electrode formed concentrically around the inner electrode 171D and spaced apart from the inner electrode 171D by a gap. The semicircular body of the outer electrode 171C is broken where the conductive wire connects the inner electrode 171D to the current-supply conductor 172B. The outer electrode 171C is connected to a current sensing conductor 172B, which in turn is connected to a current sensing plate 173 of the electrode interface 135, which transmits the sensed current to the reader device.

The cartridge 100 of fig. 1A-1D provides a stand-alone, easy-to-use device for performing amplification-based tests on a target, such as nucleic acid testing in which genomic material in a sample is copied exponentially using a molecular amplification procedure. Advantageously, since the liquid and solid components of the amplification procedure are provided in the cartridge in advance and automatically mixed with the sample, the user need only apply the sample and insert the cartridge 100 into the reader device to determine the test results in some embodiments. In some embodiments, one or both of the cartridge or the reader may include a heater and a controller configured to operate the heater to maintain the cartridge at a desired temperature for amplification. In some embodiments, one or both of the cartridge or the reader may include a motor to impart vibration or agitation to the cartridge, causing any entrapped gas to rise to the top of the liquid and be expelled from the test wells.

Fig. 2 depicts a photograph of another exemplary cassette 200 configured for detecting a target. Cartridge 200 is used to generate some of the test data described herein and represents an alternative configuration to some of the components described with respect to cartridge 100.

The cartridge 200 includes a printed circuit board layer 205 and an acrylic layer 210, the acrylic layer 210 being covered and adhered to a portion of the printed circuit board layer 205 using a pressure sensitive adhesive. Acrylic layer 210 includes a plurality of test holes 215A and a plurality of temperature monitor holes 215B formed as circular apertures extending through the height of acrylic layer 210. The printed circuit board layer 205 may be formed similarly to the circuit board 179 described above and includes a pair of electrodes 220 located within each test aperture 215A and a thermistor 225 located within each temperature monitor aperture 215B. The electrodes 220 and thermistor 225 are each connected to conductors that terminate at a plurality of leads 230 of the printed circuit board. As shown, for the signal electrodes, 6 wires are labeled "SIG" followed by the numbers 1-6; for the excitation electrodes, 6 wires are labeled "EXC" followed by the numbers 1-6; and for thermistors, 2 wires are labeled RT1 and RT 2.

During some of the tests described herein, the following exemplary protocol was followed. First, the user fills the hole 215A with the test fluid and caps the fluid with mineral oil. The test fluid may have no primer control, given the clear negative control in the absence of the primer that causes amplification.

Next, the user heats the cartridge 200 to 65 degrees celsius for 10 minutes to expand any entrapped air in the test fluid and cause it to rise as bubbles to the top of the liquid. During this initial heating, bubbles are formed in the holes 215A.

In the next step, the user uses a pipette or other tool to scrape a bubble from the surface of the liquid in the hole 215A. As described above, elimination of air bubbles may facilitate more accurate test results.

After eliminating the air bubbles, the user allows the cartridge 200 to cool to room temperature. Next, the user injects a loop-mediated isothermal amplification (LAMP) Positive Control (PC) into the bottom of each test well 215A, places the cartridge 200 on the heating block, and starts to perform the LAMP test. The signals detected from the signal electrodes are analyzed to identify positive signal cliffs, as described herein.

Fig. 3A and 3B depict another example cartridge 300 configured for detecting a target. Fig. 3A depicts a top, front, and left side perspective view of the cartridge 300, and fig. 3B depicts a perspective cutaway view showing the outline of the aperture 320 of the cartridge 300. The cartridge 300 represents an alternative configuration to some of the components described with respect to the cartridge 100.

Cartridge 300 includes a sample introduction region 305, a central channel 310, test wells 320, branches 315 that fluidly connect the test wells 320 to the central channel 310, electrodes 325A and 325B located within each test well 320, and an electrode interface 320 that includes a contact plate connected to a conductor (the contact plate in turn is connected to a respective one of the electrodes 325A, 325B and is configured to receive signals from or transmit signals to a reader device). As shown in fig. 3B, the holes 320 may have curved bottom surfaces such that each hole is generally hemispherical. The cartridge 300 is described as having an open top so as to expose its internal components, however, in use a lid or other upper layer may be provided to enclose the fluid flow paths of the cartridge 300. The lid may include a vent to allow gas to escape from the cartridge 300, for example provided with a liquid impermeable, gas permeable filter as described above with respect to fig. 1A-1D.

The fluid sample applied at the sample introduction region 305 flows down the central channel 310 (e.g., in response to pressure from a reader device that injects the sample into the cartridge 300 through a port connected above the sample introduction region 305). In some embodiments, such a reader device may be provided with a set of cartridges (e.g., stacked), and each cartridge may be provided with the same or different samples. The fluid sample may be primarily a liquid with dissolved or entrapped gas (e.g., bubbles). Fluid may flow from the central channel 310 through the branch channels 315 into the test wells 320. The branch channel 315 may enter into the top of the well and may be curved (e.g., include multiple turns with small radii) to prevent or mitigate reverse flow of fluid that may lead to cross-contamination of amplification procedures between wells.

Fig. 4A-4G depict various examples of electrode configurations that may be used in the test wells of the cartridges of fig. 1A-3B or fig. 51A-53B or in the test wells or channels of another suitable target detection cartridge described herein. The test wells shown in fig. 4A-4G are depicted as circular, but in other examples, electrodes may be used in test wells of other geometries. Unless otherwise noted, the filled circles in fig. 4A-4G indicate contact between the disclosed electrodes and conductors leading to or from the electrodes. "width" as used hereinafter refers to the dimension along the horizontal direction of the page of FIGS. 4A-4G, while "height" as used hereinafter refers to the dimension along the vertical direction of the page of FIGS. 4A-4G. Although depicted in a particular orientation, in other embodiments, the electrodes shown in fig. 4A-4G may be rotated. Further, the dimensions of the disclosed examples represent some possible implementations of the electrode configurations 400A-400G, and variations may have different dimensions that follow the same proportions between the dimensions of the examples provided. The electrodes shown in fig. 4A-4G may be made of suitable materials including platinum, gold, steel, or tin. Tin and platinum behave similarly in experimental tests and are suitable for certain test setups and test targets.

Fig. 4A depicts a first electrode configuration 400A in which a first electrode 405A and a second electrode 405B are each formed as semicircular edges. The straight edges of the first electrode 405A are positioned adjacent to the straight edges of the second electrode 405B and are separated by a gap along the width of the construction 400A. The gap is larger than the radius of the semicircular body of the electrode. Thus, the first electrode 405A and the second electrode 405B are positioned to mirror the edges of a semicircle. In one example of the first electrode configuration 400A, the gap between the proximal portions of the first and second electrodes 405A, 405B spans about 26.369mm, the height (along a straight edge) of each of the electrodes 405A, 405B is about 25.399mm, and the radius of the semicircular body of each of the electrodes 405A, 405B is about 12.703 mm.

Fig. 4B depicts a second electrode configuration 400B. Similar to the first electrode configuration 400A, the first and second electrodes 410A, 410B of the second electrode configuration 400B are each formed as semicircular edges and are positioned as congruent semicircular bodies with their straight sides facing each other. The first and second electrodes 410A, 410B of the second electrode configuration 400B may be the same size as the first and second electrodes 405A, 405B of the first configuration 400A. In the second electrode configuration 400B, the gap between the first electrode 410A and the second electrode 410B along the width of the configuration 400B is smaller than in the first configuration 400A, and the gap is smaller than the radius of the semicircular bodies of the electrodes 410A and 410B. In one example of the second electrode configuration 400B, the gap between the closest portions of the first and second electrodes 410A, 410B spans about 10.158mm, the height (along a straight edge) of each of the electrodes 410A, 410B is about 25.399mm, and the radius of the semicircular body of each of the electrodes 410A, 410B is about 12.703 mm.

Fig. 4C depicts a third electrode configuration 400C having a first linear electrode 415A and a second linear electrode 415B separated by a gap along the width of the configuration 400C, wherein the gap is approximately equal to the height of the electrodes 415A and 415B. The width of the electrodes 415A and 415B is about one-half to one-third of the height of the electrodes. In one example of the third electrode configuration 400C, the gap between the proximal portions of the first and second electrodes 415A, 415B spans about 25.399mm, the height of each of the electrodes 415A, 415B is also about 25.399mm, and the width of each of the electrodes 415A, 415B is about 10.158 mm. The ends of the first and second electrodes 415A, 415B may be rounded, for example, having a radius of about 5.078 mm.

Fig. 4D depicts a fourth electrode configuration 400D having a first rectangular electrode 420A and a second rectangular electrode 420B separated by a gap along the width of configuration 400D, wherein the gap is approximately equal to the width of electrodes 420A and 420B. In one example of the fourth electrode configuration 400D, the gap between the proximal portions of the first and second electrodes 420A, 420B spans about 20.325mm, the height of each of the electrodes 420A, 420B is also about 23.496mm, and the width of each of the electrodes 420A, 420B is about 17.777 mm.

Fig. 4E depicts a fifth electrode configuration 400E having a first linear electrode 425A and a second linear electrode 425B separated by a gap along the width of configuration 400E, wherein the gap is approximately equal to the height of electrodes 425A and 425B. The fifth electrode configuration 400E is similar to the third electrode configuration 400C in that the width of electrodes 425A and 425B is reduced to about one-half to two-thirds of the width of electrodes 415A and 415B while having the same height. In one example of the fifth electrode configuration 400E, the gap between the proximal portions of the first and second electrodes 425A, 425B spans about 25.399mm, the height of each of the electrodes 425A, 425B is also about 25.399mm, and the width of each of the electrodes 425A, 425B is about 5.078 mm. The ends of the first electrode 425A and the second electrode 425B may be rounded, for example, having a radius of about 2.542 mm.

Fig. 4F depicts a sixth electrode configuration 400F having a concentric ring electrode 430A and a concentric ring electrode 430B. Sixth electrode configuration 400F is the configuration shown in test well 175 of fig. 1D. The inner electrode 430B may be a disk-shaped electrode or a circular electrode, and may be located at the center of the test hole. The outer electrode 430A may be a semicircular electrode formed concentrically around the inner electrode 430B and separated from the inner electrode 430B by a gap. In the sixth electrode configuration 400F, the gap is approximately equal to the radius of the inner electrode 430B. The breaking of the semicircular body of the outer electrode 430A occurs where the conductive wire connects the inner electrode 430B to the current-providing conductor. In one example of the sixth electrode configuration 400F, the gap between the inner edge of the annular first electrode 430A and the outer edge of the circular second electrode 430B spans about 11.430mm, the radius of the circular second electrode 430B is about 17.777mm, and the thickness of the ring of the annular first electrode 430A is about 5.080 mm. The ends of the first electrode 430A may be rounded (e.g., having a radius of about 2.555 mm), and the gap between the open ends of the rings of the first electrode 435A may be about 28.886mm from apex to apex.

Fig. 4G depicts a seventh electrode configuration 400G having a concentric ring electrode 435A and a concentric ring electrode 435B. Similar to the embodiment of fig. 4F, inner electrode 435B may be a disk-shaped electrode or a circular electrode having the same radius as inner electrode 430B and may be located at the center of the test well. The outer electrode 435A may be a semicircular electrode formed concentrically around the inner electrode 435A and separated from the inner electrode 435A by a gap. In the seventh electrode configuration 400G, the gap is larger than the radius of the inner electrode 435B, e.g., 2 to 3 times larger. Accordingly, the outer electrode 435B has a larger radius than the outer electrode 430B. In one example of the seventh electrode configuration 400G, the gap between the inner edge of the annular first electrode 435A and the outer edge of the circular second electrode 435B spans about 24.131mm, the radius of the circular second electrode 435B is about 17.777mm, and the thickness of the ring of the annular first electrode 435A is about 5.080 mm. The ends of the first electrode 435A may be rounded, for example, having a radius of about 2.555mm, and the gap between the open ends of the rings of the first electrode 435A may be about 46.846mm from apex to apex.

In the embodiments of fig. 4A-4E, either electrode may be used as the excitation electrode and the other electrode may be used as the signal electrode. In the embodiments of fig. 4F and 4G, inner electrode 430B and inner electrode 435B are configured to function as actuation electrodes (e.g., connected to a current source), and outer electrode 430A and outer electrode 435A are configured to function as signal electrodes (e.g., provide their signals to a memory or processor). In some example tests, the sixth electrode configuration 400F exhibited the best performance of the configurations shown in fig. 4A-4G.

Fig. 5A depicts a first electrode (or excitation electrode) and a second electrode (or signal electrode) that may be spaced apart from each other within the test well of the cartridges of fig. 1A-3B or fig. 51A-53B, or within the test well or channel of another suitable target detection cartridge described herein.

Formation of aggregates, nucleic acid complexes, or polymers (e.g., during an amplification procedure within a test well of the cartridge of fig. 1A-3B or fig. 51A-53B) can affect waveform characteristics of one or more electrical signals sent through the channel. As shown in FIG. 5A, a first or excitation electrode 510A is spaced apart from a second or sense electrode 510B within the test well 505. The test wells 505 may contain a test solution that is undergoing an amplification procedure. In some of all such procedures, stimulation voltage 515 can be provided to stimulation electrode 510A, with stimulation voltage 515 being transmitted from stimulation electrode 510A into the fluid (preferably all or substantially all of the liquid) within well 505.

After passing through and being attenuated by the liquid sample (schematically represented by resistance R and reactance X), the attenuated excitation voltage is sensed or detected at sensing electrode 510B. The fluid acts as a resistor R in series with the excitation electrode 510A and the sensing electrode 510B. The fluid also acts as a series capacitor, represented by reactance X. Similar to that shown in graph 520, the raw sense signal over a portion of the duration or all of the duration of the test may be represented over time as a sinusoid with varying amplitude.

The excitation voltage 515 may be an alternating current at a predetermined drive frequency. The particular frequency selected may depend, for example, on the particular target sought to be detected, the medium of the test sample, the chemical composition of the amplification procedure components, the temperature and/or excitation voltage of the amplification procedure. In some embodiments of the cartridge of FIGS. 1A-3B or 51A-53B, the excitation drive frequency may be between 1kHz and 10kHz at as low an excitation voltage as possible. As an example, in order to identify haemophilus influenzae (h. influenza) (10) incorporated in 5% whole blood⁶Copy/reaction), the excitation sensor drive frequency was varied from 100Hz to 100,000Hz at 0.15 volts. These tests show that the desired "signal cliff" (an artifact in the portion of the signal indicative of a positive test sample described in more detail below) becomes more easily detected below 100Hz, and is most easily detected between 1kHz and 10 kHz. Furthermore, with frequencies in the range between 1kHz and 10kHz, the signal cliff may advantageously pass byThe 12 minute test time was identified before. Advantageously, faster identification of signal cliffs may result in shorter test times, thereby resulting in faster test results being provided and more tests being able to be performed per day. At frequencies below 1kHz, the reactance component of the signal (where the signal cliff may be found in a positive sample) decreases monotonically. For other tests, the sensor drive frequency may similarly be fine-tuned to optimize performance, i.e., to optimize detectability of the signal cliffs. Detectability of a signal cliff refers to the ability to consistently distinguish between positive and negative samples.

FIG. 5B depicts an example graph 525 showing an impedance signal 530 that may be extracted from the raw signal 520 provided by the sense electrode 510B. The impedance signal 530 represents the electrical impedance Z of the test well over time. The impedance Z can be expressed by the cartesian complex equation as follows:

Z＝R+jX

where R represents the resistance of the test well and is the real part of the equation above, and X represents the reactance of the test well and is the imaginary part of the equation above (noted j). Thus, the impedance of the test well can be decomposed into two components, namely a resistance R and a reactance X.

Initially, the value of resistance R can be determined by making a baseline measurement of the test well prior to or at the beginning of the amplification procedure. Although the resistance of the test fluid may deviate from this baseline value throughout the test, the current sensed through sensing electrode 510B due to the resistance of the test fluid may be in phase with the signal provided through excitation electrode 510A. Thus, a change or deviation in resistance may be identified by the value of the in-phase component of the time-varying signal 520. The reactance may result from one or both of an inductive effect in the test fluid, a capacitive effect in the test fluid, which may cause the fluid to temporarily retain current (e.g., electrons provided by the excitation electrode 510A). After a period of time, the retained current flows out of the test fluid into the sensing electrode 510B. Due to this delay, the current sensed by the sense electrode 510B due to the reactance of the test fluid may be out of phase with the current sensed from the resistance of the test fluid. Thus, the reactance value of the test fluid may be identified by the value of the out-of-phase component of the signal 520 over time. Based on the change in the chemical composition of the test fluid caused by the amplification procedure, the reactance may fluctuate throughout the test period. A signal cliff indicative of a positive sample may be found in the reactance X (e.g., the reactance increases or decreases at or above a threshold rate or threshold amplitude, and/or the reactance increases or decreases within a predetermined time window).

During testing, excitation electrode 510A may be sinusoidally excited at some amplitude and voltage. Excitation electrode 510A is in series with the test liquid in the well (which can be considered a resistor R). The resistor (e.g., test fluid) and the electrodes form a voltage divider having a voltage determined by the ratio of the resistor and electrode chemistry/impedance. The resulting voltage waveform sensed at sensing electrode 510B represents the complex impedance signal 530. In some implementations, a curve of the impedance signal 530, for example, may not be generated, but rather the raw sense signal 520 may be decomposed into its resistive and reactive components, as described herein. The impedance signal 530 is provided as an example representative of a combined curve representing the resistance of the test fluid and the reactance of the test fluid over time. The complex impedance signal 530 may be interpreted as a quadrature modulated waveform (e.g., a combination of an in-phase waveform resulting from the resistance of the test fluid and an out-of-phase waveform resulting from the reactance of the test fluid), where the in-phase and out-of-phase components vary on a time scale much larger than the modulation frequency. The in-phase waveform is in phase with the complex waveform of the complex impedance. Some embodiments may use a synchronous detector, e.g., with multipliers and low pass filters implemented in a Field Programmable Gate Array (FPGA), to extract the in-phase and out-of-phase components from the raw signal 520 and calculate their amplitudes and phases.

To decompose the impedance signal 530 (or the raw sense signal 520) into resistive and reactive components as its constituent elements, the voltage waveform 520 at the sense electrode 510B is acquired at a frequency faster than its Nyquist frequency (e.g., twice the highest frequency of the excitation voltage) and then decomposed into an in-phase component (resistance) and an out-of-phase component (reactance). The in-phase and out-of-phase voltage components can be accounted for using known series resistances (e.g., R values) to compute the real part of the impedance (resistance) and the imaginary part of the impedance (reactance).

Fig. 5C depicts a graph 541 of the resistive component 540A and the reactive component 540B extracted from the raw signal 520 generated based on an exemplary positive test over time (t 3 minutes to t 45 minutes). As shown, signal cliff 545 indicates a particular time window T_WChange Δ of reactance 540B during_R. A signal cliff 545 indicates a positive sample. The reactance curve 540B is relatively flat or stable before the signal cliff 545 occurs, and the reactance curve 540B is again relatively flat or stable after the signal cliff 545. Thus, in this embodiment, a signal cliff 545 for a particular test parameter represented by graph 541 occurs a in expected area 535 _RIs reduced.

Change delta in reactance corresponding to positive sample signal cliff 545_RAnd a particular time window T for which signal cliff 545 is expected to occur_WMay vary depending on a number of parameters of the test. These parameters include the particular target being tested (e.g., the rate at which the target is amplified), the frequency of the excitation voltage, the configuration of the excitation and sensor electrodes (e.g., their respective shapes and dimensions, the gap separating the electrodes, and the material of the electrodes), the rate of collection, the amount of amplification agent provided at the beginning of the test, the temperature of the amplification procedure, and the amount of target present in the sample. In some embodiments, the expected characteristics of the signal cliff of a positive sample (e.g., predetermined by an experiment) can be used to distinguish between a positive sample and a negative sample. In some embodiments, the expected characteristics of a signaler cliff may be used to determine the severity or progression of a medical condition, for example, by correlation between particular signaler cliff characteristics and particular initial amounts of target in a sample. The predetermined expected characteristics may be provided, stored, and then retrieved during determination of the test results by a reader device configured to receive signals from the sensing electrodes of the test cartridge.

For a given test, the electrical potential of the signal cliff 545 for a positive sample can be determined experimentally based on monitoring and analyzing the reactance curve generated by the positive control sample (and optionally, the negative control sample)Change of resistance Δ_RAnd an expected time window T_W. In some embodiments, test parameters affecting a cliff may be altered and fine-tuned to identify parameters corresponding to precisely distinguishable cliffs. Readers and cartridges as described herein may be configured to match the configuration being tested and provide the reader and cartridge with the expected signal cliff characteristics for the test.

For example, in one set of experimental tests for haemophilus influenzae, the test fluid initially included amplification primers and 1,000,000 added copies of the target, the excitation voltage was 200mV P2P, the test parameters included a 10kHz sweep start and 10MHz sweep stop for the frequency of the excitation current, and the proximal and distal electrode gaps were configured to be 2.55mm and 5mm, respectively. The amplification temperature was set to 65.5 degrees celsius and the two electrode arrangements (one for each of the near and far gaps) included platinum electrodes. At low frequencies (10kHz-100kHz), using a 5mm gap electrode configuration, a detectable signal cliff is initially identified at about 23 minutes into amplification (about 10kHz) and about 30 minutes into amplification (about 100kHz), with the magnitude of the change in reactance being about 3.5 ohms-4 ohms at 10kHz and down to about 3.25 ohms-3.5 ohms at 100 kHz. At low frequencies (10kHz-100kHz), using a 2.5mm gap electrode configuration, a detectable signal cliff begins to be identified at about 25 minutes into amplification (about 10kHz) and about 30 minutes into amplification (about 100kHz), with a change in reactance of about 3.5 ohms-4 ohms. At higher frequencies, the reactance drop of the signal cliffs is reduced and the time to identify these smaller signal cliffs is shifted to a later time in the amplification procedure. Thus in this example, the test wells in the test cartridge may be configured with 5mm gap electrodes, and the reader device may be configured to provide 10kHz excitation current to the test cartridge during amplification. Instructions may be provided to the reader device to provide this current throughout the amplification period or within a time window (e.g., 20 to 35 minutes) around the expected signal cliff time (here 23 minutes) and monitor the resulting reactance of the test well. Instructions may also be provided to the reader device to identify a positive sample based on a reactance exhibiting a change of about 3.5 ohms to 4 ohms at about 23 minutes into amplification, or within a time window around the expected time of a signal cliff.

Once identified, a_RAnd T_WIs provided to the reader device for distinguishing between positive and negative samples for that particular test. In some examples, such an apparatus may determine that reactance curve 540B is at the identified time window T_WWhether or not it has a desired value and/or slope corresponding to a signal cliff. In other embodiments, the reader device may analyze the change in the shape of the reactance curve over time to determine whether it contains a signal cliff. In some implementations, the reader can be based on the identified time window T_W(where signal cliff 545 is expected to occur) to modify its test procedure (e.g., by providing only the stimulus voltage and monitoring the resulting signal within the window), advantageously saving power and processing resources compared to continuous monitoring throughout the test time.

Fig. 5D depicts a graph 551 of the resistive and reactive components extracted from the raw sensor data of the sense electrode 510B during testing of the examples of the positive and negative controls. Specifically, graph 551 shows a curve 550A of the resistance of the positive sample, a curve 550B of the resistance of the negative sample, a curve 550C of the reactance of the positive sample, and a curve 550D of the reactance of the negative sample over a test duration of 35 minutes. As shown in fig. 5D, a positive sample signal cliff occurs at about 17 minutes into the test, leading to the signal cliff with a relatively flat and stable reactance curve 550B. In contrast, the negative sample reactance curve 550D does not exhibit a signal cliff at the same time, but rather maintains a secondary curvature from about t-8 minutes to the end of the test.

Fig. 5E depicts a graph 561 of resistance components 560A and reactance components 560B extracted over time from the raw signal 520 generated based on an exemplary positive test (t 0 min to t 60 min from the start of amplification). As shown, signal cliff 565 represents a particular time window T_WChange Δ of reactance 560B in the period_R. Signal cliff 565 indicates a positive sample. Before signal cliff 565 occurs, reactance curve 560B is relatively flat or stable, and after signal cliff 565,the reactance curve 560B is again relatively flat or stable and has a slight concavity. Signal cliff 565 for a particular test parameter represented by graph 561 appears as a peak, spike, or bell-shaped curve in expected region 535 during which the reactance value appears as an approximate parabola at Δ_RThe value rises and falls. As described herein, altering certain test parameters (e.g., test well configuration, chemistry and initial amounts of amplification components, target and excitation current characteristics) can alter the geometry of the signal cliff generated from a positive sample. Thus, in some embodiments, the geometry of the "signal cliff" in the reactance value vs time curve may vary between different tests, but for a particular test the curve geometry and/or timing signal cliff (timing signal cliff) remains consistent within the reactance variation and/or timing parameters between positive samples of that test. Fig. 6 depicts a schematic block diagram of an example reader device 600 that may be used with a cartridge (e.g., cartridge 100 or cartridge 300) described herein. The reader device 600 includes a memory 605, a processor 610, a communication module 615, a user interface 620, a heater 625, an electrode interface 630, a voltage source 635, a compressed air reservoir 640, a motor 650, and a cavity 660 into which a cartridge can be inserted.

When the test cartridge 100 is inserted into the reader device, the electrode interface 135 of the cartridge interfaces with the electrode interface 630 of the reader device 600. This may allow the reader device 600 to detect that a cartridge is inserted (e.g., by testing whether a communication path is established). In addition, such communication may enable the reader device 600 to identify a particular inserted test cartridge 100 and obtain the corresponding test protocol. The test protocol may include the duration of the test, the temperature of the test, characteristics of the positive sample impedance curve, and information output to the user based on the results of the various determinations of the test. In other embodiments, the reader device 600 may receive an indication via the user interface 620 that a cartridge is inserted (e.g., by a user entering a "start test" command, and optionally by a test cartridge identifier).

The memory 605 includes one or more physical electronic storage devices configured to store computer-executable instructions for controlling the operation of the reader device 600 and data generated during use of the reader device 600. For example, the memory 605 may receive and store data from sensing electrodes connected to the electrode interface 630.

The processor 610 includes one or more hardware processors that execute computer-executable instructions to control the operation of the reader device 600 during testing, such as by managing the user interface 620, controlling the heater 625, controlling the communication module 615, and activating the voltage source 635, compressed air 640, and motor 650. One example of a test operation is described with respect to FIG. 7A below. The processor 610 may also be configured with instructions to determine a test result based on data received from the excitation electrodes of the inserted test cartridge, for example by implementing the routine of FIG. 7B described below. The processor 610 may be configured to identify different targets in the same test sample based on signals received from different test wells of a single cartridge, or may identify a single target based on individual analysis or comprehensive analysis of signals from different test wells.

The communication module 615 may optionally be provided in the reader device 600 and include a network-enabled hardware component (e.g., a wired network component or a wireless network component) for providing network communication between the reader device 600 and a remote computing device. Suitable network components include WiFi, bluetooth, cellular modem, ethernet port, USB port, and the like. Advantageously, the networking capability may enable the reader device 600 to transmit test results and other test data over a network to identified remote computing devices (e.g., hospital information systems and/or laboratory information systems storing electronic medical records, national health agency databases, and computing devices of clinicians or other designated personnel). For example, when determining test results via a reader device, physicians may receive test results for a particular patient on their mobile device, laptop, or office desktop, enabling them to provide faster turn-around times for diagnosis and treatment planning. Further, the networking capabilities may enable the reader device 600 to receive information from remote computing devices over a network, such as updated signal cliff parameters for existing tests, new signal cliff parameters for new tests, and updated or new test protocols.

The user interface 620 may include a display for presenting test results and other test information to a user, and a user input device (e.g., buttons, a touch-sensitive display) that allows a user to input commands or test data to the user reader device 600.

A heater 625 may be positioned adjacent to the cavity 660 for heating the inserted cartridge to a desired temperature for the amplification procedure. Although depicted as being on a single side of the cavity 660, in some embodiments, the heater 625 may surround the cavity.

As described herein, voltage source 635 can provide excitation signals at a predetermined voltage and a predetermined frequency to the respective excitation electrodes of the inserted test cartridge. The compressed air reservoir 640 may be used to provide pneumatic pressure to the pneumatic interface 160 of the cartridge 100 via the channel 645, thereby facilitating the flow of liquid within the cartridge. The compressed air reservoir 640 may store previously compressed air or generate compressed air as needed by the reader device 600. In other embodiments, other suitable pneumatic pumps and pressure providing mechanisms may be used in place of the stored or generated compressed air. As described above, the motor 650 is operable to move the actuator 655 towards and away from the blister pack 140 of the inserted cartridge in order to rupture the blister pack.

Fig. 7A depicts a flowchart of an example procedure 700 for operating a reader device during testing as described herein. The procedure 700 may be performed by the reader device 600 described above.

At block 705, the reader apparatus 600 may detect that the test cartridge 100, 200, 300 has been inserted, for example in response to a user input or in response to establishing a signal path with an inserted cartridge. In some embodiments, the cartridge 100, 200, 300 may include an information element that identifies the particular test to be performed on the reader device 600, and optionally test protocol information.

At block 710, the reader apparatus 600 may heat the cartridge 100, 200, 300 to a specified temperature for amplification. For example, the temperature may be provided by information stored on the cartridge 100, 200, 300, or retrieved in an internal memory of the reader device 600 in response to identification of the cartridge 100, 200, 300.

At block 715, the reader device 600 may activate a blister pack puncturing mechanism device, such as the motor 650 and the actuator 655. Puncturing the blister package may release its liquid contents (including chemical components used to facilitate amplification) from its previously closed chamber.

At block 720, the reader device 600 can activate a pneumatic pump to move the sample and liquid from the blister pack through the fluid flow channel of the cartridge to the test wells. As described above, the test well may include a vent that makes it possible to push liquid through the fluid flow path of the cartridge and also allows any trapped air to escape. The pneumatic pump may include compressed air 640 or other suitable pressure source and may be in fluid communication with the pneumatic interface 160.

At block 725, the reader device 600 may release any entrapped air from the test wells, for example, by pushing fluid through the fluid flow channels of the cartridge until some resistance is sensed (e.g., liquid in the fluid flow channels is pushed toward a liquid impermeable, gas permeable filter of the vent). Block 725 may optionally include agitating the inserted cassette to facilitate any entrapped air or bubbles moving up through the liquid and out through the vent. Further, at block 725, the reader device 600 optionally can provide a signal to the cartridge that causes the closing of the valves located between the test wells to avoid mixing of the amplification procedure.

At decision block 730, the reader device 600 may determine whether the test is still within its specified test duration. For example, where the expected window of time for which a signal cliff should occur in a positive sample is known, the duration of the test may end at the end of the window or some predetermined period of time after the end of the window. If so, the process 700 transitions to optional decision block 735 or to block 740 (in embodiments where block 735 is omitted).

At optional decision block 735, reader device 600 determines whether to monitor for test well amplification by recording data from the test well sense electrodes. For example, instructions may be provided to the reader 600 to monitor only the impedance of the test wells during a particular window or windows of testing. If the reader device 600 determines not to monitor the test well amplification, the process 700 loops back to decision block 730.

If the reader device 600 determines to monitor the test well amplification, the process 700 transitions to block 740. At block 740, the reader device 600 provides an excitation signal to the excitation electrodes of the test wells of the inserted cartridge. As described above, the excitation signal may be an alternating current at a particular frequency and a particular voltage.

At block 745, the reader device 600 detects and records data from the sensing electrodes of the test wells of the inserted cartridge. In some embodiments, this data may be stored for later analysis, for example after testing is complete. In some embodiments, the reader device 600 may analyze this data in real time (e.g., while the test is still in progress) and may stop the test once a positive sample signal cliff is identified.

When the reader device 600 determines at block 730 that the test has not been within its specified duration, the process 700 moves to block 750 to analyze the test data and output the test results. The test result may include an indication that the sample is positive or negative for the target test, or may more specifically indicate an estimated amount of the target in the sample tested.

Fig. 7B depicts a flowchart of an example process for analyzing test data to detect a target as described herein, which may be performed by the reader device 600 as per block 750 of fig. 7A or 54.

At block 755, the reader device 600 may acquire recorded signal data received from the electrodes of the well. Even if the cartridge has multiple wells, the data from each well can be analyzed individually. The test results from the wells can be later analyzed in combination to determine a single test result for a single target or to determine multiple test results for multiple targets based on all tests performed within the cartridge.

At block 760, the reader apparatus 600 may decompose the signal into resistive and reactive components across some or all of the different points in time of the test. For example, as described above, at each point in time, the reader device 600 may determine the in-phase and out-of-phase components of the originally acquired voltage waveform, which may then be deconvolved (deconvoluted) using the known series resistance of the electrode circuit to calculate the in-phase (resistive) and out-of-phase (reactive) portions of the impedance of the test aperture.

At block 765, the reader apparatus 600 may generate a plot of reactance value versus time. Also at block 765, the reader device 600 may optionally generate a curve of resistance over time.

At block 770, the reader device 600 may analyze the reactance curve to identify a change in the signal indicative of a positive test. As described above with respect to the signal cliff of fig. 5C, the reader device 600 may look for a change in reactance that is greater than a threshold, may look for such a change within a predetermined time window, may analyze the slope of the reactance curve at a predetermined time, or may analyze the overall shape of the reactance curve to determine whether a signal cliff (e.g., a relatively more stable value before and after a rise or fall in the signal) is present.

At decision block 775, based on the analysis performed at block 770, the reader device 600 may determine whether the sought signal change is identified in the reactance curve. If so, the routine 750 transitions to block 780 to output an indication of a positive test result to the user. If not, the process 750 transitions to block 785 to output an indication of a negative test result to the user. The results may be output locally (e.g., on a display of the device), or over a network to a designated remote computing device.

Overview of additional exemplary cartridges, readers, and Signal processing

The embodiments described in this section are exemplary and are not intended to limit the scope of the present disclosure. Some embodiments are directed to systems, methods, and/or devices for sensing and/or identifying pathogens, genomic material, proteins, and/or other small molecules or biomarkers for diagnosis. In some embodiments, small, portable, low-power devices provide fast and robust sensing and identification. Such devices may utilize microfluidics, biochemistry and electronics to detect one or more targets in situ and closer to or at the bedside.

Fig. 51A-51C depict an exemplary handheld detection system 5100 for detecting a target. The system 5100 includes a reader device 5110 and a cartridge 5120 configured to fit within a cavity 5112 of the reader device 5110. The cartridge 5120 generally includes an outer portion 5122 and an inner portion 5124. When the cartridge 5120 is inserted into the reader device 5110, some or all of the inner portion 5124 is contained within the reader device 5110. The outer portion 5122 is sized and shaped to be grasped by a user, and may include one or more three-dimensional surface features (e.g., indentations 5126) to facilitate insertion and/or removal of the cartridge 5120 from the reader device 5110.

As shown in fig. 51B and 51C, the reader device 5110 and the cartridge 5120 are sized and shaped such that one or more interchangeable cartridges 5120 can be inserted and/or removed by hand at the cavity 5112. As will be described in greater detail, the reader device 5110 can include one or more heating assemblies configured to heat at least a portion of the interior portion 5124 of the cartridge 5120. The reader device 5110 may further include circuitry configured to interface with the circuitry of the cartridge 5120 to detect one or more electrical properties of a sample contained in the cartridge.

In some embodiments, some cartridges 5120 can be power cartridges. The reader device 5110 may be powered on and off by the electrical cartridge 5120, instead of or in addition to a conventional power switch or button external to the reader device 5110. The electrical cartridge 5120 can have a similar size and shape as other cartridges used with the reader device 5110. In operation, when the reader device 5110 is powered down, the electrical cartridges 5120 may remain engaged within the cavity 5112. The circuitry of electrical cartridge 5120 may be in contact with internal circuitry of reader device 5110 such that removal of electrical cartridge 5120 from reader device 5110 causes reader device 5110 to be powered on for testing. After completion of one or more tests, or at any other time the reader device 5110 is to be powered down, the electrical cartridge 5120 is inserted into the cavity 5112. When electrical cartridge 5120 is inserted, the circuitry of electrical cartridge 5120 again contacts the internal circuitry of reader device 5110 such that insertion of electrical cartridge 5120 causes reader device 5110 to be powered down. The electrical receptacle application is discussed in more detail with reference to FIG. 6.

In some embodiments, one or more external status indicators can be provided external to reader device 5110 to provide status indications to a user. For example, in one particular embodiment, the status indicator can include a light ring 5114 disposed about the cavity 5112. In other embodiments, the optional status indicator can be located at any suitable location on the reader device 5110. The light ring 5114 or other status indicator may include one or more light sources, such as Light Emitting Diodes (LEDs) or the like. The light ring may also be configured to, for example, indicate when the device is in use or not in use, or when different stages of a detection method using the device are reached, completed, or being performed (e.g., receiving a sample by the device or in a well, performing amplification, detection of aggregates in a well, or transmitting results to a receiver). Different colored lights may be used to indicate different stages of a detection method using a device such as those described above.

In some embodiments, a plurality of different colored LEDs may be provided within the light ring 5114 or other status indicator to display various status indications. For example, the light ring 5114 may include a combination of more than two colors (e.g., white, blue, and red), each of which may be activated independently. Each light source may be operated in multiple modes, such as a "steady" (solid) mode characterized by continuous activation of the light source (e.g., a steady "on" state), a "flashing" mode characterized by repeated activation and deactivation of the light source, a "flashing" mode characterized by a single activation and deactivation of the light source, a "breathing" mode characterized by repeated gradual brightening and dimming of the light source, and so forth.

A combination of color and activation mode may be used to indicate the status of the reader device 5100. For example, in some embodiments, the light ring 5114 or other status indicator may exhibit a first indication (e.g., a steady white light) when the reader device 5100 is powered on and ready to receive the detection cartridge 5120 (e.g., when the electrical cartridge is removed). Other examples of device states that may be indicated by the status indicators include, for example, the cartridge 5120 being inserted into the reader device 5110, a test having begun and running, a test completed, a cartridge removed after a test completed, an error (e.g., premature removal of the cartridge 5120, a test failure, etc.), a bluetooth pairing, or any other state of the reader device 5110. In one non-limiting example, a steady white light ring 5114 indicates that the electrical cartridge has been removed and the device has been powered on or that the test cartridge has been removed after the test is complete, a steady blue light ring 5114 indicates that the test cartridge has been inserted into the reader device 5110, a breathing blue light ring 5114 indicates that the test has begun and is running, a breathing white light ring 5114 indicates that the test has been completed and the cartridge can be removed, a steady, breathing, or flashing red light ring indicates an error, blue and red flashing at the light ring 5114 indicates that bluetooth pairing is in progress, and a smooth, flashing, or flashing blue light ring 5114 indicates that bluetooth pairing is complete. It should be understood that other embodiments may include any combination or sub-combination of the status indicator modes listed above, and/or may include further status indications, lighting colors, operating modes, and the like.

Fig. 52A-52F depict an example cartridge 5200 configured for detection of a target. As described herein, the target can be a viral target, a bacterial target, an antigenic target, a parasitic target, a microRNA target, or an agricultural analyte. Some embodiments of the cartridge 5200 may be configured for testing a single target, while some embodiments of the cartridge 5200 may be configured for testing multiple targets. The case 5200 includes a case 5210 and a cap 5240 configured to mechanically couple to the case 5210. When the case 5210 and the cap 5240 are connected together, the case 5210 forms a portion of the outer portion 5202 as well as the inner portion 5204 of the case 5200. The cap 5240 forms the remainder of the outer portion 5202.

Fig. 52A and 52B depict a complete case 5200 including a case 5210 and a cap 5240 joined together. In use, the cap 5240 and the cartridge body 5210 can operate to enclose the provided sample within the cartridge 5200, thereby preventing the test operator from being exposed to the sample and preventing any liquid from escaping into the electronics of the associated reader device. The case 5210 and cap 5240 can be connected by a friction fit, a snap fit, and/or one or more mechanical or chemical fastening means.

The case 5210 and cap 5240 can be formed from a suitable fluid-impermeable material (e.g., plastic), and can be opaque, translucent, or transparent. The cartridge 5210 can further comprise a translucent or transparent cover 5212 that partially defines a fluid flow channel within the cartridge 5210, and one or more electrode interfaces 5214. The cap 5212, fluid flow channel, and electrode interface 5214 are discussed in more detail with reference to fig. 52C and 52D. The case 5210 and/or the cap 5240 can further comprise a case identifier 5215. The cartridge identifier 5215 may include human-readable and/or machine-readable information, such as text, a barcode, a QR code, and the like. The cartridge identifier 5215 may include any suitable information associated with the cartridge, such as information specifying the type of test, target agent, sample type, cartridge serial number or other individual cartridge identifier, and the like. In addition to serving as an identifier for users having a test type associated with the cartridge 5200, the cartridge identifier 5215 may also be scanned by a user (e.g., using a user interface device in communication with the reader device) to communicate one or more test protocols to the reader device. The case 5210 and/or the cap 5240 can include ergonomic features (e.g., indentations 5216) to facilitate gripping of the case 5200.

Fig. 52C and 52D depict the cartridge body 5210 assembly of the cartridge 5200 of fig. 52A and 52B. The case 5210 includes a base 5211 and a cover 5212. The base 5211 can be formed from a fluid-impermeable material (e.g., an injection molded or milled acrylic material or a plastic material). The base 5211 comprises a receiving aperture 5218 and an assembly of cartridge flow channels, including a first section 5222, a mixing aperture 5224, a second section 5226, a test aperture 5228, a third section 5230 and a vent port 5232. It is to be understood that the specific geometric configuration or relative arrangement of the features may be varied in other embodiments. As used herein, fluid communication refers to the ability to transfer a fluid (e.g., a liquid or a gas). The cap 5212 can be formed from a fluid-impermeable material. In some embodiments, the cover 5212 is a translucent or transparent material, such as glass, plastic, or the like. The cap 5212 is closed to the base 5211 to form the cartridge 5210 and serves as a boundary that confines fluid within the cartridge flow channel assembly described above. In some embodiments, the translucent or transparent cover 5212 advantageously allows for visual inspection of the fluid within the cartridge flow channel (e.g., to verify that the test well is full prior to testing, etc.). One or more conductive components of the electrode interface 5214 are disposed on the cover 5212. The mating feature 238 is sized and shaped to receive a corresponding mating feature of the cap 5240. The receiving aperture 5218 optionally includes a chamfer 5220 to facilitate the attachment of the cap 5240 to the cartridge body 5210.

The box body flow passage includes: a section 5222, a section 5226, and a section 5230, as well as an inlet (fig. 53C) fluidly connecting the receiving well 5218 to the first section 5222, a mixing well 5224, and a testing well 5228. The first section 5222 of the cartridge flow channel leads from the inlet to the mixing bore 5224. The second section 226 of the cartridge flow channel leads from the mixing port 5224 to the testing port 5228. The third section 5230 is a test hole outlet flow path leading from the test hole 5228 to an exhaust port 5232 which allows gas to escape from the test hole 5228 and exit the case 5200.

The mixing well 5224 can include one or more reagents in dry form (e.g., powder). As the fluid sample enters the mixing well 5224, the powdered reagent and/or other dry reagents may be hydrated by the fluid sample. The reagents provided in the mixing wells 5224 may be selected based on one or more protocols of the intended test associated with the cartridge 5200. In some embodiments, uniform or homogeneous mixing of the reagent with the fluid sample can produce more accurate test results. In this regard, the mixing holes 5224 are configured to promote uniform mixing of the reagent with the fluid sample, e.g., by including cross-sectional shapes and/or curved regions that promote turbulent flow rather than laminar flow of the liquid within the mixing holes 5224. Turbulence is a flow state in fluid dynamics that is characterized by chaotic changes in the pressure and flow rate of a fluid. Turbulent flow is in contrast to laminar flow, which occurs when a fluid flows in parallel layers without disruption between the layers.

The block 5222, block 5226 and block 5230, mixing holes 224 and/or testing holes 5228 of the cartridge flow channel can be completely enclosed within the material of the base 5211, or can have three surfaces formed by the material of the base 5211 and the cap 5212 forms an upper surface that closes off these channels.

The test area or inner portion 5204 of the cartridge 5210 includes: a section 5226, a section 5230, a test well 5228, a valve 5232, an electrode 5213, an electrode 5215 and an electrode interface 5214 of the cartridge flow channel. The electrode interface 5214 comprises a plurality of contact plates 5214₁-5214₅. Although five contact plates 5214 are depicted₁-5214₅The case 5210 may likewise include more or less than five contact plates. First contact plate 5214₁Is electrically connected to the first electrode 5213 of the test hole 5228, and the second contact plate 5214₅And is electrically connected to the second electrode 5215 of the test well 5228. Contact plate 5214₁Contact plate 5214₅Is configured to connect the excitation electrode of the test well with a voltage or current source of the reader device, and the contact plate 5214₁Contact plate 5214₅Is configured for electrically connecting the signal electrode of the test well with a signal reading conductor of the test device. Contact plate 5214₁-5214₅The additional contact plate in (b) may be used in conjunction with the reader device for other purposes. For example, contact plate 5214 ₁-5214₅May be connected to circuitry of an electrode interface of the reader device to indicate one or more test protocols to the reader. In another example, an electrical receptacle as described above with reference to fig. 51A-51C may include a similar set of contact plates 5214₁-5214₅The contact pad is configured to be connected to circuitry of an electrode interface of a reader device to activate power circuitry of the reader device.

The mixing wells 5224 may be provided with dry or lyophilized components of the solid, such as primers and proteins, for use in the testing procedure. The particular selection and chemistry of these dry or lyophilized components may be tailored to the particular target or targets for which the cartridge 5200 is designed to test. These dry or lyophilized components may be hydrated with a liquid (e.g., a buffer or liquid sample flowing into the test well, such as a fluid sample within the cartridge 5200) so that they are activated for the testing procedure. Advantageously, providing dry or lyophilized solid components in the mixing well 5224 enables the cartridge 5200 to be stored prior to use to contain components required for the amplification procedure, while also delaying initiation of amplification until after application of the sample.

The test hole 5224 is depicted as a generally cylindrical hole formed as a circular opening in the material of the base 5211 and surrounded by the planar surface of the cover 5212. The test well 5224 contains two electrodes 5213 and 5215, one of which is an excitation electrode configured to apply a current to the sample in the test well 5224 and the other of which is a signal electrode configured to detect a current flowing from the excitation electrode through the liquid sample. In some embodiments, a thermistor may be provided to one or more test wells in place of an electrode to provide monitoring of the temperature of the fluid within the cartridge 5100.

In some embodiments, bubbles within the test well 5224 (particularly if the bubbles are located along the current flow path between the electrode 5213 and the electrode 5215) can generate noise in the signal picked up by the signal electrode. This noise may reduce the accuracy of test results determined based on signals from the signal electrodes. The desired high quality signal can be obtained when only liquid is present along the current flow path, or when there are minimal bubbles along the current flow path. As described above, any air originally present in the fluid flowing along the flow channels of the cartridge body can be pushed out through the vent 5232. In addition, the electrodes 5213, 5215 and/or the test wells 5224 can be shaped to mitigate or prevent the buildup of fluid samples, where air or bubbles form in the liquid sample and collect along the electrodes 5213 and 5215.

For example, in some embodiments, the electrodes 5213 and 5215 can be located at the bottom of the test well 5224. This may allow any air or gas to rise to the top of the fluid in the test well and away from the flow channel between the electrodes. As used herein, the bottom of the test well 5224 refers to the portion of the test well in which heavier liquids settle due to gravity, and the top of the test well refers to the portion of the test well in which lighter gases rise above the heavier liquids. In addition, the electrodes 5213 and 5215 are positioned away from the periphery or edges of the test well 5224 (where bubble nucleation typically occurs).

In addition, the electrodes 5213 and 5215 can be formed from a thin, flat layer of material having a minimum height relative to the lower circuit board layer that forms the bottom of the test holes 5224. In some embodiments, the electrodes 5213 and 5215 can be formed using electrodeposition and patterning to form a thin layer of metal film (e.g., about 5300nm in height). This minimum height may help prevent or mitigate entrapment of gas bubbles along the interface between the electrode and the underlying layer. In some embodiments, a layer of conductive material may be deposited on top of each electrode to create a smoother transition between the edges of the electrodes and the bottom of the test well. For example, a thin polyimide layer (e.g., about 5 microns in height) may be deposited on top of the electrodes, or the circuit board may be butter coated. Additionally or alternatively, the electrode may be located in a trench in the underlying layer, the trench having a depth approximately equal to the height of the electrode. These and other suitable methods may result in an electrode that is substantially flat or flush with the bottom surface of the hole.

Advantageously, the above-described features can help keep the electrodes 5213 and 5215 surrounded by liquid and prevent or reduce gas bubbles from being located along the current flow path between the electrodes 5213 and 5215.

Fig. 52E and 52F depict the cap 5240 assembly of the box 5200. Fig. 52F is a sectional view taken along line 2F-2F in fig. 2E to illustrate the internal structure of the cap 5240. The cap 5240 is sized and shaped to mate with the box body 5210 or otherwise mechanically connect to the box body 5210, thereby forming the complete box 5200. The cap 5240 includes mating features 5242, the mating features 5242 configured to interlock with corresponding mating features 5238 of the cartridge body when the cartridge 5200 is assembled. The cap further includes a plunger 5244 disposed about the retention aperture 5250 for retaining the capillary therein.

The plunger 5244 is sized and shaped to sealingly engage the receiving aperture 5218 of the cartridge body 5210 (fig. 52C-52D). The plunger 5244 optionally includes a groove 246, the groove 246 configured to receive an O-ring or other gasket to achieve and/or enhance a seal between the plunger 5244 and the receiving bore 5218. An optional chamfer 5248 at the distal end of the plunger 5244 can facilitate engagement of the plunger 5244 with the receiving aperture 5218 (fig. 52C-52D), either alone or in combination with the chamfer 5200 of the receiving aperture 5218. As will be described in more detail with reference to fig. 53A-53E, the plunger 5244 is thus sealingly engaged with the receiving aperture 5218 to advance the fluid sample into the cartridge body 5210.

The retention well 5250 is configured to partially surround a capillary containing a fluid sample for testing. The holding hole 5250 preferably has an inner diameter larger than the outer diameter of the capillary to be inserted. A plurality of retaining structures 5252 extend inwardly from the inner wall of the retaining bore 5250 to retain the capillary tube in a central position within the retaining bore 5250. Preferably, the distance between the opposing retaining structures 5252 is about equal to or slightly greater than the outer diameter of the capillary tube. As shown in fig. 52F, the rear portion 5254 of each retention structure 5252 extends further inward relative to the remainder of the retention structure 5252. The distance between the opposing rear portions 5254 is small enough that the capillary cannot fit between the rear portions 5254. Thus, the rear 5254 of the retention structure 5252 blocks the movement of the capillary tube along the retention hole 5250 and maintains a space between the capillary tube and the rear wall of the retention hole 5250. As will be described in more detail with reference to fig. 53A-53E, this spaced location of the capillary within the cap 5240 allows air or other fluid to flow into the retention apertures 5250 and into the rear of the capillary around the sides of the capillary between the retention structures.

The cassette 5200 of fig. 52A-52F provides a stand-alone, easy-to-use device for performing amplification-based tests on targets, such as nucleic acid tests in which genomic material in a sample is copied exponentially using a molecular amplification procedure. Advantageously, because the solid components of the amplification procedure are provided in the cartridge in advance and automatically mixed with the sample, the user need only apply the sample and insert the cartridge 5200 into the reader device to determine the test results in some embodiments. In some embodiments, one or both of the cartridge or the reader may include a heater and a controller configured to operate the heater to maintain the cartridge at a desired temperature for amplification. In some embodiments, one or both of the cartridge or the reader may include a motor to impart vibration or agitation to the cartridge, causing any entrapped gas to rise to the top of the liquid and be expelled from the test wells.

Fig. 53A-53E illustrate mechanical fluid transfer aspects of a cassette 5120, a cassette 5200 described herein. As will be described in more detail, the cartridge body 5210 and the cap 5240 are configured to generate air pressure when connected together such that the air pressure pushes the fluid sample through the fluid flow channels of the cartridge body 5210. Fig. 53A-53E illustrate the cap 5240 having translucency to reveal internal features of the cap 5240. The cartridge 5210 is shown in cross-section in fig. 3C-3E to reveal internal features of the cartridge 5210.

Referring to fig. 53A and 53B, a fluid sample can be received in the capillary 300, e.g., within the interior cavity 5305 of the capillary 5300. The cap 5240 is sized and shaped to receive the capillary 5300 as described above with reference to fig. 52E and 52F. The fluid sample can be introduced into the capillary 5300 while the capillary 5300 is within the cap 5240, or the capillary 5300 can contain the fluid sample when the capillary 5300 is placed in the cap 5240.

Fig. 53A is a front view of the cap 5240 of the case 5200. When the capillary tube 300 is disposed within the retention aperture 5250 of the cap 5240, the retention structure 5252 clamps the capillary tube 5300 in a position spaced from the walls of the retention aperture 5250. Accordingly, a plurality of air channels 5310 are formed between the inside of the holding hole 5250 and the outside of the capillary 5300.

Fig. 53B is a top view of the cap 5240 of fig. 53A. A rear portion 5310 (e.g., rear portion 5254 of fig. 52F) of some or all of the retaining structures 5310 maintains the capillary 5300 spaced from the rear portion of the retaining aperture 5250. This arrangement forms a cap fluid flow channel 5315 so that air or other fluid can flow into the retention aperture 5250 through the air channel 5310, around the rear of the capillary 5300, and out of the retention aperture 5250 through the interior cavity 5305 of the capillary 5300. Thus, application of relatively high pressure at the air channel 5310 can cause fluid within the internal cavity 5305 to flow out of the capillary 5300 along the cap fluid flow channel 5315.

Fig. 53C-53E illustrate various stages in the process of connecting the cap 5240 to the cartridge 5210, along with the associated fluid flow channels, for effecting movement of the sample into the test well 5228 and other components of the cartridge flow channel 5325. Fig. 53C depicts the cap 5240 adjacent to the case 5210 but not connected to the case 5210, fig. 53D depicts the cap 5240 connected to the case 5210, and fig. 53E depicts the cap 5240 fully connected to the case 5210.

As shown in fig. 53C, the plunger 5244, the retaining hole 5250 and the capillary 5300 are aligned with the receiving hole 5218 of the cartridge 5210. The plunger 5244 is sized and shaped to sealingly engage the receiving bore 5218. An O-ring or other seal (not shown) may be positioned in the groove 5246 of the plunger 5244 to effect and/or enhance a seal between the plunger 5244 and the receiving bore 5218. The receiving bore 5218 is fluidly connected to the mixing bore 5224 through an inlet 5221, which inlet 5221 is sized and shaped to sealingly receive one end of the capillary 5300. When the cap 5240 is aligned with the box 5210, the process continues to the configuration of fig. 53D.

As shown in fig. 53D, the plunger 5244 engages the wall of the receiving bore 5218. When the plunger 5244 (and/or an O-ring disposed on the plunger 5244) engages the walls of the receiving aperture 5218, a volume of ambient air is trapped within the walls of the receiving aperture 5218. The trapped air 5320 has a volume defined by the portion of the receiving aperture 5218 not occupied by the plunger 5244. As the cap 5240 is pressed further onto the cartridge body 210, the outer end of the capillary 5300 enters the inlet 5221 and sealingly engages the inlet 5221. Thus, as the cap 5240 and the cartridge body 5210 are pressed further together, the trapped air 5320 is compressed within the constricted volume (shrinkingvolume) of the portion of the receiving bore 5218 not occupied by the plunger 5214. Because inlet 5221 is blocked by capillary tube 5300, the compression of trapped air 5320 causes trapped air 5320 to flow along cap flow channel 5315 of fig. 53B.

Referring now to fig. 53E, fluid transfer by connecting the cap 5240 and the cartridge 5210 is described. Fig. 53E shows flow along the cap flow channel 5315 and the box flow channel 5325 with circled numbers shown as indicia at certain points along the fluid flow channel. The circled numbers will be discussed below as exemplary steps of the process of the trapped air 5320 and the fluid sample as they travel through the cap flow channel 5315 and the cartridge body flow channel 5325 within the cartridge 5200, each step including a directional arrow showing the direction of fluid travel at that step. For clarity and brevity of fig. 53E, some of the components labeled with reference numbers in fig. 52A-53D are not labeled in fig. 53E.

Prior to step (1), the user provides the fluid sample within capillary 5300. Also prior to step (1), capillary 5300 is placed within retaining aperture 5250 of cap 5240 between retaining structures 5252 to form cap flow channel 5315.

In step (1), when the plunger 5244 compresses the trapped air 5320, the trapped air 5320 is forced into the air passage 5310. The trapped air 5320 flows along the cap flow channel 5315 through the air channels 5310 between the retention structures 5252 and along the exterior of the capillary tube 5300.

In step (2), the trapped air 5320 reaches the rear of the holding holes 5218. The trapped air 5320 continues along the cap flow channel 5315 into the inner cavity 5305 of the capillary 5300. Upon entering the inner cavity 5305, the trapped air 5320 contacts and applies pressure to the fluid sample contained within the capillary 5300. Pressure is directed along the length of the capillary 5300 towards the cartridge 5210.

In step (3), the fluid sample flows out of the capillary 5300 and into the inlet 5221 of the cartridge 5210. The pressure applied at the opposite end of capillary 5300 by trapped air 5320 pushes the fluid sample into inlet 5221. Capillary or wicking action may also urge fluid sample into inlet 5221, for example, where the section fluidly connected along cartridge flow channel 5325 and the inlet are suitably narrow to cause wicking action. In step (4), the fluid sample travels through the first section 5222 of the cartridge body flow channel 5325.

In step (5), the fluid sample enters the mixing well 5224. The mixing bore may include one or more reagents. The agitation caused by the flow of the fluid sample within the relatively large volume of the mixing bore 5224 causes the reagent and sample to mix. In some embodiments, the reagent and the fluid sample are mixed into a uniform solution, wherein the reagent is uniformly distributed throughout the fluid sample. The depth, width, and/or cross-sectional profile of the mixing holes 5224 can be selected to facilitate mixing of the reagents and fluid sample.

In step (6), the mixed reagent and fluid sample (referred to as "test fluid") exits the mixing well 5224 and travels along the second section 5226 of the cartridge body flow channel 5325 into the test well 5228.

In step (7), a portion of the test fluid proceeds along the third section 5230 of the cartridge body flow path 5325 to fill any remaining open volume within the cartridge body flow path 5325. The flow path of step (7) illustrates the optional flow of gas (e.g., the test fluid or the gaseous portion of the ambient air present within the cartridge 5210) through the valve 5232. In some embodiments, valve 5232 can comprise a liquid-impermeable, gas-permeable filter, such that any gas present in the test fluid or within the cartridge 5210 can be vented through valve 5232 as the test fluid fills the space within cartridge flow channel 5325. The valve 5232 can further minimize the occurrence of air bubbles within the test well 5228. In some embodiments, the valve 5232 may not be present and/or may not be configured to vent gases.

After completion of steps (1) to (7), the case 5200 is closed and contains a test fluid within the case 5210 and the cap 5240. The closed cartridge 5200 can then be placed in a reader device (e.g., reader device 5110, reader device 600, reader device 6000 as described herein) for testing to detect one or more target agents within a test fluid. In various embodiments, the size of the fluid sample and/or the amount of reagent may preferably be selected to provide sufficient test fluid to substantially fill the enclosed fluid space within the cartridge 5200 along the cap flow channel 5315 and the cartridge body flow channel 5325. The volume of the receiving aperture 5218 and the corresponding size of the plunger 5244 can preferably be selected such that the receiving aperture 5218 contains sufficient air for conveying the fluid sample along the length of the fluid flow path and into the test aperture 5328. It will be appreciated that the advancement of the fluid sample through the capillary 5300 into and along the cartridge flow channel 5325 may occur, as described above with reference to fig. 53A-53E, due to: capillary or wicking, fluid pressure due to compression of trapped liquid or gas (e.g., air) within the receiving apertures 5218, or both.

FIG. 59 depicts a schematic block diagram of an example reader device 6000, which reader device 6000 may be used with a cartridge (e.g., cartridge 5120 or cartridge 5200) as described herein. The reader device 6000 schematically shown may be, for example, the reader device 5110 of fig. 51A-51C. Reader device 6000 includes memory 6005, processor 6010, communication module 6015, heater 6025, electrode interface 6030, voltage source 6035, and chamber 6060 into which the cartridge may be inserted. The reader device 6000 may further include a status indicator 6040. The reader device 6000 is in communication with a user interface 6020, which user interface 6020 may include a user interface of a remote computing device, such as a smartphone, tablet, or other device having a test control application executing thereon.

When the cartridge 5120, 5200 is inserted into the cavity 6060 of the reader device 6000, the electrode interface 5214 of the cartridge interfaces with the electrode interface 6300 of the reader device 6000. This may allow the reader device 6000 to detect that a cartridge is inserted (e.g., by testing whether a communication path is established). In some embodiments, the optional electrical cartridge described above with reference to fig. 51B and 51C may activate the power supply circuitry of the reader device 6000 when the electrode interface 5214 of the cartridge is connected with the electrode interface 6030 of the reader device 6000. In addition, such communication may enable reader device 6000 to identify a particular inserted cassette 5120, test cassette 5200, and obtain the corresponding test protocol. The test protocol may include the duration of the test, the temperature of the test, characteristics of the positive sample impedance curve, and information output to the user based on various determined test results. In other embodiments, the reader device 6000 may receive an indication via the user interface 6020 that the cartridge is inserted (e.g., by a user inputting a "start test" command, and optionally a test cartridge identifier).

Memory 6005 includes one or more physical electronic storage devices configured to store computer-executable instructions for controlling the operation of reader device 6000 and data generated during use of reader device 6000. For example, memory 6005 can receive and store data from sense electrodes coupled to electrode interface 6030.

Processor 6010 includes one or more hardware processors that execute computer-executable instructions to control the operation of reader device 6000 during testing, for example, by controlling heater 6025, controlling communication module 6015 to interact with user interface 6020, and activating voltage source 6035. One example of a test operation is described with respect to FIG. 7A below. Processor 6010 may also be configured with instructions to determine the test results based on data received from the excitation electrodes of an inserted test cartridge, for example by implementing the procedure of FIG. 7B described below.

The communication module 6015 includes network-enabled hardware components (e.g., wired network components or wireless network components) for providing network communication between the reader device 6000 and a remote computing device. Suitable network components include WiFi, bluetooth, cellular modem, ethernet port or USB port, etc. Advantageously, the network capabilities may enable the reader device 6000 to interact with and be controlled by a remote computing device (e.g., one or more additional handheld computing devices, such as a smartphone, a tablet, etc.). In some embodiments, the remote device may communicate with additional remote computing systems (e.g., hospital information systems and/or laboratory information systems storing electronic medical records, national health agency databases, and computing devices of clinicians or other designated personnel). In addition, the networking capabilities may enable the reader device 6000 to receive information from remote computing devices over the network, such as updated signal cliff parameters for existing tests, new signal cliff parameters for new tests, and updated or new test protocols.

The user interface 6020 may be implemented within a remote device connected to the communication module 6015 via WiFi, bluetooth, or the like. The remote device may have a test control application installed thereon to provide a test system user interface for providing control options to a user and/or presenting test results and other test information on a display of the remote device. Further details of the user interface 6020 are described with reference to fig. 55A-55D.

A heater 6025 may be positioned adjacent to the cavity 6060 for heating the inserted cartridge to a desired temperature for the amplification procedure. Although described as being on a single side of the cavity 6060, in some embodiments, the heater 6025 may surround the cavity.

As described herein, the voltage source 6035 can provide an excitation signal at a predetermined voltage and a predetermined frequency to the excitation electrodes of the inserted test cartridge.

The status indicator 6040 may include any suitable notification device, such as one or more lights, sound generators, or the like. The operation of the lamp-based status indicator is described in more detail with reference to fig. 51A-51C.

Fig. 54 depicts a flowchart of an example procedure 701 for operating a reader device during testing as described herein. The program 701 may be executed by the reader apparatus 600 or the reader apparatus 6000 described above.

At block 706, the reader device 600 or the reader device 6000 may detect that the electrical cartridge has been removed from the reader device 600 or the reader device 6000. In some embodiments, the detection of block 706 may occur based on: the electrode interface 630 or the electrode interface 6030 of the reader device 600 or the reader device 6000 and the one or more contact plates 5214 of the electrical cartridge₁-5214₅(fig. 52D) disconnection of the signal path between the two.

At block 711, the reader apparatus 600 or the reader apparatus 6000 is automatically powered on in response to detecting removal of the electrical cartridge at block 706. In some implementations, the reader device may transmit a notification to the user interface 620 or the user interface 6020 device and/or illuminate one or more status lights of the status indicator 640 or the status indicator 6040 to indicate that the reader device 600 or the reader device 6000 is powered on and ready to receive the test cartridge 120, the test cartridge 200.

At block 716, reader apparatus 600 or reader apparatus 6000 may detect that cartridge 5120, cartridge 5200 has been inserted, e.g., in response to a user input or in response to establishing a signal path with an inserted cartridge. In some embodiments, cartridge 5120, cartridge 5200 may include an information element that identifies a particular test to be performed on reader device 600 or reader device 6000, and optionally test protocol information.

At block 721, the reader apparatus 600 or reader apparatus 6000 can heat the cartridge 5120, the cartridge 5200 to a specified temperature for amplification. For example, the temperature may be provided by information stored on cartridge 5120, cartridge 5200, or retrieved in an internal memory of reader device 600 or reader device 6000 in response to identification of cartridge 5120, cartridge 5200.

At decision block 725, the reader device 600 or reader device 6000 may determine whether the test is still within its specified test duration. For example, where the expected window of time for which a signal cliff should occur in a positive sample is known, the duration of the test may end at the end of the window or some predetermined period of time after the end of the window. If so, the process 701 transitions to optional decision block 730 or to block 735 (in embodiments where block 730 is omitted).

At optional decision block 730, reader device 600 or reader device 6000 determines whether to monitor test well amplification by recording data from the test well sense electrodes. For example, instructions may be provided to the reader device 600 or the reader device 6000 to monitor the impedance of the test wells only during a particular window or windows of the test. If the reader device 600 or the reader device 6000 determines not to monitor the test well amplification, the process 701 loops back to decision block 725.

If either the reader device 600 or the reader device 6000 determines to monitor the test well amplification, the process 701 transitions to block 735. At block 735, the reader device 600 or reader device 6000 provides an excitation signal to the excitation electrodes of the test wells of the inserted cartridge. As described above, the excitation signal may be an alternating current at a particular frequency and a particular voltage.

At block 740, the reader device 600 or the reader device 6000 detects and records data from the sensing electrodes of the test wells of the inserted cartridge. In some embodiments, this data may be stored for later analysis, for example after testing is complete. In some embodiments, the reader device 600 or the reader device 6000 may analyze this data in real time (e.g., while the test is still in progress) and may stop the test once a positive sample signal cliff is identified.

When the reader apparatus 600 or the reader apparatus 6000 determines at block 725 that the test has not been within its specified duration, the procedure 701 moves to block 745 to analyze the test data and output the test results. The test result may include an indication that the sample is positive or negative for the target test, or may more specifically indicate an estimated amount of the target in the sample tested. After the test is complete, further testing of the new cartridge may be performed by returning to block 715. Alternatively, the reader device 600 or the reader device 6000 may detect the insertion and power down of the electrical cartridge in response.

55A-55D depict screens of an example graphical user interface 5500 of a user device implementing an example test program that communicates with a reader device as described herein. The user interface 5500 may be the illustrated user interface 620 connected to the reader device 600 of fig. 6 or the reader device 6000 of fig. 59, for example. User interface 5500 may be implemented with any of reader device 5110, reader device 600, and/or cartridge 5120, cartridge 5200 described herein. The screens depicted in fig. 55A-55D may be displayed by, for example, an application executing on a smartphone or other user interface device paired with (e.g., via WiFi, bluetooth, etc.) the reader device 5110, the reader device 600, the reader device 6000, so as to allow a user to control and/or monitor the reader device 5110, the reader device 600, the reader device 6000 from the user interface device.

FIG. 55A depicts an initial pre-test screen that may be displayed after detection of an inserted cassette 5120, 5200. In one example, the user scans the cartridge identifier of the cartridge (e.g., cartridge identifier 5215 of fig. 52B) prior to inserting the cartridge into the reader device. When the device is inserted, the paired reader device detects the inserted cartridge and sends a message to the user interface device that the cartridge has been inserted. Then, the application displays the initial pre-test screen described in fig. 55A.

The screen before the initial test includes a status indication area 5505, a test recognition area 5510, a progress indication area 5515 containing a digital progress indication 5517 and a graphic progress indication 5519, and an input area 5520. Status indication area 5505 may include instructions, such as requesting the user to confirm the information in test identification area 5510. The test identification area 5510 includes information relating to the test to be performed, such as the name or other identifier of the test subject, the condition or target agent to be detected, and the like. In the initial pre-test screen of FIG. 55A, input area 5520 includes "cancel" and "begin test" options that are selectable by the user to allow the user to cancel the test or confirm the details and begin the test.

Fig. 55B depicts an in-test screen that may be displayed when the reader device is performing a test on a fluid sample within the cartridge. Status indication area 5505 indicates that a test is in progress. As the test progresses, the digital progress indication 5517 and the graphical progress indication 5519 are updated to display the current progress of the test. A user-selectable cancel test option is provided in the input area to allow the user to stop the test if desired.

FIG. 55C depicts an initial test complete screen that may be displayed when the reader device has completed a test and has analyzed the recorded test data to determine a test result. Status indication area 5505, digital progress indication 5517, and/or graphical progress indication 5519 may indicate that the test is complete. In input area 5520, a user selectable option is provided to view the test results.

FIG. 55D depicts a test result display screen for communicating test results to a user. The test identification area 5510 may still display some or all of the initially displayed test identification information. The test identification area 5510 may additionally display a result 5512, such as a positive or negative, or other condition associated with the test result. The input area 5520 may provide a user selectable option to continue (e.g., conduct additional tests, or communicate results, etc.).

55A-55D depict screens of an example graphical user interface 5500 of a user device implementing an example test program that communicates with a reader device as described herein. The user interface 5500 may be, for example, the illustrated user interface 6020 coupled to the reader device 5500 of fig. 55. User interface 5500 may be implemented with any of reader device 5110, reader device 6000, and/or cartridge 5120, cartridge 5200 described herein. The screens depicted in fig. 55A-55D may be displayed by, for example, an application executing on a smartphone or other user interface device paired with the reader device 5110, the reader device 6000 (e.g., via WiFi, bluetooth, etc.) to allow a user to control and/or monitor the reader device 5110, the reader device 6000 from the user interface device.

FIG. 55D depicts a test result display screen for communicating test results to a user. The test identification area 5510 may still display some or all of the initially displayed test identification information. The test identification area 5510 may additionally display a result 5512, such as a positive or negative, or other condition associated with the test result. The input area 5520 may provide a user selectable option to continue (e.g., conduct additional tests, communicate results, etc.).

Fig. 56A and 56B depict further examples of handheld detection systems 5600 for detecting a target. Similar to the system 5100 of fig. 51A-51C, the system 5600 can be implemented in conjunction with any of the target detection procedures, systems, and devices described herein. The system 5600 includes a reader device 5610 and a cartridge 5620 configured to fit within a cavity 5612 of the reader device 5610. The cartridge 5620 is sized and shaped to be grasped by a user to facilitate insertion and/or removal of the cartridge 5620 from the reader device 5610. The reader device 5610 may further include a light ring 5614 disposed about the cavity 5612. Light ring 5614 may include any or all of the light sources, colors, modes of operation, etc., described above with reference to light ring 5114 of fig. 51A-51C.

Fig. 57A-57J depict an exemplary cassette 5700 configured for detecting a target. As described herein, a target can be a viral target, a bacterial target, an antigen target, a parasite target, a microRNA target, an agricultural analyte, a nucleic acid target, a DNA target, a human DNA target, a genomic target, or a human genomic target. Some embodiments of the cartridge 5700 may be configured to test a single target, while some embodiments of the cartridge 5700 may be configured to test multiple targets. The cartridge 5700 includes a cartridge body 5710 and a cap 5750 configured to be mechanically coupled to the cartridge body 5710. When joined together, the cartridge body 5710 and cap 5750 may form an assembled cartridge 5700 for insertion into a reader device (e.g., the reader device 5600 of fig. 56A and 56B). As will be described in greater detail below, the cartridge 5710 may include a plurality of test wells therein such that a single cartridge 5700 may be configured for testing a single sample for a plurality of targets.

Fig. 57A and 57B depict a completed cassette 5700 that includes a cassette body 5710 and a cap 5750 that are connected together. In use, the cap 5750 and the cartridge body 5710 may operate to enclose a sample provided within the cartridge 5700, thereby preventing the test operator from being exposed to the sample and any liquid from escaping into the electronics of the associated reader device. The cartridge body 5710 and cap 5750 may be connected by a friction fit, a snap fit, and/or one or more mechanical or chemical fastening means. The connection of the cartridge 5710 and cap 5750 is discussed in more detail with reference to fig. 58A-58D.

The body 5710 and cap 5750 may be formed of a suitable fluid impermeable material (e.g., plastic, metal, etc.) and may be opaque, translucent, or transparent. The cassette 5710 may also include a transparent, translucent, or opaque cover surface (e.g., a Printed Circuit Board (PCB)5714 or other surface that partially defines a fluid flow path within the cassette 5710), and one or more electrode interfaces 5735. The PCB 5714, fluid flow channel, and electrode interface 5735 are discussed in more detail with reference to fig. 57E-57J. The cartridge body 5710 and/or cap 5750 may further include a cartridge identifier 5711. The cartridge identifier 5711 may include human-readable and/or machine-readable information, such as text, a barcode, a QR code, and so forth. The cartridge identifier 5711 may include any suitable information associated with the cartridge, such as information specifying the type of test, target agent, sample type, cartridge serial number or other individual cartridge identifier, and the like. In addition to serving as an identifier for a user having a test type associated with the cartridge 5700, the cartridge identifier 5711 may also be scanned by the user (e.g., using a user interface device in communication with the reader device) to communicate one or more test protocols to the reader device.

The cartridge body 5710 and/or cap 5750 may include ergonomic features (e.g., indentations, etc.) to facilitate gripping of the cartridge 5700. In the depicted example cartridge 5700, the cartridge body 5710 further includes an alignment groove 5712 positioned to align with the alignment groove 5752 of the cap 5750. The alignment groove 5752 of the cap 5750 terminates in a stop 5754 that is configured to engage a protrusion within a corresponding reader device (e.g., the reader device 5610 of fig. 56A and 56B) to define a fully inserted position of the cartridge 5700 within the reader device. The cap 5750 may further include a sample receiving area cap 5756, the sample receiving area cap 5756 being sized and shaped to close off an opening in the cap 5750 for receiving a swab or other sample carrier carrying a sample to be analyzed.

Fig. 57C and 57D depict the cap 5750 assembly of the cartridge 5700 of fig. 57A and 57B. The cap 5750 includes an elongated body that is at least partially hollow to receive a sample carrier, such as a swab or the like. The opening in the cap 5750 for receiving a sample carrier may be closed by a sample receiving area cap 5756, which sample receiving area cap 5756 may include one or more O-rings or other resilient structures to sealingly block the opening in the cap 5750.

The cap 5750 further includes a collar 5758 protruding from the cap 5750. Collar 5758 is sized and shaped to facilitate connection with cartridge body 5710. Collar 5758 generally comprises a hollow cylinder defining a plunger receiving aperture 5760 through which a fluid sample may pass from cap 5750 into cartridge body 5710. The collar 5758 includes interlocking fins 5762 that extend radially outward from the outer surface of the collar 5758, and receiving channels 5764 within the inner surface of the collar 1058. Each receiving channel 5764 terminates in a widened portion 5765 such that the receiving channels 5764 are configured to receive and retain one or more snap-fit connectors of the cassette 5710, as will be described with reference to fig. 58A-58D.

The cap 5750 may also include one or more liquid components therein to be mixed with the received sample. For example, the liquid component may include one or more amplification reagents used in the test procedure, buffer solutions, water, mucin-mitigating agents (mucin mitigling agents), or other desired liquid components. The particular selection and chemistry of these liquids can be tailored to suit the particular target or targets for which the cartridge 5700 is designed for testing. In some embodiments, the liquid component can be contained within a blister package in cap 5750. The blister pack may be pierced, for example, by insertion of a sample carrier, connection of the cap 5750 to the cartridge 5710, and the like.

Fig. 57E-57J depict the cartridge body 5710 components of the cartridge 5700 of fig. 57A and 57B. Fig. 57E-57G are external views of the cassette body 5710. Fig. 57H-57J depict a partially translucent cassette 5710 to illustrate fluid flow passages integrally formed therein. Referring to fig. 57E-57G, a cartridge 5710 includes a base 5716 and a hollow plunger 5718 rotatably connected within a receiving hole 5726 of the base such that the plunger 1018 may be rotated about its longitudinal axis while being retained within the receiving hole 5726.

The plunger 5718 includes a generally cylindrical body that is sized and shaped to fit within the plunger receiving bore 5760 of the cap 5750 (fig. 57C and 57D). The closure portion 5720 of the plunger 5718 is disposed at a distal end of the plunger 5718 and may include one or more resilient structures (e.g., one or more O-rings, integrally formed elastomeric structures, etc.) of suitable diameter to sealingly engage an inner wall of the plunger receiving bore 5760 of the cap 5750. A sacrificial seal 5724 (e.g., a layer of metal foil or other thin material) may be provided to prevent the interior of the case 5710 from being exposed to the atmosphere prior to use. The plunger 5718 additionally includes one or more snap-fit clips 5722 that extend along the exterior of the plunger 5718 parallel to the longitudinal axis of the plunger. Snap-fit clip 5722 is sized and shaped to engage and be retained within receiving channel 5764 of plunger receiving bore 5760 of cap 5750. The plunger bottom plate 5719 is rotationally fixed to the plunger 5718 (e.g., may be integrally formed with the plunger 5718). The diameter of the plunger bottom plate 5719 may be substantially equal to or slightly larger than the outer diameter of the collar 5758 of the cap 5750.

In some embodiments, one or more liquid components can be included within the plunger 5718 in place of or in addition to the liquid components included within the cap 5750. For example, the sacrificial seal 5724 may contain a liquid component within the plunger 5718, and/or the liquid component may be contained within a blister package in the plunger 5718. The liquid component contained within the plunger may include one or more amplification reagents, buffer solutions, water, mucin-reducing agents, or other desired liquid components for the testing procedure. The particular selection and chemistry of these liquids can be tailored to suit the particular target or targets for which the cartridge 5700 is designed for testing. The blister package may be pierced, for example, by inserting a sample carrier, attaching the cap 5750 to the carton 5710, and the like.

The receiving hole 5726 is coaxial with the plunger 5718 and has a generally cylindrical profile with a diameter substantially equal to or slightly larger than the collar 5758 of the cap 5750 and/or the plunger bottom plate 5719. The receiving hole 5726 further includes a cutout 5728, the cutout 5728 being sized to receive the interlocking fin 5762 of the cap 5750. A stop 5730 within the cutout 5728 is disposed within the cutout 5728 to prevent longitudinal movement of the interlocking fin 5762 in certain rotational positions, as will be described in greater detail with reference to fig. 58A-58D.

A Printed Circuit Board (PCB)5714 or other generally planar layer is disposed along a surface of the case 5710 opposite the receiving aperture 5726. In some embodiments, the PCB 5714 may perform heating and/or electrode interface functions and may further serve as a boundary for one or more fluid flow passages within the cartridge body 5710. While the example PCB 5714 described herein includes heating and electrode interface functions, these functions may be performed by more than two discrete elements in the cartridge body 5710 as well. In some embodiments, heating may be achieved by heating elements located within the respective reader devices, instead of or in addition to heating elements disposed on or in the cartridge 5700.

The PCB 5714 includes one or more layers of a generally planar surface on which one or more traces (tracks) are disposed. For example, the PCB 5714 may include one or more flexible circuits, rigid printed circuit boards, or any other suitable circuit, including one or more current paths disposed on a generally planar substrate. One or more heating traces 5732 electrically connect test hole heating element 5733 to heating current plate 5734. When the cartridge 5700 is inserted into a reader device, the heating current plate 5734 may be in contact with contacts of a current source of the reader device such that a current may be provided to the test well heating element 5733 to heat a fluid sample in one or more test wells of the cartridge 5710.

The PCB 5714 further includes a pair of electrodes 5736, 5738 (e.g., excitation electrodes and sensor electrodes) corresponding to each test well. In some embodiments, if the PCB 5714 serves as a boundary of a test well, the electrodes 5736, 5738 may be in direct contact with the fluid sample in each test well. Each electrode 5736, 5738 is electrically connected to electrode interface plate 5735 by electrode trace 5737, 5739 of PCB 5714.

In various embodiments, the PCB 5714 may include one or more layers. For example, in some embodiments, the PCB is a flexible circuit that includes an electrode layer and a heating layer that is separate from the electrode layer. Electrode layers may include electrode 5736, electrode 5738, and electrode trace 5737, electrode trace 5739, and/or electrode interface plate 5735. Heating layer may include heating elements 5733, heating traces 5732, and/or heating current plates 5734. In some aspects, the electrode layer and the heating layer may be disposed on opposite sides of a common substrate, or provided on separate substrates. Preferably, the PCB 5714 may be arranged such that the electrode layer containing the electrodes 5736, 5738 is adjacent to the cartridge 5710 and the electrodes 1036, 1038 are fluidly connected to the test wells 5740.

Fig. 57H-57J depict additional views of the cartridge body 5710, wherein the base portion 5716 is shown as transparent to show the fluid flow channels contained therein. The base 5716 may comprise any suitable fluid impermeable material, such as plastic or metal. The base 5716 may be formed by one or more processes (e.g., injection molding, die casting, milling, etc.) such that the described fluid flow passages may be integrally formed therein.

The base portion 5716 of fig. 57H-57J includes eight substantially identical fluid flow channels, each fluid flow channel including a test hole 5740. Various embodiments may include less than eight or more than eight fluid flow channels and test holes 5740 without departing from the scope of the present disclosure. For example, the base 5716 may include 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, or more fluid flow channels. Multiple identical or similar fluid flow channels within the base may cater for simultaneous testing of multiple different targets and/or multiple simultaneous tests of the same target (e.g., to improve reliability of results).

Each fluid flow path includes an inlet channel 5742, a lateral channel 5744, a test hole 5740, and an outlet channel 5746. Each inlet channel 5742 extends vertically through the base 5716 to fluidly connect a first end adjacent the plunger bottom plate 5719 to an opposite end adjacent the PCB 5714. Each lateral channel 5744 fluidly connects an inlet channel 5742 to a respective test well 5740. Each outlet channel 5746 extends perpendicularly from the test hole 5740 to fluidly connect the test hole 5740 to the bottom of the plunger bottom plate 5719. In the cartridge 5710 of fig. 57H-57J, the PCB 5714 forms a boundary that partially defines each lateral channel 5744 and each test hole 5740. In this example embodiment, the PCB 5714 may be oriented with the electrodes 5736, 5738 on a side adjacent the cartridge 5710 such that the electrodes 5736, 5738 are in contact with the interior of the test hole 5740.

As shown in fig. 57J, each inlet channel 5742 is disposed radially outward from the longitudinal axis of the plunger floor 5719 at substantially the same distance as the array of sample inlets 5741 of the plunger floor 5719. Similarly, each outlet channel 5746 is disposed radially outward from the longitudinal axis of the plunger floor 5719 at substantially the same distance as the outer ends of the array of J-shaped sample outlets 5748 of the plunger floor 5719. The inner end of the J-shaped sample outlet 5748 and the sample inlet 5741 are fluidly connected to one or more interior spaces within the plunger 5718 by a plunger floor 5719. Thus, as will be described with reference to fig. 58B and 58C, when the plunger bottom plate 5719 is rotated to the engaged position, the sample inlet 5741 is aligned with the inlet channel 5742 and the sample outlet 5748 is aligned with the outlet channel 5746 so that the fluid sample within the plunger 5718 can flow into and fill the fluid flow path within the base 5716 of the cartridge 5710.

Fig. 58A-58D illustrate mechanical fluid transfer aspects of cassettes 5620, 5700 described herein. Similar to the fluid transfer aspects described with reference to fig. 53A-53E, the cartridge body 5710 and cap 5750 are configured to generate air pressure when coupled together such that the air pressure pushes the fluid sample through the fluid flow channels of the cartridge body 5710. Cap 5750 is shown in semi-transparent in fig. 58A-58D and cassette 5710 is shown in semi-transparent in fig. 58C and 58D to show the internal features of cap 5750 and cassette 5710. The cartridge body 5710 is depicted in fig. 58C with the PCB 5714 removed.

Referring to fig. 58A, a fluid sample can be received within the cap 5750. For example, a swab or other sample carrier can be inserted into an opening of the cap 5750 opposite the box body 5710, which can then be closed by the sample receiving area cap 5756 to contain a sample within the cap (e.g., within the plunger receiving aperture 5760 or other interior space within the cap 5750). When the fluid sample has been enclosed within the cap 5750, the procedure of fig. 58A-58D may be used to mechanically couple the cartridge 5710 to the cap 5750 and move the fluid sample into the cartridge 5710.

As shown in fig. 58A, the mechanical coupling of the cartridge body 5710 and cap 5750 is initiated by inserting the plunger 5718 of the cartridge body 5710 into the plunger receiving hole 5760 of the cap 5750. A closure portion 5720 of plunger 5718 may sealingly engage the interior of plunger receiving hole 5760 to trap and begin compressing a volume of air within plunger receiving hole 5760. As plunger 5718 slides into plunger receiving aperture 5760, snap-fit clips 5722 slide within receiving channel 5764 until they enter widened portion 5765 of receiving channel 5764, where they are retained longitudinally. The retention of the snap-fit clip 5722 within the receiving channel 5764 prevents the cap 5750 from being removed from the cartridge body 5710 and further rotationally locks the cap 5750 with the plunger 5718, allowing a user to rotate the plunger 5718 and plunger floor 5719 by rotating the cap 5750 (which may be relatively large and easy to manually operate). The cap 5750 and the carton body 5710 can be slid together until the collar 5758 of the cap 5750 is partially within the receiving hole 5726 of the carton body 5710 and the interlocking fins 5762 of the collar 5758 contact the stops 5730 within the cut-outs 5728 (fig. 57F).

As shown in fig. 58B, the cap 5750, plunger 5718, and plunger floor 5719 can then be rotated about the longitudinal axis. Because the snap-fit clips 5722 rotationally secure the plunger 5718 to the cap 5750, rotation of the plunger 5718 and plunger bottom plate 5719 may be achieved by rotating the cap 5750. Cap 5750 may be rotated until interlocking fins 5762 are blocked by the sides of cutout 5728 of receiving hole 5726. In the example cartridge 5700, the cutouts 5728 and the interlocking fins 5762 are sized to allow a total rotation of about 22.5 ° when the interlocking fins 5762 are within the cutouts 5728. However, other exemplary cartridges may function within different ranges of rotational motion, such as between about 5 ° and about 90 °, between about 10 ° and about 45 °, between about 15 ° and about 30 °, between about 20 ° and about 25 °, or any angle, or a sub-range of angles therebetween. In some embodiments in a blister package in which the liquid component is contained within the plunger 5718 and/or cap 5750, rotation of the components in fig. 58B may cause the blister package to be punctured releasing the liquid component for mixing with the sample.

Referring to fig. 58C, the cap 5750 can be moved downward along the longitudinal axis such that the collar 5758 is moved further into the receiving hole 5726. Because the stop 5730 extends along only a portion of the cutout 5728, the rotational motion described with reference to fig. 58B moves the interlocking fin 5762 away from the stop 5730 so that the interlocking fin 5762 can move to the inner portion 5729 of the cutout 5728. As shown in fig. 58C, the rotated position of the plunger floor 5719 substantially aligns the inlet and outlet tunnels 5742 and 5746 with the sample inlet 5741 and sample outlet 1048, respectively. When the cap 5750 is pressed further onto the cartridge body 5710 to the position of fig. 11C, the sample carrier and/or one or more internal structures within the cap 5750 may mechanically contact and puncture a seal (e.g., the sacrificial seal 5724 of fig. 57F) on the plunger 5718 or within the plunger 5718, thereby allowing trapped air compressed by the plunger 5718 to flow into the interior of the plunger and push the fluid sample through the plunger floor 5719 and into the fluid flow path of the cartridge body 5710. In some embodiments in a blister package in which the liquid component is contained within the plunger 5718 and/or cap 5750, longitudinal movement of the components in fig. 58C may cause the blister package to be punctured releasing the liquid component for mixing with the sample.

The flow of fluid sample through the fluid flow channels of the cartridge body 5710 will now be described with continued reference to fig. 58C. Fig. 58C illustrates the flow of a portion of a fluid sample through a single exemplary fluid flow channel 5805 within a cartridge 5710, with circled numbers shown as labels at certain points along the fluid flow channel. Circled numbers are discussed below as example steps of the progression of the fluid sample as it travels through the flow cell 5805 in the cartridge 5710, each step including a directional arrow showing the direction of fluid travel for that step.

In step (1), when compressed air is allowed to flow into the plunger 5718, a fluid sample is forced through the sample inlet 5741 into the inlet passage 5742. The fluid sample travels along the inlet channel 5742 toward the lateral channel 5744.

At step (2), the fluid sample reaches the PCB 5714 boundary of the fluid flow channel and begins to travel parallel to the PCB 5714 within the lateral channel 5744. At step (3), the fluid sample continues through the curved lateral channel 5744 toward the test hole 5740.

At step (4), the fluid sample enters the test well 5740. The test wells 5740 may contain one or more reagents. The agitation caused by the turbulent flow of the fluid sample within the relatively large space of the test hole 5740 causes the reagent and sample to mix. In some embodiments, the reagent and the fluid sample are mixed into a uniform solution, wherein the reagent is uniformly distributed throughout the fluid sample. The depth, width, shape, and/or cross-sectional profile of the test wells 5740 may be selected to promote mixing of reagents and fluid sample.

In step (5), any excess fluid sample is pushed from the test hole 5740 into the outlet channel 5746 and into the outer end of the J-shaped sample outlet 5748. When the excess fluid sample reaches the inner end of the sample outlet 5748, it passes through a corresponding opening in the plunger floor 5719 and is discharged into the plunger 5718 at step (6). In some embodiments, the internal volume of the plunger 5718 and/or cap 5750 is separated from the internal volume fluidly connected to the sample inlet 5741 so as to produce a directional flow of the fluid sample along the fluid flow path 5805.

As shown in fig. 58C, after the cap 5750 is fully pressed onto the cartridge body 5710, fluid sample flow through the fluid flow channels within the cartridge body is initiated, as compressed air forces the fluid sample into the fluid flow channels 5805, the cartridge 5700 can reach a pressure equilibrium, the fluid flow channels 5805 are filled with the fluid sample, and a portion of the sample is discharged back into the plunger 5718 and/or cap 5750. Pressure equilibrium can be reached relatively quickly, for example, in 10 seconds, 5 seconds, 2 seconds, 1 second, or less.

Referring now to fig. 58D, as fluid flow path 5705 is filled, cap 5750, plunger 5718, and plunger bottom plate 5719 may again rotate relative to cartridge body 5710. In the example routine of fig. 58A-58D, the assembly is rotated in the opposite direction by the same angular displacement relative to the rotation of fig. 58B. Thus, as shown in fig. 58D, plunger floor 5719 is rotated relative to fluid flow path 5805 such that inlet and outlet channels 5742, 5746 are no longer aligned with sample inlets 5741 and sample outlets 5748, thereby closing fluid flow path 5805 and retaining the fluid sample in test hole 5740 for testing. This final rotation step additionally causes interlocking fins 5762 of cap 5750 to remain under stops 5730 within inner portion 5729 of receiving bore cutout 5728, completing and securing the mechanical connection of cassette 5710 and cap 5750. In addition, the final rotation step substantially aligns the outer profiles of the cap 5750 and cartridge body 5710 such that the assembled cartridge 5700 may be inserted into a reader device to perform one or more tests on a fluid sample contained therein.

Fig. 60A-60I illustrate further embodiments of a cartridge 1200 configured for detecting a target. As described herein, the target can be a viral target, a bacterial target, an antigenic target, a parasitic target, a mciroRNA target, or an agricultural analyte. Some embodiments of the cartridge 1200 may be configured to test a single target, while some embodiments of the cartridge may be configured to test multiple targets. The cartridge 1200 includes a cartridge body 1202 and a swab assembly 1220, the swab assembly 1220 configured to mechanically couple to the cartridge body 1202 at a swab assembly insertion point 1208.

The cartridge 1202 includes a thin film test assembly 1204 and an ergonomic frame 1206 configured to be grasped by a user. The thin film test assembly 1204 generally includes a plurality of test wells 1258, a pinch valve 1214 for isolating fluid within the test wells 1258, a gas permeable filter 1212 (e.g., a membrane, etc.), and an electrode interface 1210 for electrically connecting the electrodes to circuitry of a reader device at the test wells. The cartridge further includes a fluidic piston 1218 and a transition point 1216 for introducing a fluid sample from the swab assembly 1220 into the membrane test assembly 1204. Features of the thin film test assembly 1204 are discussed in more detail with reference to fig. 60H and 60I.

Referring now to fig. 60C-60G, the swab assembly 1220 includes a tube 1222, a slide 1224, and a cap 1234 that are configured to fit together to form a substantially enclosed swab assembly 1220. The tube 1222 includes a tube passage 1226, the tube passage 1226 being sized and shaped to receive a shaft 1236 of the cap 1234. The tube passage 1226 can be enclosed, such as with a foil seal or the like, to contain one or more liquid reagents, buffers, or the like, during transport and/or prior to use of the swab assembly 1220. The tube passage 1226 may have an hourglass profile, a bivalve profile, or other shape configured to promote mixing of fluids therein. In some embodiments, the tube passage 1226 can further include internal threads or other protruding features further configured to promote mixing of fluids in the tube passage 1226. The slide 1224 includes a hollow structure configured to fit around the engagement end 1223 of the tube 1222. The tube 1222 further includes one or more snap-fit clips 1232, the snap-fit clips 1232 configured to interlock with the first and second snap-fit openings 1228, 1230 of the slider 1224.

Fig. 60E and 60F show the cap 1234. Fig. 60E is a perspective view of the cap. Fig. 12F is a cross-sectional view of the cap showing its internal components. Cap 1234 includes a hollow shaft 1236 surrounding a cap passageway 1238 and a hollow upper portion 1244 surrounding the metering volume 1240. The upper portion 1244 may further include a closure portion 1242 comprising a resilient material (e.g., rubber, resilient plastic, or other elastomeric material) that is sized and shaped to sealingly engage the interior of the slide 1224. The foil seal 1246 may enclose one or more liquid or dried reagents (e.g., amplification reaction solution, RPA reagent solution, or reagent solution configured for a first or second isothermal amplification reaction) within the cap 1234. Vent 1248 may be fluidly connected to metering volume 1240 to allow any gases trapped within cap 1234 to vent. The shaft 1236 terminates at a cap inlet 1250 fluidly coupled to the cap passage 1238, the cap inlet 1250 may contain one or more portions of a filter 1252, the filter 1252 configured to allow fluid to flow into the cap passage 1238. In some embodiments, an additional seal may be provided over cap inlet 1250 to enclose any liquid or dried reagents within cover 1234 prior to use.

Referring to fig. 60C-60G together, and in particular to fig. 60G, an exemplary process of introducing a sample into a swab assembly 1220 is now described. The cap 1234 is separated from the tube 1222 and slide 1224 prior to introduction of the sample. Tube 1222 contains a liquid, including one or more liquid reagents, buffers, etc., enclosed within tube 1222 by a seal at the engagement end 1223 of tube 1222. The cap 1234 includes one or more additional liquid reagents, buffers, etc., enclosed within the cap 1234 by a foil seal 1246. In the initial configuration, the snap-fit clip 1232 is engaged in the first snap opening 1228.

A sample (e.g., a nasal swab or other swab-collected sample) may be received on the swab. Prior to insertion of the swab into the swab assembly 1220, the slide 1224 is moved in the first direction 1254 relative to the tube 1222 such that the snap-fit clips 1232 of the tube 1222 engage the second snap-fit openings 1230. This movement may cause the inner structure 1231 of slide 1224 to break the foil seal at the tube opening to expose the liquid reagent or buffer contained within the tube passage 1226.

When the tube passage 1226 is exposed, a swab can be introduced into the tube passage 1226 such that a sample on the swab mixes with the liquid reagent within the tube passage 1226. The internal profile and/or other mixing features of the tube passage 1226 can facilitate mixing of the sample with the liquid reagent to form the test fluid. In some embodiments, the swab can be snapped off the handle such that the portion of the swab containing the sample remains within the tube channel 1226.

After the sample has been introduced into the tube channel 1226 to form the test fluid, the cap 1234 may be mechanically connected to the tube 1222 and slide 1224 to complete the swab assembly 1220. If a seal is provided around the cap inlet 1250, the seal may be removed. The cap shaft 1236 is inserted through the slide 1224 and the cap 1234 is pushed into the slide 1224 and the tube passage 1222 so that the closing portion 1242 sealingly engages the interior of the slide 1242. As cap 1234 continues to move along direction 1254, air and fluid are compressed within tube channel 1222 to drive the mixed test fluid into metered volume 1240 through cap inlet 1250 and cap channel 1238. Any gas (e.g., air) present within the metered volume 1240 can be vented outward through the vent 1248. A filter 1252 at the cap inlet 1250 prevents solids (e.g., solids within a swab sample or debris of the swab itself) from entering the cap 1234. The location of the cap inlet 1250 at the end of the tube passage 1226 distal to the metering volume 1240 may advantageously allow the inlet 1250 to receive an optimal portion of the test fluid if the test fluid does not completely reach a homogeneous mixture. When the cap 1234 has been sealingly inserted into the tube 1222 and the slide 1224, the swab assembly 1220 is fully assembled, and any liquid therein is retained within the swab assembly 1220 by the foil seal 1246 of the cap 1234. The swab assembly 1220 may then be placed into the swab assembly insertion point 1208 of the cartridge 1200 to introduce the test fluid into the thin film test assembly 1204.

Referring now to fig. 60B, 60H, and 60I, the thin film test assembly 1204 includes a substrate 1207 surrounding a plurality of test holes 1258. One boundary of the test aperture 1258 is formed by a cover film 1205 containing a plurality of pinch valves 1214 that form part of the fluid flow path into the thin film test assembly 1204. Within each test hole 1258 is provided a pair of electrodes 1260, which electrodes 1260 may be formed in correspondence with any of the electrodes described elsewhere herein, and which electrodes 1260 are electrically connected to an electrode connection plate 1211 for connection to an electrode interface 1210 of a reader device.

When the swab assembly 1220 is inserted into the swab assembly insertion point 1208 of the cartridge 1200, fluid within the metering volume 1240 of the swab assembly 1220 flows through the transition point 1216 and along the fluid flow path within the thin film test assembly 1204 to fill the test well 1258. Any gas (e.g., air) within the membrane test assembly 1204 may be displaced from the test aperture 1258 and vented at the vent 1262 and/or the gas permeable filter 1212.

The cartridge 1200 may then be inserted into a reader device that is sized and shaped to receive the cartridge 1200. When the cartridge body 1200 is inserted into the reader device, the electrical contacts within the reader device contact the electrode connection plate 1211 of the cartridge 1200. Further, the opening in the reader device for receiving the cartridge 1200 has a width selected such that an inner surface of the reader device compresses and/or squeezes (crushes) the pinch valve 1214 preventing fluid flow therethrough after the cartridge 1200 has been inserted into the reader device. In some embodiments, the pinch valve 1214 may comprise a thermoformed plastic or other material selected such that the pinch valve 1214 may be squeezed and closed without rupturing and allowing the test fluid to escape. When the pinch valves 1214 are compressed and/or squeezed, the test fluid within each test hole 1258 is fluidly isolated within the test hole 1258 for testing. Heating and testing of the test fluid in the test wells 1258 may then be performed as described above with reference to cartridges 200, 1000. In some embodiments, the test wells 1258 may be pre-loaded with one or more primers (e.g., spot-dried, powdered, or other non-liquid primers) or other reagents corresponding to the test to be performed and/or the target reagents to be detected in each test well 1258. Some or all of the test wells 1258 may contain the same or different primers as primers present in other test wells 1258, depending on the individual test to be performed and/or the target agent to be detected in each test well 1258.

Overview of an exemplary device

Some embodiments of the methods, systems, and compositions provided herein include devices comprising an excitation electrode and a sensor electrode. In some embodiments, the excitation electrode and the sensor electrode measure an electrical property of the sample. In some embodiments, the electrical property comprises complex admittance, impedance, conductivity, resistivity, resistance, or permittivity.

In some embodiments, the electrical property is measured for a sample having an electrical property that does not change during the measurement. In some embodiments, the electrical property is measured for a sample having a dynamic electrical property. In some such embodiments, the dynamic electrical property is measured in real time.

In some embodiments, the excitation signal is applied to the excitation electrode. The excitation signal may comprise a direct current or a direct voltage, and/or an alternating current or an alternating voltage. In some embodiments, the excitation signal is capacitively coupled to/through the sample. In some embodiments, the excitation electrodes and/or the sensor electrodes are passivated to prevent direct contact between the sample and the electrodes.

In some embodiments, the parameters are optimized for the electrical properties of the sample. In some such embodiments, the parameters may include applied voltage, applied frequency, and/or electrode configuration relative to sample volume size and/or geometry.

In some embodiments, the voltage and frequency of the excitation voltage may be fixed or varied during the measurement. For example, the measurements may involve sweeping the voltage and frequency during detection, or selecting a particular voltage and a particular frequency that may be optimized for each sample. In some embodiments, the excitation voltage induces a current on the signal electrode that may vary with the admittance of the device and/or the sample characteristics.

In some embodiments, the detection parameters are optimized by modeling the admittance, device, and sample with a lumped parameter equivalent circuit consisting of an electrode-sample coupling impedance, a sample impedance, and an inter-electrode parasitic impedance. The parameters of the lumped parameter equivalent circuit are determined by measuring the admittance of the electrode-sample system at one or more excitation frequencies of the apparatus. In some embodiments, both amplitude sensitive detection techniques and phase sensitive detection techniques are used to measure the complex (digital with both real and imaginary components) admittance of the electrode-sample system. In some embodiments, the frequencies corresponding to transitions between frequency regions are determined by measuring admittances across a wide range of frequencies to optimize detection parameters. In some embodiments, the frequencies corresponding to transitions between frequency regions are determined by calculation from values given in a lumped parameter model to optimize the detection parameters.

In some embodiments, the admittance of the capacitively coupled electrode-sample system comprises three frequency regions: a low frequency region dominated by the electrode-sample coupling impedance, a medium frequency region dominated by the sample impedance, and a high frequency region dominated by the parasitic inter-electrode impedance. The admittance in the electrode-sample coupling region is capacitive in nature and is characterized by an amplitude that increases linearly with frequency, with a phase of 90 degrees. The admittance in the sample region is electrically conductive in nature and is characterized by no significant change in admittance with respect to frequency, with a phase of about 0 degrees. The admittance interelectrode region is capacitive in nature and is characterized by an amplitude that increases linearly with frequency and a phase of 90 degrees.

In some embodiments, the induced current at the pickup electrode is related to the excitation voltage and the complex admittance by the relationship:

current ═ (complex admittance) × (voltage)

In some embodiments, the device measures both the excitation voltage amplitude and the induced current amplitude to determine the amplitude of the complex admittance. In some embodiments, the device is calibrated to a known excitation voltage and the magnitude of the induced current is measured. To determine the phase of the complex admittance, a device may measure the relative phase difference between the excitation voltage and the induced current.

In some embodiments, the amplitude and phase are measured directly.

In some embodiments, the amplitude and phase are measured indirectly, for example by using both synchronous and asynchronous detection. The synchronous detector gives an in-phase component of the induced current. Asynchronous detectors give orthogonal components of the induced current. The two components may be combined to determine the complex admittance.

In some embodiments, the electrodes are not passivated.

In some embodiments, the excitation electrode and/or the detection electrode are passivated. The excitation electrode and/or the detection electrode may be passivated to prevent, for example, undesired adhesion, contamination, adsorption, or other detrimental physical interaction between the electrodes and the sample or component therein. In some embodiments, the passivation layer comprises a dielectric material. In some embodiments, passivation enables efficient capacitive coupling from the electrode to the sample. The efficiency of the coupling is determined by measuring characteristics of the electrode/sample system (which may include, for example, the dielectric properties of the passivation layer, the thickness of the passivation layer, the area of the passivation/sample interface, the passivation surface roughness, the double layer at the sample/passivation interface, the temperature, the applied voltage and applied frequency, the electrical properties of the sample, the electrical properties of the electrode material, and/or the chemical properties).

In some embodiments, electrode construction and fabrication is optimized to mitigate undesirable parasitic coupling between electrodes. This may be accomplished by electric field shielding, the use of a varying dielectric constant electrode substrate (dielectric constant electrode substrate), layout optimization, and/or a ground plane.

Overview of an example apparatus for detecting biomolecules

Some embodiments of the methods, systems, and compositions provided herein include a device for detecting a target (e.g., a biomolecule). In some such embodiments, the measurement of the electrical properties of the sample is used as a detection strategy for a biomolecular assay.

In some embodiments, the target is a nucleic acid, protein, small molecule, drug, metabolite, toxin, parasite, whole virus, bacterium, or spore, or any other antigen that can be recognized and/or bound by the capture probe moiety and/or the detection probe moiety. In some embodiments, carbohydrates can be detected by carbohydrate binding proteins (e.g., galectins or lectins).

In some embodiments, the target is a nucleic acid. In some embodiments, the method comprises nucleic acid amplification. In some embodiments, the amplification comprises isothermal amplification (e.g., LAMP). In some embodiments, the nucleic acid amplification reaction is quantified by measuring an electrical property of the reaction solution or a change therein. In some embodiments, the electrical property of the amplification reaction is measured in real time during the reaction, or a comparative measurement using the electrical property measurements before and after the reaction.

In some embodiments, the target antigen is detected via specific binding of a detection probe (e.g., an antibody, aptamer, or other molecular recognition moiety and/or binding moiety) to the antigen. In one exemplary embodiment, a detection antibody is linked to a nucleic acid sequence to form an antibody-nucleic acid chimeric complex. For the purpose of detecting the antigen, the chimeric complex is synthesized prior to the assay. Many different nucleic acids can be conjugated to a single antibody, thereby increasing the sensitivity of detection of chimeric complex binding to antigen. As described herein, after removing any excess chimeric complex that is not bound to the antigen, the nucleic acid portion of the chimeric complex is amplified and the amplification reaction is quantified by measurement of the electrical properties of the reaction solution (or changes therein). In this way, the degree of amplification of the nucleic acid (bound to the antigen by the chimeric complex) is indicative of the presence of the target antigen and allows quantification of the antigen. Secondary amplification, which represents antigen recognition, used in conjunction with electrical detection allows greater ease, sensitivity, and dynamic range than other antigen detection methods.

In some embodiments, the capture probes (e.g., antibodies, aptamers, or other molecular recognition and/or binding moieties for antigens) are bound to the surface by conjugation or ligation. The immobilization of the capture probes to the surface allows for the removal of excess, unbound reagents and/or antigens by washing. The chimeric complex binds to the surface-captured antigen, allowing unbound chimeric complex to be removed by washing. In this way, only the captured antigen is retained for detection of the chimeric complex. An example implementation is depicted in fig. 8. In some embodiments, the capture probe and the detection antibody are the same.

In some embodiments, the capture probes are immobilized to the surface by covalent conjugation, the use of streptavidin-biotin linkages, or other bioconjugation and molecular immobilization methods commonly used and familiar to those skilled in the art. In some embodiments, the surface is a planar surface, a scaffold, a filter, a microsphere, a particle of any shape, a nanoparticle or bead, or the like. An example implementation is depicted in fig. 9.

Overview of an exemplary magnetic bead

Some embodiments of the methods, systems, and compositions provided herein include magnetic beads or uses thereof. In some embodiments, the microsphere, particle or bead is magnetic and/or magnetizable. In these embodiments, the use of a magnetic support may facilitate washing of the beads to remove excess antigen and/or non-specifically adsorbed chimeric complex from the surface. Methods, including the use of magnetic particle supports, may include Magnetic Amplified Immunoassays (MAIA). An exemplary embodiment is depicted in fig. 10.

In some embodimentsMagnetic beads are used to capture targets and for magnetophoretic manipulation (magnetophoretic manipulation) in the context of pure electrical (MEMS) sample processing and/or amplification/detection cartridges and to reduce or eliminate the dependence on flow/pressure-driven mobility in vivo. In some embodiments, magnetic beads are used to extract and/or concentrate target genomic material from a sample. See, e.g., Tekin, HC., et al, Lab Chip DOI:10.1039/c3lc50477h, which is incorporated by reference herein in its entirety. Automated microfluidic processing platforms for use in embodiments provided herein are described in Sasso, LA., et al, microfluidic nanofluidics.13: 603-. Examples of beads for use in embodiments provided herein include for Nucleic Acid IVD (ThermoFisher Scientific), orSILANE Viral NA Kit(ThermoFisher Scientific)。

⁴Overview of exemplary fCD excitation and detection

In some embodiments, the disclosed devices, systems, and/or methods utilize fC-based⁴Strategy D to monitor nucleic acid amplification in real time. Thus, the one or more phase sensitive conductivity measurements may be indicative of one or more targets within the sample.

In some aspects, the method includes a fast scan frequency at a particular drive voltage value to determine an optimal excitation frequency (f) at which sample conductivity associated with amplification is maximum_opt). At f_optIn the following, the sensor outputs a minimum value corresponding to the relative phase difference between the excitation voltage and the induced current, thereby making it possible to perform highly sensitive bio-molecular quantification by conductivity measurement.

In some embodiments, fC⁴The detection system employs at least two electrodes. Two electrodes are placed in relatively close proximity to the microchannel in which nucleic acid amplification is performed. To one of the two electrodesAn AC signal is applied. The electrode to which the signal is applied can be capacitively coupled to the second of the two electrodes through the microchannel. Thus, in some aspects, the first electrode is a signal electrode and the second electrode is a signal electrode.

Typically, the signal detected at the signal electrode has the same frequency as the AC signal applied to the signal electrode, but is smaller in amplitude and has a negative phase shift. Subsequently, the pickup current may be amplified. In some aspects, the pickup current is converted to a voltage. In some aspects, the voltage is a rectified voltage. In some aspects, the rectified voltage is converted to a DC signal using a low pass filter. The signal may be biased to zero before being sent to the DAQ system for further processing.

The system described above may be represented by a series of capacitors and resistors. The change in conductivity that occurs during nucleic acid amplification within the channel can cause the overall impedance of the system to decrease, thereby causing an increase in the level of the pickup signal generated. Such a change in the level of the resulting output signal may appear as one or more peaks on the DAQ system.

The signal generation and demodulation electronics are implemented with circuitry. For example, a printed circuit board ("PCB"), ASIC device, or other integrated circuit ("IC") is fabricated using conventional fabrication and assembly techniques. In some aspects, such electronic devices are designed, in whole or in part, as single-use components and/or disposable components. The physical geometry and electrical properties (passivation layer thickness, electrode plate area, channel cross-sectional area and length, and dielectric strength) of such circuits are varied to achieve the desired results.

An exemplary nucleic acid detection system includes at least one channel and detects one or more physical properties (e.g., pH, optical properties, electrical properties, and/or characteristics) along at least a portion of the length of the channel to determine whether the channel contains a particular nucleic acid of interest and/or a particular nucleotide of interest.

The detection systems of the examples can be configured to include one or more channels for holding a sample and one or more sensor compounds (e.g., one or more nucleic acid probes), one or more input ports for introducing the sample and sensor compounds into the channels, and in some embodiments, one or more output ports through which the contents of the channels can be removed.

One or more sensor compounds (e.g., one or more nucleic acid probes) may be selected such that direct or indirect interaction between the nucleic acid and/or nucleotide of interest (if present in the sample) and the particles of the sensor compound causes the formation of aggregates that alter one or more physical properties, such as pH, optical or electrical properties and/or characteristics, of at least a portion of the length of the channel.

In some cases, the formation of aggregates, nucleic acid complexes, or polymers inhibits or prevents fluid flow in the channel and thus causes a significant decrease in conductivity and current measured along the length of the channel. Similarly, in these cases, the formation of aggregates, nucleic acid complexes, or polymers causes a measurable increase in resistivity along the length of the channel. In certain other cases, the aggregate, nucleic acid complex, or polymer is electrically conductive, and formation of the aggregate, nucleic acid complex, or polymer enhances the electrical flow path along at least a portion of the length of the channel, thereby causing a measurable increase in conductivity and current measured along the length of the channel. In these cases, the formation of aggregates, nucleic acid complexes, or polymers causes a measurable decrease in resistivity along the length of the channel.

In some cases, the formation of aggregates, nucleic acid complexes, or polymers affects the waveform characteristics of one or more electrical signals sent through the channel. As shown, for example in fig. 11, a first electrode (or excitation electrode) 1116 and a second electrode (a "pick-up" or "sensor" electrode) 118 are spaced apart from each other along the channel 1104. Fig. 11 represents an alternative or supplemental approach to that described above with respect to fig. 5A-5D. The first electrode 1116 and the second electrode 1118 may not be in contact with the measured solution contained within the channel 1104. In this sense, the first electrode 1116 and the second electrode 1118 are capacitively coupled to the solution within the channel 1104. The strength of the capacitive coupling depends on the electrode geometry, the passivation layer thickness and the passivation layer material, in particular its relative dielectric strength.

In some aspects, the solution is confined to the channel 1104. The channels may have cross-sectional areas in the order of microns. In this regard, the solution behaves as a resistor whose resistance depends on the conductivity of the solution and the geometry of the channel 1104.

In some embodiments, an alternating current/voltage is applied to excitation electrode 1116 and the induced current is measured at signal electrode 1118. The induced current is proportional to the impedance between the electrodes, which can vary with the conductivity of the solution. As shown, an excitation voltage 1400 is applied to the excitation electrode 1116, and a sense current 1410 is detected by the signal electrode 1118.

In some embodiments, the detector sensitivity is at least partially dependent on the excitation frequency. Thus, in some aspects, maximum sensitivity occurs when the absolute value of the phase of the induced current is at a minimum. In this region, the chip impedance is dominated by the fluid impedance. The fluid impedance is a function of the fluid conductivity and the chip geometry. Complex impedance information is important to ensure maximum detector sensitivity and correct detector operation.

Analysis of the lumped parameter model of the equivalent circuit shows the detector sensitivity and the coupling capacitance C_WALLSolution resistance R_LAMPAnd parasitic capacitance C_XIs closely related to the intensity of (c). Specifically, when the excitation frequency f satisfies the following condition, the change in the impedance between the electrodes with respect to the change in the conductivity is the largest:

1/(πR_LAMP C_WALL)<<f<<1/(πR_LAMP C_X)

as shown in fig. 12, the impedance of the signal depends on the excitation frequency and changes after the LAMP reaction occurs in the channel 1104. As also shown in fig. 12, the left-side inequality may define a frequency region below which the coupling impedance dominates and the change in solution impedance becomes virtually invisible. The non-uniformity on the right may define a frequency region above which parasitics dominate and electrode 1116 and electrode 1118 are actually shifted together.

As shown in fig. 13, in the two extreme regions, the impedance is capacitor-like and out of phase (nearly 90 °) with the excitation voltage. Between the two regions, the impedance begins to approach the limit of a simple resistor, and the impedance flattens out with respect to the frequency response. In practice, the maximum detector sensitivity corresponds to the phase minimum of the impedance.

To address the need for synchronous detection, two parallel channels for current can be considered in a simplified model: current and parasitic or geometric capacitance through the chip via the fluid channel. Given an excitation signal V at a given frequency f, the induced current I will be:

I(t)＝(Y+2πfC_xj)V(t)

wherein Y is the admittance of the chip due to coupling to the fluid flow channel, C_xIs the parasitic capacitance, and j is the imaginary unit. Multiplying by j indicates that the current through the parasitic channel is 90 out of phase with the excitation voltage. The measured impedance of the sample chip with respect to the excitation frequency is shown in fig. 14.

In a synchronous detector, the pick-up current is multiplied by an in-phase square wave m, and then low-pass filtered.

It is directly shown that the contribution of the signal 90 ° out of phase with the modulated signal will be zero, so we can ignore the parasitic capacitances in this analysis. To see the synchronous detection effect on the current through the fluid flow channel, the induced current (minus the parasitic contribution) can be multiplied by the modulation wave:

Where | Y | is the amplitude of the admittance, anAnd h.f.t. represents a high frequency term (e.g. greater than f). After low pass filtering, the DC term of the synchronous output can be left:

by noting the following, this expression can be simplified as follows:

the results were:

alternatively, by noting the following equation, it can be expressed in impedance by Z:

wherein the bars represent complex conjugates. The synchronized detector output thus becomes:

the impedance is calculated explicitly, taking into account a simple circuit model of the chip, and the output of the synchronized detector is predicted.

A simple equivalent circuit model comprises two capacitors C in series with a resistor R. As discussed above, the resistance R is primarily a function of the microfluidic geometry and solution conductance. The capacitance is primarily a function of the electrode area, the dielectric used for the passivation layer, and the passivation layer thickness. The impedance Z of the simplified circuit is given by:

the square of the magnitude of the impedance is:

|Z|²＝R²+(πfC)^-2

and the outputs of the synchronized detectors are:

where the numerator and denominator are multiplied by the conductance G1/R squared.

For conductivity meters, the cell constant k can be defined as:

where k has units of opposite length. The cell constant k depends primarily on the electrode position, area, and fluid flow path, and may not be a simple linear relationship. The synchronized detector outputs are then:

To assist in this analysis, dimensionless conductivity parameters can be introducedWherein:

so that:

detector output versus dimensionless conductivityThe dependence of (a) is significant.

1) For theDetector response andasymptotically proportional.

2) In thatAt which the detector response reaches a local maximum s_max＝|V|fC。

3) For theDetector response andasymptotically proportional.

Considering the dependence of the detector response on the dimensionless conductance, it is important to closely relate the chip and detector designs. The previously proposed points are explained in terms of actual conductance, with the following results:

1) for theThe detector response is asymptotically proportional to σ.

2) For theDetector response andasymptotically proportional.

3) At σ ═ pi kfC, the detector response becomes non-monotonic.

In other words, increasing the excitation frequency extends the conductivity range over which the synchronized detector output is linear. In fig. 15, the synchronized detector response is plotted against the dimensionless conductivity.

To evaluate the effectiveness of the lumped parameter model, the detector response of a known conductive solution of KCl was measuredShould be used. The channel of the chip is 2mm, and the cross-sectional area is 0.01mm². Two electrodes each 9mm²It was passivated with a layer of SU8 photoresist of 10 μm. The cell constant and capacitance were estimated and the excitation frequency was chosen so that the conductivity corresponding to the non-linearity in the detector output was about 5 mS/cm. The experiment was repeated at excitation frequencies of 10kHz, 15kHz and 20 kHz.

The conductivity of the chemistry before LAMP has been measured to be about 10 mS/cm. Table 1 below shows the estimated values of the minimum detector frequency controlled by the constraints found previously, namely:

TABLE 1

The results of the model are shown in fig. 16, demonstrating good frequency consistency with a wide range of conductivities and detector outputs at a given step. It is important to note that the same two parameters k and C are used at each frequency. The model predicts the qualitative behavior of the detector response. I.e. the functional form of the response, the dependence of the critical conductivity (where non-linearity occurs) on the excitation frequency. The model overestimates the difference in frequency-dependent behavior of conductivity over critical conductivity.

As a tool for fast estimation of conductance and wall capacitance, surface conductivity and capacitance effects can be neglected in addition to fringing field effects. The rough estimate can be further refined using a geometry-specific finite element model.

The electrode is modeled as having an area A_EOf a parallel plate capacitor of having a relative dielectric strength s_rSpaced from the dielectric of thickness t. The capacitance is then approximated as:

wherein epsilon₀Is the dielectric constant.

The fluid can be modeled as having a cross-sectional area A _FLength l and conductivity σ. Thus, the conductance of the fluid flow channel can be approximated as

Thereby, the battery constant can also be approximated.

In some aspects, the device is configured to determine an "impedance spectrum" after introduction of the chip. The device may include a numerically controlled excitation frequency. The device may have fast frequency sweep capability. The apparatus may comprise an in-phase component and a quadrature component of the induced signal from which the complex impedance may be determined. A fitness (fitness) of the impedance spectrum is determined based at least in part on curve fitting or other heuristic means to determine proper chip insertion and/or proper sample introduction. In some aspects, the device is first tested by excitation at a frequency determined by the initial sweep. In some embodiments, the device includes a detector that utilizes synchronous detection. In this way, the measured induced current (at the phase minimum) attributable to the fluid flow channel can be detected in real time.

Overview of an exemplary channel

In some embodiments, the channel or conduit has the following dimensions: a length measured along its longest dimension (y-axis) and extending along a plane parallel to a substrate of the detection system; a width measured along an axis perpendicular to its longest dimension (x-axis) and extending along a plane parallel to the substrate; and depth measured along an axis (z-axis) perpendicular to a plane parallel to the substrate. An exemplary channel may have a length substantially greater than its width and depth. In some cases, an example ratio between length to width may be: 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1 or within a range defined by any two of the aforementioned ratios.

In some embodiments, the channel or conduit is configured to have a depth and/or width that is substantially equal to or less than the diameter of an aggregate, nucleic acid complex, or polymer formed in the channel (preferably, when suspended in the channel due to interaction between the nucleic acid of interest and particles of a sensor compound (e.g., one or more nucleic acid probes) used to detect the nucleic acid of interest).

In some embodiments, the channels are configured to have a width along the x-axis ranging from about 1nm to about 50,000nm, or a width within a range defined by any two numbers within the above range, but are not limited to these exemplary ranges. Exemplary channels or conduits have lengths along the y-axis ranging from about 10nm to about 2cm, or lengths within a range defined by any two numbers within the above-noted range, but are not limited to these exemplary ranges. Exemplary channels have a depth along the z-axis ranging from about 1nm to about 1 micron, or a depth within a range defined by any two numbers within the above ranges, but are not limited to these exemplary ranges.

In some embodiments, the channel or conduit has any suitable cross-sectional shape (e.g., a cross-section taken along the x-z plane), including but not limited to circular, oval, rectangular, square, D-shaped (due to isotropic etching), and the like.

In some embodiments, the channel or conduit has a length in the range from 10nm to 10cm, for example at least or equal to 10nm, 50nm, 100nm, 200nm, 400nm, 600nm, 800nm, 1 μm, 10 μm, 50 μm, 100 μm, 300 μm, 600 μm, 900 μm, 1cm, 3cm, 5cm, 7cm, or 10cm, or a length in the range defined by any two of the aforementioned lengths. In some embodiments, the channel has a depth in the range from 1nm to 1 μm, e.g., at least or equal to 1nm, 5nm, 7nm, 10nm, 50nm, 100nm, 200nm, 400nm, 600nm, 800nm, 1 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 100 μm, 500 μm, or 1mm, or a depth in the range defined by any two of the aforementioned depths. In some embodiments, the channel has a width in the range from 1nm to 50 μm, such as, for example, 1nm, 5nm, 7nm, 10nm, 50nm, 100nm, 200nm, 400nm, 600nm, 800nm, 1 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 100 μm, 500 μm, or 1mm, or a width in the range defined by any two of the aforementioned widths.

In some embodiments, the channel or conduit is formed in a cartridge that is later inserted into the device. In some aspects, the cartridge may be a disposable cartridge. In some aspects, the cartridge is made of a cost-effective plastic material. In some aspects, at least a portion of the cartridge is made of paper and a thin layer-based material for the fluid.

An embodiment of a detection system 2100 for detecting the presence or absence of a particular nucleic acid and/or a particular nucleotide in a sample is illustrated in fig. 17A-17B. Fig. 17A is a top view of the system and fig. 17B is a cross-sectional side view of the system. The detection system 2100 includes a base panel 2102 extending substantially along a horizontal x-y plane. In some embodiments, the substrate 2102 can be comprised of a dielectric material (e.g., silicon dioxide). Other example materials for the substrate 2102 include, but are not limited to, glass, sapphire, or diamond.

The substrate 2102 supports or includes a channel 2104, the channel 2104 having at least an inner surface 2106 and an interior space 2108 for containing a fluid. In some cases, channels 2104 are etched in the top surface of the substrate 2102. Exemplary materials for inner surface 2106 of channel 2104 include, but are not limited to, glass or silica.

In certain embodiments, the channel 2104 and the substrate 2102 are constructed of glass. Biological conditions represent an obstacle to the use of glass-derived implants due to the slow dissolution of glass into biological fluids and the adhesion of proteins and small molecules to the glass surface. In certain non-limiting embodiments, surface modification using self-assembled monolayers provides methods for modifying glass surfaces for nucleic acid detection and analysis. In certain embodiments, at least a portion of the inner surface 2106 of the channel 2104 is pretreated or covalently modified to include or coated with a material that enables the sensor compound to specifically covalently bind to the inner surface. In certain embodiments, the coverslip 2114 covering the channel can also be covalently modified with a material.

Example materials for modifying the inner surface 2106 of channel 2104 include, but are not limited to, silane compounds (e.g., trichlorosilane, alkylsilane, triethoxysilane, perfluorosilane), zwitterionic sultone, poly (6-9) glycol (Peg), perfluorooctyl, fluorescein, aldehyde, or graphene compounds. Covalent modification of the inner surface of the channel reduces non-specific absorption of certain molecules. In one example, the covalent modification of the inner surface can enable covalent bonding of sensor compound molecules to the inner surface while preventing non-specific absorption of other molecules to the inner surface. The inner surface 2106 of the channel 2104 is modified, for example, with poly (ethylene glycol) (Peg) to reduce non-specific adsorption of material to the inner surface.

In some embodiments, channels 2104 are fabricated at a nanometer or micrometer scale to have a well-defined and smooth inner surface 2106. Sumita Pennathur and Pete Crisallai (2014), "Low Temperature Fabrication and Surface Modification Methods for Fused Silica Micro-and Nanochannels," MRS Proceedings,1659, pp 15-26.doi: l 0.1557/op.2014.32, the entire contents of which are expressly incorporated herein by reference.

The first end of the channel 2104 includes or is in fluid communication with the input port 2110, and the second end of the channel 2104 includes or is in fluid communication with the output port 2112. In certain non-limiting embodiments, port 2110 and port 2112 are disposed at the end of channel 2104.

In some embodiments, the top surface of the substrate 2102 having the channel 2104 and the ports 2110, 2112 is covered and closed with a cover slip 2114. In some embodiments, rigid plastic is used to define the channel (including the roof), and a semi-permeable membrane may also be used.

The first electrode 2116 is electrically connected at a first end of the channel 2104 (e.g., at or near the input port 2110). The second electrode 2118 is electrically connected at a second end of the channel 2104 (e.g., at or near the output port 2112). The first 2116 and second 2118 electrodes are electrically connected to a power or voltage source 2120 to apply a potential difference between the first and second electrodes. I.e. a potential difference is applied across at least part of the length of the channel. When fluid is present in channel 2104 and is affected by the applied potential difference, electrode 2116, electrode 2118 and the fluid create a complete electrical flow path.

A power or voltage source 2120 is configured to apply an electric field in a reversible manner such that a potential difference is applied in a first direction along the length of the channel (along the y-axis) and also in a second opposite direction (along the y-axis). In one example where the direction of the electric field or potential difference is in the first direction, the positive electrode is connected at a first end of the channel 2104 (e.g., at or near the input port 2110) and the negative electrode is connected at a second end of the channel 2104 (e.g., at or near the output port 2112). In another example where the direction of the electric field or potential difference is in a second, opposite direction, the negative electrode is connected at a first end of the channel 2104 (e.g., at or near the input port 2110) and the positive electrode is connected at a second end of the channel 2104 (e.g., at or near the output port 2112).

In some embodiments, a power or voltage source 2120 is configured to apply an AC signal. The frequency of the AC signal may be dynamically changed. In some aspects, power or voltage source 2120 is configured to provide a voltage having a frequency of 10Hz-10⁹Electrical signals of frequencies between Hz. In some aspects, power or voltage source 2120 is configured to provide a voltage having a value of 10⁵Hz-10⁷Electrical signals of frequencies between Hz.

Electrically connecting the first and second ends of the channel 2104 (e.g., at or near the input port 2110 and the output port 2112) to the nucleic acid detection circuit 2122, the nucleic acid detection circuit 2122 is programmed or configured to detect a value of one or more electrical properties of the channel 2104 for determining the presence or absence of a particular nucleic acid and/or nucleotide in the channel 2104. The electrical property value is detected at a single time period (e.g., certain time periods after the sample and the one or more sensor compounds are introduced into the channel) or at a plurality of different time periods (e.g., before and after the sample and the one or more sensor compounds are introduced into the channel). In some aspects, the electrical property value is detected continuously over a set period of time from sample introduction to throughout LAMP amplification. Exemplary electrical properties detected include, but are not limited to, current, conductance voltage, resistance, frequency, or waveform. The nucleic acid detection circuitry 2122 of certain examples includes or is configured as a processor or computing device (e.g., device 1700 shown in fig. 18). Certain other nucleic acid detection circuits 2122 include, but are not limited to, an ammeter, voltmeter, ohmmeter, or oscilloscope.

In one embodiment, the nucleic acid detection circuitry 2122 includes measurement circuitry 2123 programmed or configured to measure one or more electrical property values along at least a portion of the length of the channel 2104. The nucleic acid detection circuit 2122 also includes an equilibration circuit 2124 programmed or configured to periodically or continuously monitor one or more values of the electrical property of the channel over a period of time, and/or to select individual ones of the values after the values have reached equilibrium (e.g., have ceased to exceed a certain threshold change in variance or tolerance).

The nucleic acid detection circuitry 2122 can also include comparison circuitry 2126 programmed or configured to compare two or more electrical property values of the channel, such as a reference electrical property value (e.g., measured prior to a state in which the sample and all of the sensor compounds are introduced into the channel) and an electrical property value (e.g., measured after the sample and all of the sensor compounds are introduced into the channel). The comparison circuit 2126 can use the comparison to determine the presence or absence of nucleic acid in the channel. In one embodiment, the comparison circuit 2126 calculates a difference between the measured electrical property value and the reference electrical property value and compares the difference to a predetermined value indicative of the presence or absence of nucleic acid in the channel and uses this information to diagnose or predict a disease state or the presence or absence of infection in the subject.

In certain embodiments, when both the sample and the sensor compound are introduced into the channel, the comparison circuit 2126 is programmed or configured to compare a first value of the electrical property (e.g., the magnitude of the current) when an electric field or potential difference is applied across the channel in a first direction along the length of the channel and a second value of the electrical property (e.g., the magnitude of the current) when an electric field or potential difference is applied across the channel in a second, opposite direction along the length of the channel. In one embodiment, the comparison circuit 2126 calculates a difference between the magnitudes of the first and second values and compares the difference to a predetermined value indicative of the presence or absence of nucleic acid in the channel (e.g., whether the difference is substantially zero). For example, if the difference is substantially zero, this indicates that no nucleic acid is present in the channel (which may be in dispersed form, in polymeric form, or in aggregate form). If the difference is not substantially zero, this indicates the presence of nucleic acid (which may be in dispersed form, in polymeric form, or in aggregate form) in the channel.

In certain embodiments, the nucleic acid detection circuit 2122 is programmed or configured to determine the absolute concentration of nucleic acids in the sample, and/or the relative concentration of nucleic acids relative to one or more additional substances in the sample.

In some embodiments, the comparison circuit 2124 and the balancing circuit 2126 are configured as separate circuits or modules, while in other embodiments they are configured as a single integrated circuit or module.

Nucleic acid detection circuitry 2122 has an output 2128, and in some embodiments, output 2128 can be coupled to one or more external devices or external modules. For example, the nucleic acid detection circuit 2122 can communicate the reference electrical property value and/or the one or more measured electrical property values to one or more of: a processor 2130 (for further computation, processing, and analysis), a non-transitory storage device or memory 2132 (for storage of values), and/or a visual display device 2134 (for displaying the values to a user). In some embodiments, the nucleic acid detection circuitry 2122 generates an indication of whether the sample comprises nucleic acids and transmits the indication to the processor 2130, the non-transitory storage device or memory 2132, and/or the visual display device 2134.

In an exemplary method of using the systems of fig. 17A and 17B, one or more sensor compounds (e.g., one or more nucleic acid probes) and a sample are introduced into a channel sequentially or simultaneously. When the flow of fluid and/or the flow of charged particles in the fluid is not inhibited (e.g., due to the absence of aggregates), conductive particles or conductive ions in the fluid travel along at least a portion of the length of the channel 2104 from the input port 2110 to the output port 2112. The movement of the conductive particles or conductive ions produces or generates a first or "reference" electrical property value or range of values (e.g., current, conductivity, resistivity, or frequency) that is detected by the nucleic acid detection circuit 2122 along at least a portion of the length of the channel 2104. In some embodiments, the balancing circuit 2124 periodically or continuously monitors the electrical property value for a period of time until the electrical property value reaches equilibrium. The balancing circuit 2124 then selects one of the values as a reference electrical property value to avoid the influence of transient changes in the electrical property.

As used herein, a "reference" electrical property value refers to a value or range of values of an electrical property of a channel prior to introducing a sample and all sensor compounds (e.g., one or more nucleic acid probes) into the channel. That is, the reference value is the value that characterizes the channel prior to any interaction between the nucleic acid in the sample and all of the sensor compounds. In some cases, the reference value is detected at a time period after the sensor compound is introduced into the channel, but before the sample and additional sensor compound are introduced into the channel. In some cases, the reference value is detected at a time period after the sensor compound and sample are introduced into the channel, but before additional sensor compound is introduced into the channel. In some cases, the reference value is detected at a time period prior to introduction of the sample or sensor compound into the channel. In some cases, the reference value is predetermined and stored on a non-transitory storage medium from which the reference value is obtainable.

In some cases, the formation of a conductive aggregate, polymer, or nucleic acid complex in the channel (e.g., due to interaction between a nucleic acid of interest in the sample and one or more nucleic acid probes) enhances an electrical flow path along at least a portion of the length of channel 2104. In this case, the nucleic acid detection circuit 2122 detects the second electrical property value or range of second electrical property values (e.g., current, conductivity, resistivity, or frequency) along at least a portion of the length of the channel 2104. In some embodiments, the nucleic acid detection circuit 2122 schedules a wait time period or an adjustment time period after introducing the sample and all sensor compounds into the channel before detecting the second electrical property value. The waiting or adjusting period allows the aggregate, polymer, or nucleic acid complex to form in the channel (preferably when suspended in the channel) and allows the aggregate, polymer, or nucleic acid complex to form to alter the electrical properties of the channel (preferably when suspended in the channel).

In some embodiments, the equilibration circuit 2124 periodically or continuously monitors the electrical property value for a period of time after the sample and all sensor compounds are introduced until the value reaches equilibrium. The balancing circuit 2124 can then select one of the values as the second electrical property value to avoid the effects of transient changes in the electrical property.

The comparison circuit 2126 compares the second electrical property value to a reference electrical property value. If it is determined that the difference between the second value and the reference value corresponds to a predetermined range of increase in current or conductivity (or decrease in resistivity), the nucleic acid detection circuit 2122 determines that an aggregate, polymer, or nucleic acid complex is present in the channel, and thus, a nucleic acid target is present or detected in the sample. Based on this, the target and the presence or absence of a disease state or infection state in the subject can be diagnosed or identified.

In certain other embodiments, when the flow of fluid in the channel and/or the flow of charged particles in the fluid is partially or completely blocked (e.g., by forming aggregates, polymers, or nucleic acid complexes), the conductive particles or conductive ions in the fluid are unable to freely travel along the y-axis along at least a portion of the length of the channel 2104 from the input port 2110 to the output port 2112. The impeded or stopped movement of the conductive particles or conductive ions produces or generates a third electrical property value or range of third electrical property values (e.g., current or signal, conductivity, resistivity, or frequency) that is detected by the nucleic acid detection circuit 2122 along at least a portion of the length of the channel 2104. A third electrical property value is detected in addition to or in place of the second electrical property value. In some embodiments, the nucleic acid detection circuit 2122 can wait for a waiting period or an adjustment period after introducing the sample and all sensor compounds into the channel before detecting the third electrical property value. The waiting period or the adjusting period allows the aggregate, the polymer, or the nucleic acid complex to form in the channel and allows the aggregate, the polymer, or the nucleic acid complex to form to change the electrical property of the channel.

In some embodiments, the equilibration circuit 2124 periodically or continuously monitors the electrical property value for a period of time after the sample and all sensor compounds are introduced until the value reaches equilibrium. The balancing circuit 2124 then selects one of the values as the third electrical property value to avoid the effect of transient changes in the electrical property.

The comparison circuit 2126 compares the third electrical property value to the reference electrical property value. If it is determined that the difference between the third value and the reference value corresponds to a predetermined decrease in current or conductivity (or increase in resistivity) range, the nucleic acid detection circuit 2122 determines that an aggregate, polymer, or nucleic acid complex is present in the channel, and thus identifies that the target nucleic acid is present in the sample.

Fluid flow along the length of the channel depends on the size of the aggregates, polymers or nucleic acid complexes associated with the dimensions of the channel, and the formation of an Electric Double Layer (EDL) at the inner surface of the channel.

In general, an EDL is a region of net charge between a charged solid (e.g., the interior surface of a channel, an analyte particle, an aggregate, a polymer, or a nucleic acid complex) and an electrolyte-containing solution (e.g., the fluid contents of a channel). The EDLs are present around the inner surface of the channel as well as around any nucleic acid particles and aggregates, polymers or nucleic acid complexes within the channel. Counter ions from the electrolyte are attracted to the charge on the inner surface of the channel and induce a net charge region. EDLs affect ion flow within the channel and around the analyte particles and aggregates, polymers or nucleic acid complexes of interest, creating diode-like behavior by not allowing any counter ions to pass through the length of the channel.

To mathematically solve for the characteristic length of the EDL, the Poisson-Boltzmann ("PB") equation and/or the Poisson-Nemst-Plank equation ("PNP") are solved. These solutions are coupled with a Navier-Stokes (NS) equation for fluid flow to create a nonlinear set of coupled equations that are analyzed to understand the operation of the exemplary system.

The exemplary channels are configured and constructed with carefully selected dimensional parameters that ensure that the flow of conductive ions along the length of the channel is substantially inhibited when aggregates, polymers or nucleic acid complexes of a certain predetermined size are formed in the channel, taking into account the dimensional interactions between the channel surface, the EDL and the aggregates, polymers or nucleic acid complexes. In some cases, the exemplified channels are configured to have a depth and/or width that is substantially equal to or less than the diameter of aggregate particles formed in the channel during nucleic acid detection. In certain embodiments, the size of the EDL is also considered in selecting the dimensional parameters of the channel. In certain instances, the exemplified channels are configured to have a depth and/or width that is substantially equal to or less than the dimension of the EDL generated around the inner surface of the channel and around aggregates, polymers, or nucleic acid complexes in the channel.

In certain embodiments, the channel is free of sensor compounds (e.g., one or more nucleic acid probes) prior to use of the detection system. That is, the manufacturer of the detection system may not pre-treat or modify the channels to contain the sensor compounds. In this case, during use, a user introduces one or more sensor compounds (e.g., in an electrolyte buffer) into the channel and detects a reference electrical property value of the channel with the sensor compounds in the absence of the sample.

In certain other embodiments, the channel is pretreated or modified prior to use of the detection system such that at least a portion of the interior surface of the channel comprises or is coated with a sensor compound (e.g., one or more nucleic acid capture probes). In one example, the manufacturer detects a reference electrical property value for a channel modified with a sensor compound, and during use, the user can use the stored reference electrical property value. That is, the manufacturer of the detection system may pre-treat or modify the channels to contain the sensor compounds. In this case, the user needs to introduce the sample and one or more additional sensor compounds into the channel.

Certain example detection systems include a single channel. Certain other exemplary detection systems include multiple channels provided on a single substrate. Such detection systems may include any suitable number of channels, including but not limited to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 channels, or a plurality of channels within a range defined by any two of the aforementioned numbers.

In one embodiment, the detection system comprises a plurality of channels, wherein at least two channels operate independently of each other. The channels 2104 and associated components of the example of fig. 17A-17B are reproduced on the same substrate to obtain such a multi-channel detection system. Multiple channels are used to detect the same nucleic acid in the same sample, different nucleic acids in the same sample, the same nucleic acid in different samples, and/or different nucleic acids in different samples. In another embodiment, the detection system comprises a plurality of channels, wherein at least two channels operate in cooperation with each other. In some aspects, the channel is shaped differently depending on the target sought to be detected.

Overview of an exemplary device for bedside use

In some embodiments, the device is portable and configured to detect one or more targets in a sample. As shown in FIG. 19, the device includes a controller configured to control fC ⁴A processor 900 of a D circuit 905. fC⁴D circuit 905 includes a signal generator 907. The signal generator 907 is configured to provide one or more signals through the channel 2104 or test wells as described above. The signal generator 907 is connected to a preamplifier 915 to amplify one or more signals from the signal generator 907. One or more signals are passed through multiplexer 909 and through channel 2104. The signal from channel 2104 is amplified by post amplifier 911 and demodulated by signal separator 913. The analog-to-digital converter 917 recovers the signal and transmits the digital signal to the processor 900. The processor 900 includes a processor configured to measure, balance, compareEtc. to determine whether the desired target is detected in the sample. In some embodiments, the analog-to-digital conversion may occur first. In some such embodiments, the inductive waves may be collected as a whole and digitally demodulated in software.

In some embodiments, processor 900 is also coupled to one or more heating elements 920. The one or more heating elements 920 may be resistive heating elements. One or more heating elements 920 are configured to heat the sample and/or solution in channel 2104. In some embodiments, the sample is heated to a temperature greater than or equal to 0 ℃, 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, 85 ℃, 90 ℃, 95 ℃, 100 ℃, 105 ℃, 110 ℃, 115 ℃, 120 ℃, 130 ℃, 140 ℃, 150 ℃, 160 ℃, 170 ℃, 180 ℃, 190 ℃, 200 ℃, 210 ℃, 220 ℃, 230 ℃, 240 ℃, 250 ℃, 260 ℃, or any temperature range or any temperature between two of the aforementioned numbers. In some embodiments, the sample is cooled to a temperature less than or equal to 40 ℃, 35 ℃, 30 ℃, 25 ℃, 20 ℃, 15 ℃, 10 ℃, 5 ℃, 0 ℃, -5 ℃, -10 ℃, -15 ℃, -20 ℃, or any temperature range or any temperature between two of the above numbers. In view of the foregoing, the processor 900 and/or other circuitry is configured to read the temperature 925 of the sample and/or the channel 2104 and control the one or more heating elements 920 until a desired heating set point 930 is reached. In some aspects, the entire channel 2104 is configured to be heated by one or more heating elements 920. In other aspects, only a portion of the channel 2104 is configured to be heated by the one or more heating elements 920.

The processor 900 is configured to receive user input 940 from one or more user inputs (e.g., a keypad, touch screen, buttons, switches, or a microphone, etc.). Data is output 950 and recorded 951, reported to a user 953, pushed to a cloud-based storage system 952, and so on. In some embodiments, the data is sent to another device for processing and/or further processing. For example, fC can be⁴D data push to cloud and then proceedProcessing to determine the presence or absence of the target in the sample.

In some aspects, the device is configured to consume relatively low power. For example, a device may only require 1-10 watts of power. In some aspects, the device requires less than 7 watts of power. The device is configured to process data, communicate wirelessly with one or more other devices, send and detect signals through the channel, heat the sample/channel, and/or detect and display input/output with a touchable display.

In some embodiments, the sample collector, sample preparation device and fluidic cartridge are formed as separate physical devices. Thus, a sample is collected using the first sample collector device. The sample may comprise saliva, mucus, blood, plasma, stool, or cerebrospinal fluid. The sample is then transferred to a second sample preparation device. The sample preparation device includes components and reagents required for nucleic acid amplification. After preparation of the sample, it is transferred to a third apparatus comprising a fluidic cartridge, where amplification, fC, is performed ⁴D excitation and measurement. In some embodiments, sample collection and sample preparation is accomplished by a single device. In some embodiments, the sample preparation and fluidic cartridge are contained within a single device. In some embodiments, a single device is configured to collect a sample, prepare the sample, amplify at least a portion of the sample, and use fC⁴And D, analyzing the sample.

Overview of an exemplary compact fluidic Cartridge

In some aspects, the device includes a removable fluid cartridge that is connectable to another companion device. The removable fluid cartridge is configured as a disposable, single-use cartridge. In some embodiments, the cartridge comprises a plurality of channels. The channels may be shaped differently. In some aspects, 4 shapes of channels are used and repeated to ensure accuracy. In some aspects, more than 4 shapes of channels are used and repeated to ensure accuracy. In some aspects, each channel is configured to detect a unique target. In other aspects, each channel is configured to detect the same target. In some embodiments, the cartridge includes one or more heating elements. In general, flowThe body case may include a configuration for fC⁴D at least one channel of analysis.

In some aspects, the cartridge comprises a multilayer laminate structure. One or more channels are embossed and/or laser cut into the substrate. In some embodiments, the substrate comprises a polypropylene film. One or both sides of the film are coated with an adhesive. The channel layer is secured to a polyamide heater coil to heat all or a portion of the channel. The channels are at least partially covered by a hydrophilic PET layer. The printed electrodes may be disposed below the PET layer. In some aspects, each channel provides at least one thermistor for temperature feedback.

In other aspects, the cartridge comprises injection molded plastic. One or more channels are provided in the injection molded plastic. A PET layer or PET film is coated on all or part of the channels by laser welding PET to the IM plastic. Injection molding can provide the benefits of rigidity and 3D structure, and also allows for features such as valves and frames that are convenient to manipulate. Depending on the particular design, the cartridge may or may not include printed electronics and/or heating elements and/or thermistors.

An exemplary embodiment of a fluid cartridge 500 is depicted in fig. 20. As shown, cartridge 2500 includes 4 layers. PCB/PWB layer 2501 has electrodes 2505 shown on it. The electrodes can be passivated with a 30nm titanium dioxide layer using methods such as atomic deposition. The PCB/PWB layer may include access points 2506 for screws or other retaining devices to hold the 4 layers together. The power supply and detection circuitry may be connected to the PCB/PWB layer. Liner layer 2510 has cuts 2513 and 2514, and an entry point 2506. The cushion layer may be made of a material such as fluorosilicone rubber. The lower rigid substrate layer 2520 includes an entry point 2506 and an entry port 2522. Upper rigid layer 2530 includes an entry point 2506 and an entry port 2522. The lower and upper rigid layers may each be made of a material such as acrylic. When the 4 layers are assembled together via securing screws or other retaining devices through the multiple access points 2506 of the multiple layers, 4 channels are formed. The notch 2513 and the notch 2514 form the sides of the channel. The cutout 513 forms a channel with two trapezoidal ends, and the cutout 2514 forms a channel with substantially straight sides. Portions of the PCB/PWB layer 2501 (including the electrodes 2505) form the bottom of the vias. Lower rigid layer 2520 forms the top of the channel, and inlet port 2522 provides inlet and outlet ports to the channel. The inlet port 2522 of the upper layer and the inlet port of the upper rigid layer provide a means of providing reagents to each channel. In some embodiments, a channel with two trapezoidal ends can have a volume of about 30 μ L to about 50 μ L. In some embodiments, a channel with substantially straight sides can have a volume of about 20 μ L to about 30 μ L. This volume can be adjusted by varying the compression of at least the cushion layer. Fig. 21 depicts a top view of the fluidic cartridge 2500 of fig. 20 and shows an access point 506 for a screw or other retaining device, an access port 2522 in communication with a channel 2550, and an electrode 2505. Fig. 22 provides example dimensions for two electrodes 2505. Fig. 23 provides dimensions of an example of a channel 2550 having two trapezoidal ends. In some embodiments, the channel is heated to a temperature of 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃, or 75 ℃, or a temperature within a range defined by any two of the above numbers, and pressurized. In some aspects, the channels may be pressurized to 1, 2, 3, 4, 5, or 6 atmospheres or within a range defined by any two of the above pressures.

In some embodiments, the channel of the fluidic device can be adapted or configured to hold a sample volume of greater than or equal to 1 μ L, 2 μ L, 3 μ L, 4 μ L, 5 μ L, 6 μ L, 7 μ L, 8 μ L, 9 μ L, 10 μ L, 20 μ L, 30 μ L, 40 μ L, 50 μ L, 60 μ L, 70 μ L, 80 μ L, 90 μ L, 100 μ L, 200 μ L, 300 μ L, 400 μ L, 500 μ L, 600 μ L, 700 μ L, 800 μ L, 900 μ L, or 1000 μ L or a volume between any two of the foregoing volumes or any range between any two of the foregoing volumes. In some embodiments, the channel of the fluidic device may be adapted to be pressurized. In some embodiments, the sample in the channel can be pressurized to a pressure greater than or equal to 1 atmosphere, 2 atmospheres, 3 atmospheres, 4 atmospheres, 5 atmospheres, 6 atmospheres, 7 atmospheres, 8 atmospheres, 9 atmospheres, 10 atmospheres, or any range between any two of the foregoing pressures. In some embodiments, the channel of the fluidic device may be adapted to be maintained at a temperature greater than or equal to-20 ℃, -15 ℃, -10 ℃, -5 ℃, 0 ℃, 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 85 ℃, 80 ℃, 85 ℃, 90 ℃, 95 ℃, 100 ℃, 105 ℃, 110 ℃, 115 ℃, 120 ℃, 130 ℃, 140 ℃, 150 ℃, 160 ℃, 170 ℃, 180 ℃, 190 ℃, 200 ℃, 210 ℃, 220 ℃, 230 ℃, 240 ℃, 250 ℃, 260 ℃, or any temperature between any two of the aforementioned temperatures or any range between any two of the aforementioned temperatures.

Overview of sample Collection of examples

In some embodiments, the methods, systems, and devices disclosed herein utilize a simplified and direct sample collection procedure. In this way, the number of steps from sample collection to analysis is reduced. In other words, in some embodiments, it is desirable to minimize the number of times a user transfers and/or manipulates a sample to avoid contamination of the sample. In some aspects, the devices disclosed herein are configured to be compatible with multiple sample collection methods to suit all types of testing environments. Thus, in some aspects, a uniform vial-to-chip interface is utilized. By adjusting the sample collection system, the detection hardware remains unchanged regardless of the type of sample collected and analyzed.

Overview of exemplary assays

Some embodiments of the methods, systems, and compositions provided herein include simple lysis/amplification/detection of a target from a crude sample in a single container. Some embodiments include immune-based amplification for detection of non-nucleic acid targets. Some embodiments include a reagent added to the reaction that causes an increased conductivity change. Some embodiments include isothermal amplification methods, such as LAMP, SDA, and/or RCA. In some embodiments, the target for detection is a biomarker (e.g., a protein), a small molecule (e.g., a drug or anesthetic), or a biological weapon (e.g., a toxin). Detection of such targets can be achieved by conjugating an immune-based binding reagent (e.g., an antibody or aptamer) to the nucleic acid that will participate in the isothermal amplification reaction. In some embodiments, the additive of the amplification reaction can increase the solution conductivity change, which is correlated with the quantification of the target. The use of additives may provide greater sensitivity and dynamic range of detection. The use of additives may also help reduce the formation of non-specific amplification products.

Some embodiments of the methods provided herein allow for sample collection and processing with one or more of the following desirable characteristics: the device is non-centrifugal, portable, cheap, disposable, can not need wall socket electrical appliances (wall outlet electrical appliances), and can be used easily or visually; only relatively low technical skill may be required to use, RNA and/or DNA may be extracted from a small volume of sample (e.g., 70 μ L), RNA and/or DNA may be able to be stabilized until amplification, thermostable reagents may be used that are not required for cold-stranded storage, are assay compatible for low levels of test sample (e.g., samples having 1,000 copies/mL or less), and/or have a dynamic range that is able to detect viral loads spanning, for example, at least 4 orders of magnitude, may improve reproducibility of low copy target samples.

As described herein, some embodiments of the provided methods, systems, and compositions include the collection and processing of samples for diagnostic devices. Examples of collected samples (also referred to as biological samples) may include, for example, plants, blood, serum, plasma, urine, saliva, ascites fluid, spinal fluid, semen, lung lavage fluid, saliva, sputum, mucus, feces, liquid media containing cells or nucleic acids, solid media containing cells or nucleic acids, tissue, and the like. The method of obtaining a sample may comprise using: finger prick, heel prick, venipuncture, adult nasal aspirate, pediatric nasal aspirate, nasopharyngeal wash, nasopharyngeal aspirate, nasal swab, bulk collection in a cup, tissue biopsy, or lavage of a sample. Further examples include environmental samples, such as soil samples and water samples.

Overview of exemplary amplifications

Some embodiments of the methods, systems, and compositions provided herein include amplification of nucleic acid targets. Methods of nucleic acid amplification are well known and include methods that vary the temperature during the reaction (e.g., PCR).

Further examples include isothermal amplification, wherein the reaction may occur at a substantially constant temperature. In some embodiments, isothermal amplification of the nucleic acid target causes a change in the conductivity of the solution. There are various types of isothermal nucleic acid amplification methods, such as nucleic acid sequence-based amplification (NASBA), Strand Displacement Amplification (SDA), loop-mediated amplification (LAMP), Invader assay, Rolling Circle Amplification (RCA), signal-mediated RNA amplification technique (SMART), helicase-dependent amplification (HDA), Recombinase Polymerase Amplification (RPA), Nicking Endonuclease Signal Amplification (NESA), and nicking endonuclease assisted nanoparticle activation (NENNA), rolling circle Replication (RCA), nickase amplification reaction (NEAR), nucleic acid sequence-based amplification (NASBA), exonuclease-assisted target recycling, linker (Junction) or Y-probe, split DNAZyme and deoxyribozyme amplification strategies, template-directed chemical reactions that generate amplified signals, non-covalent DNA catalytic reactions, hybrid strand reactions (HCR), and detection via self-assembly of DNA probes to generate supramolecular structures. See, e.g., Yan l., et al, mol.biosyst., (2014)10: 970-.

In the example of LAMP, the two primers in the forward primer set were named inner primer (F1c-F2, with the c strand representing "complementary") and outer primer (F3). At 60 ℃, the F2 region of the inner primer first hybridizes to the target and is extended by DNA polymerase. The outer primer F3 then binds to the same target strand at F3c, and the polymerase extends F3 to displace the newly synthesized strand. Due to hybridization of the F1c and F1 regions, the displaced strand forms a stem-loop structure at the 5' end. At the 3' end, a reverse primer set can hybridize to the strand and a new strand with stem-loop structures at both ends is generated by the polymerase. The dumbbell-structured DNA enters an exponential amplification cycle, and a strand having a plurality of inverted repeats of the target DNA can be prepared by repeating extension and strand displacement. In some embodiments of the methods provided herein, the components of the LAMP include 4 primers, DNA polymerase, and dntps. Examples of LAMP applications include viral pathogens including dengue (m.parida, et al, j.clin.microbiol.,2005,43, 2895-.

In the case of an SDA, the probe comprises two parts: a Hinc II recognition site at the 5' end, and another segment comprising a sequence complementary to the target. The DNA polymerase can extend and incorporate the deoxyadenosine 5' - [ α -thio ] triphosphate (dATP [ α S ]). Then, the restriction endonuclease, Hinc II, nicks the probe strand at the recognition site for Hinc II, since the endonuclease cannot cleave the other strand containing the phosphorothioate modification. Endonuclease cleavage exposes the 3' -OH, followed by extension by DNA polymerase. The newly formed strand still contains a cleavage site for Hinc II. The newly synthesized double strand is subsequently nicked, then repeated several times for DNA polymerase mediated extension and this results in an isothermal amplification cascade. In some embodiments of the methods provided herein, the components of SDA include 4 primers, DNA polymerase, release hindii, dGTP, dCTP, dTTP, and dATP α S. Examples of the use of SDA include mycobacterium tuberculosis genomic DNA (m.vincent, et al, EMBO rep.,2004,5, 795-.

In the case of NASBA, the forward primer 1(P1) consists of two parts, one of which is complementary to the 3' end of the RNA target and the other of which is complementary to the T7 promoter sequence. When P1 binds to the RNA target (RNA (+)), Reverse Transcriptase (RT) extends the primer into the complementary DNA of the RNA (DNA (+)). RNase H then degrades the RNA strand of the RNA-DNA (+) hybrid. Reverse primer 2(P2) then binds to DNA (+), and Reverse Transcriptase (RT) produces double stranded DNA (dsdna) containing the T7 promoter sequence. After this initial phase, the system enters an amplification phase. T7 RNA polymerase generates many RNA strands (RNA (-), based on dsDNA, and a reverse primer (P2) binds to the newly formed RNA (-). The RT extends the reverse primer and RNase H degrades RNA of the RNA-cDNA double strand into ssDNA. The newly generated cDNA (DNA (+)) then becomes the template for P1, and the cycle is repeated. In some embodiments of the methods provided herein, the components of the NASBA include 2 primers, reverse transcriptase, RNase H, RNA polymerase, dntps, and rntps. Examples of NASBA applications include HIV-1 genomic RNA (D.G.Murphy, et al., J.Clin.Microbiol.,2000,38, 4034-. Each of the foregoing references is hereby expressly incorporated by reference herein in its entirety.

Further examples of isothermal amplification methods include: self-sustained sequence replication reaction (3SR), 90-I, BAD Amp, cross-primer amplification (CPA), isothermal index amplification reaction (EXPAR), isothermal chimeric primer-primed nucleic acid amplification (ICAN), Isothermal Multiple Displacement Amplification (IMDA), ligation-mediated SDA, multiple displacement amplification, polymerase helix reaction (PSR), restriction cascade index amplification (RCEA), smart amplification program (SMAP2), Single Primer Isothermal Amplification (SPIA), transcription-based amplification system (TAS), transcription-mediated amplification (TMA), Ligase Chain Reaction (LCR) and/or multiple cross-displacement amplification (MCDA), rolling circle Replication (RCA), and nickase amplification reaction (NEAR).

Overview of exemplary immuno-isothermal amplification

Some embodiments of the methods, systems, and compositions provided herein include the use of immuno-isothermal amplification to detect non-nucleic acid targets. In some such embodiments, primers for use in the isothermal amplification method are linked to the antibody or fragment thereof or aptamer. An "aptamer" as used herein may include a peptide or oligonucleotide that specifically binds to a target molecule. In some embodiments, the antibody or aptamer may be linked to a primer used in an isothermal amplification method by a covalent or non-covalent bond. In some embodiments, primers used in isothermal amplification methods can be linked to an antibody or aptamer through a biotin and streptavidin linker. In some embodiments, primers used in the isothermal amplification method can be linked to an antibody or aptamer using THUNDER-LINK (Expedeon, UK).

In some embodiments, the target antigen binds to an antibody or aptamer, and the primer linked to the antibody or aptamer is a substrate for isothermal amplification and/or priming of isothermal amplification. See, for example, Pourhassan-Moghaddam et al, Nanoscale Research letters,8: 485-. In some embodiments, the target antigen is captured in a sandwich format between two antibodies or aptamers (Abs; capture antibody and detection antibody) that specifically bind to the target antigen. Capture abs pre-immobilized on a solid support surface capture target Ag, and detection abs attached to primers or targets used in the isothermal amplification method attach to the captured Ag. After washing, isothermal amplification is performed and the presence of the amplification product indirectly indicates the presence of the target Ag in the sample.

Overview of exemplary recombinant polymerase amplification

Some embodiments include a method of nucleic acid amplification, referred to as Recombinant Polymerase Amplification (RPA). In some embodiments, the RPA comprises the following steps. First, a recombinase reagent is contacted with a first nucleic acid primer and a second nucleic acid primer to form a first nucleoprotein primer and a second nucleoprotein primer. Next, a first and a second nucleoprotein primer are contacted with the double stranded target sequence to form a first double-stranded structure on a first portion of the first strand and a double-stranded structure on a second portion of the second strand, such that the 3' ends of the first and the second nucleic acid primer are oriented towards each other on a given template nucleic acid or DNA molecule. Third, the 3' ends of the first and second nucleoprotein primers are extended by a DNA polymerase to generate first and second double-stranded nucleic acids, and first and second displaced strands of nucleic acid. Finally, the second and third steps are repeated until the desired degree of amplification is achieved.

Some embodiments include methods of nested RPA. In some embodiments of the nested RPA, a first region of the nucleic acid is amplified by the RPA to form a first amplified region. In some embodiments, a second region of the nucleic acid that is entirely within the first amplified region is then amplified using RPA to form a second amplified region. This process can be repeated as often as necessary. For example, a third region of the nucleic acid that is entirely within the second region can be amplified from the second amplified region by RPA. In addition to the one-, two-, and three-wheeled RPAs discussed above, at least 4-or 5-wheeled nested RPAs are also contemplated.

Some embodiments include methods of using RPA to detect genotypes. This may be useful for genotyping, for detecting normal or diseased conditions, lack of predisposition to diseased conditions, or susceptibility to disease conditions. In addition, RPA can be used to detect the presence of a genome (e.g., of a pathogen). In this use, the method is useful for diagnosis and detection.

Some embodiments include recombinases, single-stranded binding proteins, polymerases, and nucleotides that facilitate the establishment of efficient amplification reactions. Some embodiments include the use of additional components, or modified components, that help to create a recombinant polymerase amplification system that is sensitive, robust, and has optimal signal-to-noise properties. Some embodiments include the recombinases engineered or modified analogs of the E.coli recA or T4 phages uvsX, polymerases (e.g., E.coli DNA polymerase I Klenow fragment, Bst polymerase, Sau polymerase, Phi-29 polymerase, Bacillus subtilis Pol I (Bsu)), and/or single-stranded DNA binding proteins from E.coli and T4 (gp32 protein).

Some embodiments include forms of gp32 with altered synergistic and/or chain assimilation properties. Some embodiments include the use of T4 uvsY proteins and/or molecular crowding agents (e.g., PEG) to help establish an optimal reaction environment. Some embodiments include the use of other enzymes (including topoisomerases, helicases and/or nucleases) involved in DNA metabolism to improve amplification behavior. Some embodiments include using conditions optimized for repeated invasion/extension of primers targeting supercoils or linear templates to produce linear amplification, and using this method for DNA sequencing. Some embodiments include the use of a recombinase in the detection of a particular amplification product of a reaction by directing an oligonucleotide labeled in some way to a particular product species and thereby measuring a change in the appearance or nature of the reaction.

Some embodiments include implementing RPA for diagnostic applications. Some embodiments include methods of combining oligonucleotides with different activities into nucleoprotein filaments to increase signal-to-noise ratio. Some embodiments include product detection methods other than gel electrophoresis or fluorescent molecules (e.g., SYBR Green). Some embodiments include compositions of active lyophilizates that can be stored at ambient temperature for at least 10 days and retain amplification activity when reconstituted with only buffered samples.

Some embodiments include methods of controlling an RPA reaction by controlling the presence of a supporting nucleoside triphosphate cofactor (such as ATP) for a limited period of time during the reaction. If chemically caged (caged) nucleotide triphosphates are used, free ATP pulses can be generated by a defined burst of light irradiation corresponding to the uncaging wavelength of the photo-protecting group. The released ATP may allow binding of the recombinase protein to single-stranded dna (ssDNA), and subsequent homology searching and strand exchange activity of the recombinase-ssDNA complex. In some embodiments, ATP may be added to the reaction periodically. Over time, the concentration of ATP may decrease due to hydrolysis as well as ADP, whether by recombinant enzyme hydrolysis or by hydrolysis by other reaction components specifically added to hydrolyze excess ATP. Thus, after a period of time defined by a decrease in ATP concentration and/or an increase in ADP concentration, the recombinase molecule may cease functioning and separate from the DNA. Subsequent light pulses at the caged release wavelength can be delivered to release fresh ATP, or fresh ATP can be added to restart the recombinase activity. In this way, a controlled series of cycles of homology searching and triggering is achieved, thereby enabling extensions to be initiated in stages.

Some embodiments include methods of controlling the invasive stage of the RPA reaction, such as methods of separating one primer-driven activity from another. In some embodiments, the method utilizes recombinase-mediated invasion at a primer target site and synthesis is accomplished from the primer. In some embodiments, this results in a single-stranded displaced DNA that can serve as a template for a second facing primer (fascing primer) that is modified and therefore unable to support recombinase-mediated initiation of synthesis. This avoids collisions caused by polymerase collisions.

Some embodiments include methods of assessing polymorphisms of amplification products without size fractionation. In some embodiments, the amplification reaction products are allowed to form double-stranded hybrids with immobilized probes having either the original single-stranded or double-stranded characteristics by the action of a recombinase and/or single-stranded DNA binding protein and other accessory molecules. Some embodiments include methods that destabilize the imperfect hybridization formed between the product and the probe, which occurs in a dynamic environment of recombinase action and supports the activity of a variety of additional enzyme components. In some embodiments, the production hybrid is detected by one of many standard methods for revealing the presence or absence of molecular interactions.

Some embodiments include a combination of defined in vitro conditions that support a stable, persistent dynamic recombination environment with other enzymatic activities, allowing strand invasion and pairing between ssDNA and duplexes to occur continuously and in the presence of other metabolic enzymes (especially those non-thermophilic enzymes that are necessary in traditional methods), as well as other processes that are not equivalent or attainable in systems that employ thermal or chemical melting.

Some embodiments include RPA, a method for amplifying a target nucleic acid polymer. Some embodiments include a general in vitro environment, wherein high recombinase activity is maintained in a highly dynamic recombination environment, supported by ATP. One benefit of RPA is that it can be performed without the need for thermal melting of the double stranded template. Thus, the need for expensive thermal cyclers is also eliminated.

Front chain RPA (lsRPA)

In some embodiments, the RPA comprises leading strand recombinase-polymerase amplification (lsRPA). In some embodiments of lsRPA, the single-stranded or partially single-stranded nucleic acid primer is targeted to the homodouble-stranded or partially double-stranded sequence using a recombinase agent, which will form a D-loop structure. In some embodiments, an invaded single-stranded primer (part of the D-loop) is used to initiate the polymerase synthesis reaction. In some embodiments, a single primer species amplifies a target nucleic acid sequence by multiple rounds of double-stranded invasion and subsequent synthesis. If two primers are used in opposite directions, amplification of the fragment can be achieved. Examples of lsRPA are briefly described in fig. 37, 38A, and 38B.

In some embodiments, the target sequence to be amplified is double-stranded DNA. However, the target sequence to be amplified is not limited to double-stranded DNA, as other nucleic acid molecules (e.g., single-stranded DNA or RNA) can be converted to double-stranded DNA by those skilled in the art using known methods. Suitable double stranded target DNA may be genomic DNA or cDNA. The RPA of the invention can amplify a target nucleic acid by at least 10-fold, at least 100-fold, at least 1,000-fold, at least 10,000-fold, or at least 1,000,000-fold.

In some embodiments, the target sequence is amplified with the aid of a recombinase reagent. Recombinase agents include enzymes that can coat single-stranded dna (ssdna) to form filaments that can then scan regions of sequence homology of double-stranded dna (dsdna). In some embodiments, when the homologous sequences are located, the nucleoprotein filament (comprising recombinase agent) strand invades the dsDNA, creating a replacement vesicle called a D-loop and a short hybrid. Suitable recombinase reagents include the E.coli RecA protein, the T4 uvsX protein or any homologous protein or protein complex from any phylum. The eukaryotic RecA homolog is generally designated Rad51 as the first member of the panel identified. Other non-homologous recombinase agents, such as RecT or RecO, may be used. Recombinase reagents typically require the presence of ATP, ATP γ S, or other nucleoside triphosphates and analogs thereof. In some embodiments, recombinase reagents are used in a reaction environment in which regeneration of the targeting site may occur shortly after the turn of D-ring stimulated synthesis. The completed recombination events involving recombinase breakdown can avoid amplification stagnation or inefficient linear amplification of ssDNA due to oscillatory single-sided synthesis from one end to the other.

In some embodiments, the derivative or/and functional analog of the recombinase agent itself functions as the recombinase agent. For example, small peptides from recA may be used, which have been shown to retain certain aspects of the recombinant properties of recA. The peptide comprises residues 193 to 212 of recA of E.coli and mediates pairing of single stranded oligonucleotides.

Since the use of ATP γ S may lead to the formation of stable recombinase reagent/dsDNA complexes (which may not be compatible with efficient amplification), some embodiments use ATP and/or helper enzymes to load and/or maintain the recombinase reagent/ssDNA primer complexes. In some embodiments, the limitation of using ATP γ S may be overcome by using additional reaction components capable of stripping recA bound to ATP γ S from the exchange complex. In some embodiments, the effect is exerted by a helicase (e.g., RuvA/RuvB complex).

Some embodiments include a method of performing RPA, the method comprising two steps. In a first step, the following reagents are combined in a reaction: (1) at least one recombinase; (2) at least one single-stranded DNA binding protein; (3) at least one DNA polymerase; (4) dNTPs or a mixture of dNTPs and ddNTPs; (5) a crowding agent; (6) a buffer solution; (7) a reducing agent; (8) ATP or ATP analogs; (9) at least one recombinase loading protein; (10) a first primer and optionally a second primer; and (11) a target nucleic acid molecule. In a second step, the reagents are incubated until a desired degree of amplification is achieved.

In some embodiments, the recombinase is uvsX, recA, or a combination of both. In some embodiments, the recombinase comprises a C-terminal deletion of an acidic residue to increase its activity. In some embodiments, the recombinase concentration is in a range of, e.g., 0.2 μ M-12 μ M, 0.2 μ M-1 μ M, 1 μ M-4 μ M, 4 μ M-6 μ M, and 6 μ M-12 μ M.

In some embodiments, the single-stranded DNA binding protein is e.coli SSB or T4 gp32 or a combination or derivative of these proteins. The gp32 derivatives may include at least gp32(N), gp32(C), gp32(C) K3A, gp32(C) R4Q, gp32(C) R4T, gp32K3A, gp32R4Q, gp32R4T, and combinations thereof. In some embodiments, the DNA binding protein is present at a concentration between 1 μ Μ and 30 μ Μ.

In some embodiments, the DNA polymerase is a eukaryotic polymerase. Examples of eukaryotic polymerases include pol- α, pol- β, pol- δ, pol- ε, and derivatives and combinations thereof. In some embodiments, the DNA polymerase is a prokaryotic polymerase. Examples of prokaryotic polymerases include E.coli DNA polymerase I Klenow fragment, bacteriophage T4 gp43 DNA polymerase, Bacillus stearothermophilus polymerase I large fragment, Phi-29 DNA polymerase, T7 DNA polymerase, Bacillus subtilis Pol I, E.coli DNA polymerase II, E.coli DNA polymerase III, E.coli DNA polymerase IV, E.coli DNA polymerase V, and derivatives and combinations thereof. In some embodiments, the concentration of DNA polymerase is between 10,000 units/mL to 10 units/mL, such as between 5000 units/mL to 500 units/mL. In some embodiments, the DNA polymerase lacks 3'-5' exonuclease activity. In some embodiments, the DNA polymerase comprises strand displacement properties.

Some embodiments include derivatives of the proteins mentioned herein. For example, some embodiments include derivatives of recombinases, polymerases, recombinase loading proteins, single-stranded DNA binding proteins, adjuvants, RecA/ssDNA nucleoprotein filament stabilizers, and the like. Derivatives of these proteins include at least fusion proteins comprising a C-terminal tag, an N-terminal tag, or a C-terminal and an N-terminal tag. Non-limiting examples of suitable sequence tags include 6-histidine (6 x-His; HHHHHHHHHHHH), c-myc epitope (EQKLISEEDL), FLAG octapeptide (DYKDDDDK), protein C (EDQVDPRLIDGK), Tag-100(EETARFQPGYRS), V5 epitope (GKPIPNPLLGLDST), VSV-G (YTDIEMNRLGK), Xpress (DLYDDDDK), and hemagglutinin (YPVPYDDYA). Non-limiting examples of suitable protein tags include β -galactosidase, thioredoxin, His-patch (patch) thioredoxin, IgG binding domain, intron-chitin binding domain, T7 gene 10, glutathione-S-transferase (GST), Green Fluorescent Protein (GFP), and Maltose Binding Protein (MBP). Those skilled in the art will appreciate that sequence tags and protein tags may be used interchangeably, for example, for purification and/or identification purposes.

In some embodiments, dntps include, for example, dATP, dGTP, dCTP, and dTTP. In the leading and following strands, RPA, ATP, GTP, CTP and UTP may also be included for the synthesis of RNA primers. In addition, ddntps (ddATP, ddTTP, ddGTP, and ddGTP) may be used to generate fragment ladders (ladders). dNTPs can be used at a concentration of between 1. mu.M and 200. mu.M per NTP species. A mixture of dNTPs and ddNTPs can be used at 1/100 to 1/1000 where the ddNTP concentration is the dNTP concentration (1. mu.M to 200. mu.M).

In some embodiments, crowding agents used in RPA include polyethylene glycol (PEG), dextran, and Ficoll. The concentration of the crowding agent can be 1 vol% to 12 vol% or 1 wt% to 12 wt% of the reaction. Examples of PEG include PEG1450, PEG3000, PEG8000, PEG10000, PEG compounds with a molecular weight of 15000 to 20,000 (also known as Carbowax 20M), and combinations thereof.

The buffered solution in the RPA may be Tris-HCl buffer, Tris-acetate buffer, or a combination thereof. The buffer may be present at a concentration between 10mM and 100 mM. The buffered pH may be between 6.5 and 9.0. The buffer may further comprise Mg ions (e.g. in the form of magnesium acetate) at a concentration between 1mM and 100mM, preferably with a concentration between 5mM and 15 mM. One preferred Mg concentration is 10mM (Mg concentration or Mg acetate concentration).

In some embodiments, the reducing agent to be used comprises DTT. In some embodiments, the concentration of DTT is between 1mM and 10 mM.

In some embodiments, the ATP or ATP analog is ATP, ATP- γ -S, ATP- β -S, ddATP, or a combination thereof. In some embodiments, the concentration of ATP or ATP analog is between 1mM and 10 mM.

Recombinase loading proteins can include, for example, T4uvsY, E.coli recO, E.coli recR, and derivatives and combinations of these proteins. One preferred concentration of these proteins is between 0.2. mu.M and 8. mu.M.

The primers used may be made of DNA, RNA, PNA, LNA, morpholino backbone nucleic acid, phosphorothioate backbone nucleic acid, or a combination thereof. In this case, their combination refers to a single nucleic acid molecule, which may comprise one or more of one base linked to one or more of the other bases. In some embodiments, the concentration of these molecules ranges between 25nM and 1000 nM. In some embodiments, the primer comprises a non-phosphate ester linkage between two bases at its 3' end and is resistant to 3' to 5' nuclease activity. In some embodiments, the primer comprises a locked nucleic acid at its 3 'final base or 3' penultimate base. For example, in a nucleic acid of sequence 5'-AGT-3', T is the 3 'final base and G is the 3' penultimate base. In some embodiments, the primer may be at least 20 bases in length or at least 30 bases in length. In some embodiments, the primer is between 20 and 50 bases in length. In some embodiments, the primer is between 20 and 40 bases in length, for example between 30 and 40 bases in length.

In some embodiments, the primer comprises a 5' sequence that is not complementary to the target nucleic acid. These 5' sequences may comprise, for example, a restriction endonuclease recognition site. The primer may be double-stranded with the single-stranded 3' end portion.

In some embodiments, the nucleic acid is labeled with a detectable label. Detectable labels include, for example, fluorescent dyes, enzymes, fluorescence quenchers, enzyme inhibitors, radioactive labels, and combinations thereof. In some embodiments, the nucleic acid does not have a detectable label or dye or marker.

The target nucleic acid may be single-stranded or double-stranded. In some embodiments, a single-stranded nucleic acid is converted to a double-stranded nucleic acid. In some embodiments, the target nucleic acid is supercoiled or linear. The sequence to be amplified (target nucleic acid) may be located between other sequences. The sequence to be amplified may also be located at one end of the linear nucleic acid. In some embodiments, the target nucleic acid is linear and is not linked to a non-target nucleic acid. In other words, when the target nucleic acid is linear, it can be in any of the following forms:

[ non-target nucleic acid ] - [ non-target nucleic acid ]

[ non-target nucleic acid ] - [ target nucleic acid ]

[ target nucleic acid ] - [ non-target nucleic acid ]

[ target nucleic acid ]

The above arrangement is intended to represent both single-stranded nucleic acids and double-stranded nucleic acids. A "1" can be described as a linear target nucleic acid molecule having two ends and wherein both ends are attached to a non-target nucleic acid molecule. A "2" can be described as a linear target nucleic acid molecule having two ends and one of the ends is attached to a non-target nucleic acid molecule. A "3" can be described as a target nucleic acid molecule, which is a linear nucleic acid molecule (without non-target nucleic acids).

In some embodiments, the target nucleic acid is a single-stranded nucleic acid that is converted to a double-stranded nucleic acid by a polymerase or a double-stranded nucleic acid that is denatured by the action of heat or chemical treatment.

In the reaction, the target nucleic acid can be any concentration, for example, less than 10,000 copies, less than 1000 copies, less than 100 copies, less than 10 copies, or 1 copy. The reaction volume may be 5. mu.L, 10. mu.L, 20. mu.L, 30. mu.L, 50. mu.L, 75. mu.L, 100. mu.L, 300. mu.L, 1mL, 3mL, 10mL, 30mL, 50mL, or 100 mL.

The reaction may be incubated for 5 minutes to 16 hours, for example 15 minutes to 3 hours or 30 minutes to 2 hours. Incubation may be performed until a desired degree of amplification is achieved. The desired degree of amplification may be 10-fold, 100-fold, 1000-fold, 10,000-fold, 100,000-fold, or 1,000,000-fold amplification. The incubation temperature may be between 20 ℃ and 50 ℃, between 20 ℃ and 40 ℃, for example between 20 ℃ and 30 ℃. An advantage of the methods described herein is that temperature is not critical in some embodiments, and in some embodiments, precise control is not absolutely necessary. For example, in some embodiments, in a field environment, it is sufficient to maintain the RPA near body temperature (35 ℃ to 38 ℃) or at room temperature by placing the sample in a body gap. Furthermore, in some embodiments, RPA may be performed without temperature-induced melting of the template nucleic acid.

In some embodiments, the RPA includes an adjuvant. Examples of adjuvants include helicases, topoisomerases, lytic enzymes, and combinations thereof, which have helicase, relaxant, and lytic activities, respectively, on DNA. Adjuvants may include RuvA, RuvB, RuvC, RecG, PriA, PriB, PriC, DnaT, DnaB, DnaC, DnaG, DnaX clamp loader (clamp loader), polymerase core complex, DNA ligase or slide clamp or combinations thereof. The slide clamp can be an escherichia coli beta-dimer slide clamp, an eukaryotic PCNA slide clamp or a T4 slide clamp gp45, and combinations thereof. In addition, adjuvants may include a DNA polymerase III holoenzyme complex consisting of β -Clamp, DnaX Clamp-loading protein and polymerase core complex. In some embodiments, these latter adjuncts will allow for the progression of leading and trailing RPAs.

In some embodiments, RPA is performed in the presence of RecA/ssDNA nucleoprotein filament stabilizers. Examples of such stabilizers include RecR, RecO, RecF, and combinations thereof. These stabilizers may be present at a concentration of between 0.01 μ M and 20 μ M. Other examples of stabilizers include T4 uvsY protein that stabilizes the uvsX/ssDNA nucleoprotein complex.

In some embodiments, other components of RPA include systems for ATP regeneration (conversion of ADP to ATP). Such systems may be, for example, phosphocreatine and creatine kinase.

The RPA reaction may also include a system to regenerate ADP from AMP and convert pyrophosphate to phosphate (pyrophosphatase).

In some embodiments, the RPA reaction as listed above is performed entirely with the e.coli components by using recA, SSB, recO, recR, and/or e.coli polymerase.

In some embodiments, the RPA reaction is performed with a T4 component by using uvsX, gp32, uvxY, and/or T4 polymerase.

In some embodiments, RPA is performed by combining some or all of the following reagents: (1) uvsX recombinase at a concentration between 0.2 μ Μ and 12 μ Μ; (2) gp32 single-stranded DNA binding protein at a concentration of between 1. mu.M and 30. mu.M; (3) bacillus subtilis DNA polymerase I large fragment (Bsu polymerase) at a concentration of 500 units to 5000 units per mL; (4) dNTP or a mixture of dNTP and ddNTP with a concentration of between 1 μ M and 300 μ M; (5) polyethylene glycol at a concentration of 1% to 12% (by weight or volume); (6) tris-acetate buffer at a concentration between 1mM and 60 mM; (7) DTT at a concentration between 0.025mM and 1mM or between 0.025mM and 10 mM; (8) ATP at a concentration of between 1mM and 10 mM; (9) uvsY at a concentration between 0.2. mu.M and 8. mu.M; (10) a first primer and optionally a second primer, wherein the concentration of said primers is between 50nM and 1 μ Μ; and/or (11) at least one copy of a target nucleic acid molecule. In some embodiments, after the assembly (assembled) reaction, it is incubated until a desired degree of amplification is achieved. In some embodiments, this is within 2 hours, or within 1 hour, for example within 50 minutes.

An advantage of the methods described herein is that the reagents for RPA (optionally in addition to the crowding agent and the buffer) can be freeze-dried (e.g., lyophilized) prior to use. In some embodiments, freeze-dried reagent supplies are desirable because they do not require refrigeration to maintain activity. For example, tubes of RPA reagent may be stored at room temperature. This feature may be useful in field conditions where access to refrigeration is limited.

In some embodiments, RPA reagents may be freeze-dried onto the bottom of a tube or onto beads (or another type of solid support). In some embodiments, to perform the RPA reaction, the reagents are reconstituted in a buffer solution with a crowding agent, or with only buffer solution or water, depending on the composition of the lyophilized reagents. In some embodiments, a target nucleic acid or a sample suspected of containing a target nucleic acid is added. The reconstitution fluid may also contain sample DNA. In some embodiments, the reconstituted reaction is incubated for a period of time and the amplified nucleic acids (if present) are detected.

In some embodiments, the reagents are assembled by combining the reagents such that, when constituted, they will have the following concentrations: (1) uvsX recombinase at a concentration between 0.2 μ Μ and 12 μ Μ; (2) gp32 single-stranded DNA binding protein at a concentration of between 1. mu.M and 30. mu.M; (3) t4 gp43 DNA polymerase or Bsu polymerase at a concentration between 500 units and 5000 units per mL; (4) dNTP or a mixture of dNTP and ddNTP at a concentration of between 1. mu.M and 300. mu.M; (5) DTT at a concentration between 1mM and 10 mM; (6) ATP at a concentration of between 1mM and 10 mM; (7) uvsY at a concentration between 0.2. mu.M and 8. mu.M. Optionally, a first primer and optionally a second primer may be added, wherein their concentration upon reconstitution will be between 50nM and 1. mu.M. In some embodiments, the reagents are freeze-dried prior to use. Stabilizers, such as trehalose, may be included in the lyophilization mixture, for example, 20mM to 200mM and most preferably 40mM to 80mM in the reconstitution reaction, to improve lyophilization performance and shelf life. If desired, the lyophilized reagents may be stored for 1 day, 1 week, 1 month, or 1 year or more prior to use.

In use, the reagents may be reconstituted with the following buffers: (a) tris-acetate buffer at a concentration between 1mM and 60 mM; (b) polyethylene glycol at a concentration of 1% to 12% (by weight or volume), or (c) water. In some embodiments, if the primers are not added prior to lyophilization, they may be added at this stage. In some embodiments, a target nucleic acid or a sample suspected of containing a target nucleic acid is added to initiate a reaction. Due to earlier extraction or processing steps, the target or sample nucleic acid may be contained in the reconstitution buffer. In some embodiments, the reaction is incubated until a desired degree of amplification is achieved.

Any of the RPA reaction conditions discussed herein may be freeze-dried. For example, the following reagents may be assembled by combining each reagent such that they have the following concentrations at the time of constitution: (1)100 ng/. mu.L-200 ng/. mu.L of uvsX recombinase; (2)600 ng/. mu.L gp 32; (3) bsu polymerase or T4 polymerase at 20 ng/. mu.L; (4) 200. mu.M dNTP; (5)1mM DTT; (6)3mM ATP or ATP analog; (7)16 ng/. mu.L to 60 ng/. mu.L uvsY; (8) a first primer of 50nM to 300nM and a second primer of 50nM to 300 nM; (9)80mM potassium acetate; (10)10mM magnesium acetate; (11)20mM creatine phosphate; (12)50 ng/. mu.L to 100 ng/. mu.L creatine kinase. The reagents may be freeze-dried into the bottom of the tube or into the wells of the porous container. The reagents may be dried or attached to a mobile solid support (e.g., beads or strips) or wells.

As another example, the following reagents may be assembled by combining each reagent such that they will have the following concentrations when constituted: (1)100 ng/. mu.L-200 ng/. mu.L of uvsX recombinase; (2)300 ng/. mu.L-1000 ng/. mu.L gp 32; (3)10 ng/. mu.L-50 ng/. mu.L Bsu polymerase or T4 polymerase; (4) 50-500. mu.M dNTPs; (5)0.1mM to 10mM DTT; (6)3mM ATP or ATP analog; (7)16 ng/. mu.L to 60 ng/. mu.L uvsY; (8) a first primer of 50nM to 1000nM and a second primer of 50nM to 1000 nM; (9)40mM to 160mM potassium acetate; (10)5mM to 20mM magnesium acetate; (11)10mM to 40mM creatine phosphate; (12)50 ng/. mu.L to 200 ng/. mu.L creatine kinase. In some embodiments, these reagents are freeze-dried and stored. In some embodiments, in use, the reagents are reconstituted with Tris-acetate buffer at a concentration between 1mM and 60mM and polyethylene glycol at a concentration between 1% and 12% (by weight or volume). The primer of item 8 above may be omitted before lyophilization and added after reconstitution. In some embodiments, to initiate RPA, a target nucleic acid or a sample suspected of containing a target nucleic acid is added. In some embodiments, the reaction is incubated until a desired degree of amplification is achieved.

Some embodiments include reagents for performing RPA. The kit may comprise any of the reagents for RPA discussed above and may be at the concentrations described above. The reagents of the kit may be lyophilized. For example, the kit may comprise: (1)100 ng/. mu.L-200 ng/. mu.L of uvsX recombinase; (2)300 ng/. mu.L to 1000 ng/. mu.L gp 32; (3)10 ng/. mu.L to 50 ng/. mu.L of Bsu polymerase or T4 polymerase; (4)50 μ M to 500 μ M dNTPs; (5)0.1mM to 10mM DTT; (6)1mM to 5mM ATP or ATP analog; (7)16 ng/. mu.L to 60 ng/. mu.LuvsY; (8) a first primer of 50nM to 1000nM and a second primer of 50nM to 1000nM (optional); (9)40mM to 160mM potassium acetate; (10)5mM to 20mM magnesium acetate; (11)10mM to 40mM creatine phosphate; (12)50 ng/. mu.L to 200 ng/. mu.L creatine kinase.

In some embodiments, RPA is performed with several helper enzymes that can promote efficient breakdown of recombinase agent/dsDNA complexes after initiation of DNA synthesis. These include those capable of stimulating 3 'to 5' decomposition and those capable of supporting 5 'to 3' decomposition.

The helper enzymes may include several polymerases that can displace RecA in the 3 'to 5' direction and can stimulate 3 'to 5' decomposition of the recombinase agent/dsDNA complex. These DNA polymerases can include Escherichia coli PoIV and other species of homologous polymerases. In some embodiments, in the life cycle of e.coli, 3 'to 5' direction RecA replacement occurs as part of the SOS damage targeted synthesis in conjunction with SSB, a slipping clamp, and DNA polymerase. In some embodiments, the polymerase involved in this activity in e.coli is Poly, and members of the polymerase superfamily include UmuC, DinB, Rad30, and Rev1, whose in vivo function may be to replicate a DNA damage template. In some embodiments, in vitro 3 'to 5' cleavage of RecA filaments cannot be catalyzed by PolI, poiiiii, or PolIV alone. In some embodiments, only PolV in concert with SSB has measurable ATP-independent 3 'to 5' RecA/dsDNA lytic activity. Indeed, in some embodiments, PolV pushes and removes RecA from DNA in a 3 'to 5' direction ahead of the polymerase. The inclusion of PolV or functional homologues may increase the efficiency of amplification.

Other helper enzymes may include a class of enzymes known as helicases, which may be used to promote the breakdown of RecA from dsDNA. In some embodiments, these promote decomposition in both the 5 'to 3' and 3 'to 5' directions. In some embodiments, helicases function to move the branch point of a recombinant intermediate from one place to another to separate strands and to break down and recycle components bound to DNA. In some embodiments, after a first round of invasion/synthesis occurs in RPA, the two new DNA duplexes are "tagged" by the presence of RecA bound at the site to which the primer must bind for an additional round of synthesis. In this case, the dsDNA may tend to occupy a high affinity site in RecA or homologues until it is actively displaced, either by ATP hydrolysis-dependent dissociation in the 5 'to 3' direction (possibly limiting), or by 3 'to 5' dissociation by some other active process. In some embodiments, the helicase complex used to stimulate the breakdown of RecA from the intermediate comprises the e. In some embodiments, the RuvAB complex promotes branch migration and dissociates RecA proteins, allowing RecA to be recycled. In some embodiments, the RuvAB complex is targeted to a recombinant intermediate, particularly a Holliday linker-like structure. Upon its action, the RuvAB complex may surround the DNA and force RecA from DNA in ATP-driven translocations. In some embodiments, the RuvAB complex can recognize branching structures within RecA-coated DNA. Incorporation of RuvAB into RPA mixtures can promote dissociation of RecA from dsDNA after strand exchange and displacement, allowing resynthesis of replica templates from the same site. In addition, the ruva complex can act in concert with RuvC, which ultimately cleaves and cleaves the Holliday linker. In some embodiments, complex structures (e.g., Holliday linkers) formed at the site of invasion can be resolved as RuvC is added to the RPA reaction mixture. In some embodiments, a lytic enzyme activity (e.g., provided by RuvC) is useful when the targeting oligonucleotide is partially double-stranded. In some embodiments, the reverse branch migration can generate a Holliday linker that can then be cleaved by the RuvABC complex to generate a clean, isolated amplification product.

In some embodiments, the helper enzyme comprises an e.coli RecG protein. In some embodiments, RecG can stimulate the breakdown of the branching structure. In vivo, the protein may function to turn the replication fork at the site of DNA damage by derotating both the leader and trailer strand to drive the replication fork back to generate a 4-way junction. In some embodiments, in vivo, this linkage functions as a substrate for chain switching to allow for lesion bypassing. In vitro, RecG can bind to the D-ring and cause a reduction in D-ring structure by driving reverse branch migration. In some embodiments, RecG prefers ligation with double stranded elements on either side, and therefore partially double stranded targeting oligonucleotides homologous to the targeting site in both single and double stranded regions may be useful. This may stimulate reverse branch migration and the formation of a Holliday linker, which can be resolved by the RuvABC complex. In vivo, RecG and ruva may compete to produce different recombination outcomes because branch migration will be driven in both directions. In both cases, the protein can target the ligated DNA coated with RecA and break it down in an active manner.

In some embodiments, the helper enzyme used in the RPA reaction mixture allows for the continuous production of RecA nucleoprotein filaments in the presence of ATP and SSB. To allow for the removal of RecA at the appropriate time, some embodiments use ATP instead of ATP γ S in the RPA reaction. In some embodiments, RecA/ssDNA filaments formed with ATP spontaneously disaggregate in the 5 'to 3' direction and do not repolymerize at a significant rate in the presence of SSB. In some embodiments, a solution to this problem is the use of RecO, RecR, and/or possibly RecF proteins. In some embodiments, the uvsY protein is used to stabilize the T4 uvsX nucleoprotein filament in a similar manner. In some embodiments, the RecA/ssDNA filament dissociates in the presence of SSB and ATP. In some embodiments, such dissociation does not occur if RecA/ssDNA is incubated in the presence of RecO and RecR proteins. In some embodiments, the RecR protein remains bound to the silk and stabilizes the structure for an indefinite period of time. In some embodiments, in the presence of RecR and RecO, even if ssDNA is bound by SSB, the filaments of RecA can reassemble in place of SSB. In the T4 phage system, similar properties may be attributed to the uvsY protein. Thus, in some embodiments, ATP γ S may be avoided by using ATP to maintain RecA/ssDNA filament integrity in the presence of RecO and RecR, or uvsY to maintain uvsX/ssDNA filament integrity. In some embodiments, the RecF protein interacts with the RecO and RecR systems in a seemingly opposite manner. RecF competes with RecR, tending to drive silk breakdown in vitro. In some embodiments, all three components work together in vivo to control the production of invasive structures while limiting the extent of RecA coating of ssDNA. In some embodiments, RecF is included in the RPA reaction at a suitable concentration to reproduce the kinetics of the in vivo process. RecF may facilitate dissociation of RecA-coated intermediates after invasion has occurred.

In some embodiments, RPA allows for the formation of short extension fragments of double-stranded nucleic acids with free 3' -OH for extension from double-stranded templates without the need for thermal melting. In some embodiments, this is achieved by using RecA proteins from e.coli (or RecA homologues from other phyla, including the T4 uvsX protein). RecA or uvsX may form nucleoprotein filaments around single-stranded DNA in the presence of ATP, dATP, ddATP, UTP, ATP γ S and possibly other types of nucleoside triphosphates and their analogs. In some embodiments, the silk scans double-stranded DNA. In some embodiments, when the homologous sequences are located, the recombinase will catalyze the strand invasion reaction and pairing of the oligonucleotide with the homologous strand of the target DNA. In some embodiments, the original paired strand is displaced by strand invasion, leaving a bubble of single-stranded DNA in this region.

In some embodiments, the RecA protein is obtained from a commercial source. In some embodiments, it is purified according to standard protocols. In some embodiments, the RecA homolog is purified from a thermophilic organism, including Thermococcus kodakaraensis, Thermotoga maritima (Thermotoga maritima), liquid producing pyrobacteria (Aquifex pyrophilus), Pyrococcus furiosus (Pyrococcus furiosus), Thermus aquaticus (Thermus aquaticus), pyrobacum islandicum, or Thermus thermophilus (Thermus thermophilus). In some embodiments, RecA is purified from prokaryotes such as Salmonella typhimurium (Salmonella typhimurium), bacillus subtilis, Streptococcus pneumoniae (Streptococcus pneumoniae), Bacteroides fragilis (Bacteroides fragilis), Proteus mirabilis (Proteus mirabilis), Rhizobium meliloti (Rhizobium meliloti), Pseudomonas aeruginosa (Pseudomonas aeruginosa), including vertebrates (e.g., human Rad51 or Xenopus laydis), or from plants such as broccoli, in some embodiments, escherichia coli a and T4 uvsX proteins are purified from the over-expressed culture using a C-terminal hexahistidine tag and retain biological activity.

In some embodiments, the leader recombinase-polymerase amplification method (lsRPA) can be divided into four stages.

1) Sequence targeting

In some embodiments, RPA is initiated by using a targeting sequence of a synthetic oligonucleotide coated with RecA or a functional homolog (e.g., T4 uvsX protein). In some embodiments, to allow for exponential amplification, two such synthetic oligonucleotides are used in such a way that their free 3' -ends face each other. In some embodiments, the nucleoprotein filaments comprising these oligonucleotides and recombinase proteins rapidly and specifically recognize a target in the composite DNA. In some embodiments, once targeted, the recombinase protein catalyzes strand exchange, forming a D-loop structure. Some embodiments use ATP instead of ATP γ S in the procedure for efficient amplification. If ATP, RecO, RecR and/or RecF molecules are used, the usefulness for efficient amplification may be demonstrated, or if uvsX recombinase is used, the uvsY protein may be demonstrated to be useful.

2) Initiation of DNA Synthesis

In some embodiments, the DNA polymerase detects and binds to the hybrid between the invasive oligonucleotide and the template DNA and initiates DNA synthesis from the exposed free 3' -hydroxyl group in the hybrid. Some embodiments include the disassembly of recombinase proteins from double-stranded hybrids formed by strand exchange after exposure of the 3' -hydroxyl group, and subsequent DNA synthesis. In some embodiments, to achieve this breakdown, ATP is used which can support spontaneous breakdown of the recombinant enzyme from the invading complex. Furthermore, the decomposition may be stimulated/enhanced by using other proteins contained in the reaction mixture, such as RuvA, RuvB, RuvC, recG, other helicases or other stimulating components that may act to strip the recombinase from the strand exchange products.

3) Strand displacement DNA synthesis and replicon isolation.

In some embodiments, when the DNA polymerase synthesizes a complementary copy of the template DNA or a partial extension product thereof using the free 3' -hydroxyl group of the invasive oligonucleotide, the polymerase replaces single-stranded DNA, which may be coated with a single-stranded binding protein (SSB) included in the reaction. In some embodiments, invasion of the oligonucleotides across the target nucleic acid sequence occurs within a similar time frame such that the two polymerases on the same template nucleic acid initially progress toward each other. In some embodiments, when these extension complexes meet each other, the original template simply fans out and the polymerase continues to synthesize without strand displacement, now copying the SSB-bound ssDNA template. In some embodiments, due to steric hindrance, when the polymerases meet, the polymerase can temporarily dissociate from the template to allow separation of the two template strands.

4) Synthesis and re-invasion were completed.

In some embodiments, once the template strands have been separated, the polymerase completes extension to the template end (or, if the initial template is longer than the desired product, extends through the sequence serving as the second-facing targeting site). In some embodiments, the new product is targeted and replicated in a manner similar to the original template (i.e., from both targeting ends) to allow for exponential amplification. In some embodiments, for targeting recombinase/oligonucleotide filaments, newly synthesized targeting sites are freely available. In some embodiments, the site initially used to prime synthesis is vacated due to the use of conditions in the reaction that favor the resolution of the recombinase from the strand exchange product. In some embodiments, single-stranded DNA will not be the primary product and exponential amplification will occur, provided that re-invasion at the latter site occurs at a time shorter than the time required for polymerase synthesis to pass through the second targeting site, prime at the second site, and return to the first site. In some embodiments, having multiple synthetic complex runs on the same template allows for shorter amplification times.

RPA synthesized using synchronized leader and trailer chains

In some embodiments, lsRPA comprises a multi-component system with the ability to regenerate targeting sequences, thereby allowing exponential amplification of double stranded DNA. In some embodiments, lsRPA avoids linear production of single stranded DNA. Another approach that completely avoids the possibility of single stranded products and the requirement for simultaneous end-initiation in some embodiments involves more complex reaction mixtures. In some embodiments, the system replicates events that occur during the normal replication cycle of a cell to allow for the synthesis of the coupled leader and trailer chains. As an example of this approach, the leader/trailer chain RPA is described briefly in fig. 37 and fig. 39A-39D.

For clarity of description, the RPA process may be divided into four stages, but in some embodiments, all stages occur simultaneously in a single reaction.

1) Sequence targeting

In some embodiments, RPA is initiated by using a targeting sequence of a synthetic oligonucleotide coated with RecA, or T4 uvsX, or a functional homolog. In some embodiments, such nucleoprotein filaments rapidly and specifically recognize a target in the complexed DNA. In some embodiments, once targeted, RecA or uvsX proteins catalyze strand exchanges, forming D-loop structures. Some embodiments include the use of ATP instead of ATP γ S in the procedure for efficient amplification. In some embodiments, ligation of the leader and trailer strand synthesis avoids rapid recombinase stripping after synthesis initiation. In some embodiments, if ATP is used, either the RecO, RecR, and RecF are used with bacterial recA recombinase, or the T4 uvsY protein and the T4 uvsX protein are used for efficient amplification.

2) Initiator (Primosome) assembly

In some embodiments, the initiator is assembled at the D-ring. In some embodiments, the D-loop structure is formed by RecA in e.coli as part of a mechanism to salvage damaged DNA in vivo or during other forms of recombination. In some embodiments, the combined effect of RecA-mediated strand exchange and priming assembly is to generate a replication fork. An example of a replication fork is a nucleoprotein structure comprising an isolated template DNA strand and a replicator. In some embodiments, the replicons include a polymerase holoenzyme complex, a priming entity, and other components for simultaneously replicating both strands of template DNA. In some embodiments, the priming entity provides DNA helication and an okazaki fragment priming function for replication of fork processes. In some embodiments, similar priming assembly occurs at recombinant intermediates in the T4 phage directed by the gp59 and gp41 proteins.

In some embodiments, the initiator assembly proteins are PriA, PriB, PriC, DnaT, DnaC, DnaB and DnaG. In some embodiments, these proteins can assemble a priming complex on bacteriophage phiX174 DNA in vitro. In some embodiments, the PriA binds to a priming body assembly site (PAS) on the phiX174 chromosome. In some embodiments, PriB, DnaT and PriC bind to the PriA-DNA complex sequentially. In some embodiments, PriB stabilizes PriA at PAS and promotes binding of DnaT. In some embodiments, omitting PriC from the reaction reduces priming by a factor of 3 to 4. In some embodiments, the function of PriC in the bacterium is genetically redundant to PriB. In some embodiments, DnaC loads DnaB into the complex in an ATP-dependent manner. In some embodiments, the PriABC-DnaBT complex is capable of translocating along a chromosome. In some embodiments, the DnaG primase transiently interacts with the complex to synthesize an RNA primer.

In some embodiments, DnaB and DnaG function as helicase and primase, respectively, during replication in e. In some embodiments, these two components are combined with PolIII holoenzyme to synthesize primers for okazaki fragments. In some embodiments, the other initiator components described participate in assembly of the initiator onto DNA, as well as in association with dimeric polymerases. In some embodiments, the priming body assembly protein participates in the reconstitution of a replication fork at the recombinant intermediate formed by RecA and strand exchange. In some embodiments, PriA initiates the assembly of a replicate capable of DNA synthesis on a recombinant intermediate. In some embodiments, the D-ring can be targeted in vitro with a mixture of PriA, PriB, and DnaT, which is then able to incorporate DnaB and DnaC. In some embodiments, once the priming entity is formed at the D-ring, what remains to initiate replication is to load the holoenzyme complex into the site. In some embodiments, in the phage T4 system, the gp59 helicase loading protein recruits and assembles gp41 to replicate the helicase into a D-ring structure.

3) Fork assembly and initiation of DNA synthesis

In some embodiments, the replication fork will assemble at the priming body assembly site. In some embodiments, in e.coli, the presence of a free 3' -end on the invasive strand of the D-loop stimulates the DnaX clamp carrier complex detailed previously to assemble a β -dimer at this site to act as a sliding clamp. In some embodiments, the holoenzyme and 2 core units are linked together by a scaffold T subunit. In some embodiments, the r subunit has a DnaB helicase component for the elicitor, an interaction surface for the β -dimer, and for a clamp vector (clamp loader). In some embodiments, these multiple interactions coordinate the synthesis of the leading and trailing strands using 2 asymmetrically linked core polymerase complexes. In some embodiments, in the T4 phage, the gp59/41 protein initiates replicon assembly with uvsY and gp32 proteins and with other components that coordinate the assembly of the sliding clamp gp45 aided by gp44 and gp62 proteins.

In some embodiments, in E.coli, the primosome-primase DnaG synthesizes short RNA primers on the unraveled followed strand DNA template. In some embodiments, the clip loader recognizes an RNA/DNA duplex and loads a second β -dimer clip onto the site in the presence of a holoenzyme. In some embodiments, the presence of active elicitors and the interaction of the r subunit with DnaB ensures synchronous leader/trailer chain synthesis.

In some embodiments, the replication fork is now assembled. In some embodiments, synthesis of both the leader and the trailer strand occurs simultaneously, and DnaB helicase separates the template strand immediately prior to the full enzyme. In some embodiments, okazaki fragments ranging in length from 1 kilobase to 2 kilobases are generated following the strand holoenzyme core. In some embodiments, once the preceding RNA primer is encountered by the following strand polymerase, it separates from the β -clamp and synthesis begins with a newly assembled clamp loaded near the front of the leading strand. In some embodiments, the same trailing strand holoenzyme core is reused because it is physically tethered to the leading strand core.

In some embodiments, there is a dynamic interaction between the β -dimer clamp, the core subunit, and the clamp loader. In some embodiments, their affinity switches according to the physical environment. In some embodiments, the β -dimers that have been "discarded" at the end of the okazaki fragment are recovered by active removal of the excess 6 subunits or bracketing vector that may be present.

In some embodiments, the RNA primer at the end of the okazaki fragment is removed by the 5 'to 3' exonuclease activity of DNA polymerase I. DNA ligase then ligates the okazaki fragments together to form a continuous, successor strand.

4) Fork meet and terminate

In some embodiments, in RPA, replication is initiated at two distal sites and replication forks are directed towards each other. In some embodiments, as the replication forks converge, the two original template strands will separate from each other because they are completely separated behind and in front of each fork. In some embodiments, the lead strand core of each fork is completely synthesized, the remaining RNA primers are processed, and the final product is two double-stranded molecules. In some embodiments, nucleic acids are amplified by this method on the order of megabases (Mb). In the present disclosure, megabases also include megabase pairs. In some embodiments, based on the known synthesis rate of PolIII holoenzymes, the replication fork proceeds at a rate of about 1Mb/1000 seconds, i.e., about 15 to 20 minutes per cycle for the immb fragment.

Some embodiments allow for efficient re-invasion of the target site through the use of helicases, resolvases, and mixtures of RecO, RecR, and RecF proteins. In some embodiments, re-invasion and priming of body assembly is possible shortly after removal of the holoenzyme from the fork assembly site under appropriate conditions. In some embodiments, the DNA branches at several points, and each branch naturally breaks down when encountering an upcoming fork. Under these conditions, it is possible to achieve a huge amplification in a time similar to the time it takes for DNA to replicate only once. Some embodiments limit the concentration of targeting oligonucleotides to avoid nucleotide depletion before synthesis is complete.

In addition to the holoenzyme complex, in some embodiments, the replicator utilizes another complex called a primer, which is synthesized to follow the strand and move the replication fork forward. In some embodiments, the elicitor complex comprises a helicase encoded by DnaB and a primase encoded by DnaG. In some embodiments, replication includes the activities of single-stranded DNA binding protein (SSB), e.coli DNA polymerase I, and DNA ligase, in addition to the proteins of the holoenzyme and the priming entity.

Nest type RPA

In some embodiments, the RPAs comprise nested RPAs. In some embodiments, the nested RPA involves a first RPA of a first region of DNA. In some embodiments, the reaction mixture is diluted, e.g., 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, or 100-fold or more to reduce the concentration of the first primer pair, and the second primer pair is introduced into the reaction mixture and the RPA is repeated. According to one embodiment of the invention, the second primer pair is designed to be internal to the first primer pair to amplify a subsequence of the first RPA product. In some embodiments, the method increases specific amplification, i.e., reduces non-specific background amplification products and thus increases sensitivity.

In some embodiments, nested RPAs are not limited to the use of two sets of primers. In some embodiments, more sets of primers may be used to increase specificity or sensitivity. Thus, three, four or five pairs of primers may be used. In addition, as another embodiment of the present invention, different primer sets may share a common primer as shown in fig. 40.

In the example of fig. 40, the primer sets are designed for sequential use. For example, the first RPA is performed with the primer set 1, the second RPA with the primer set 2 using the amplification product of the first RPA, the third RPA with the primer set 3 using the amplification product of the second RPA, the fourth RPA with the primer set 4 using the amplification sequence of the third RPA, and finally, the fifth RPA with the primer set 5 using the amplification product of the fourth RPA. In this case, the primer sets 1, 2 and 3 share a common primer, primer (a). Primer sets 3, 4 and 5 share a common primer, primer (b).

Nested RPA can be performed using either of the two RPA methods described, as well as a combination of the two methods in any particular order. That is, RPA may be performed by the leading strand RPA alone, by the leading and trailing strands RPA alone, or by a combination of the leading strand RPA and the leading and trailing strands RPA in any particular order.

RPA reagent solution

Some embodiments of the methods described herein relate to RPA reagent solutions. In some embodiments, the RPA reagent solution comprises a target nucleic acid, primers each complementary to a region of the target nucleic acid, a buffer, dntps, a recombinase, a single-stranded DNA binding protein (SSB), and/or a strand-displacing DNA polymerase.

In some embodiments, the recombinase enzyme comprises UvsX or RecA. In some embodiments, the SSB comprises gp32 or e. In some embodiments, the strand displacement DNA polymerase includes Bst DNA polymerase large fragment, Bst 2.0 polymerase, Bst 3.0 polymerase, Gsp polymerase, Sau polymerase, Bsu DNA polymerase large fragment, Deep VentR DNA polymerase, Deep VentR (exo-) DNA polymerase, Klenow fragment (3'→ 5' exo-), DNA polymerase I large (Klenow) fragment, phi29 DNA polymerase, VentR DNA polymerase, or VentR (exo-) DNA polymerase. Other recombinases, SSBs, and/or strand displacing polymerases, such as those described herein, can also be used.

In some embodiments of the methods described herein, the RPA reagent solution or amplification reaction solution comprises RPA primers. In some embodiments, the RPA primers comprise conventional PCR primers that are about 20 bases long. In some embodiments, the RPA primers comprise longer primers (30 bases to 45 bases) than traditional PCR primers. RPA with longer primers may produce faster amplification than RPA with traditional PCR primers.

Other RPA agents (e.g., those described herein) may also be used in the methods described herein. In some embodiments, the RPA reagent solution comprises a reagent selected from the group consisting of: tris-acetate, polyethylene glycol, trehalose, potassium acetate, creatine kinase, phosphocreatine, adenosine triphosphate, or a recombinase-loaded protein (e.g., uvsY), or wherein the RPA reagent solution or the amplification reaction solution does not contain a primer with a detectable label or dye. In some embodiments, the amplification reaction solution comprises DTT at a concentration of 0.5mM to 1mM, 0.25mM to 0.5mM, about 0.25mM, 0mM to 0.25mM, or 0 mM. In some embodiments, the amplification reaction solution comprises DTT at a concentration of 1mM-10mM, about 5mM, or 5mM, and calcium chloride at a concentration of 5mM-15mM, calcium chloride at a concentration of about 0.9mM, calcium chloride at a concentration of 0.9mM, Ca at a concentration of 5mM-15mM²⁺Ca of about 0.9mM²⁺Or 0.9mM Ca²⁺. In some embodiments, the amplification reaction solution comprises a concentration of 1mM-20mM, 1mM-5magnesium or magnesium ions in mM, 5mM-10mM, 7mM-9mM, 8mM, about 8mM, 10mM-20mM, about 13mM, or 13 mM. In some embodiments, the amplification reaction solution comprises a dNTP mixture at a concentration of 1mM-10mM, 1mM-3mM, about 1.8mM, 5mM-6mM, about 5.6mM, or 5.6 mM. In some embodiments, the amplification reaction solution further comprises a blocking region oligonucleotide comprising a nucleic acid sequence that is the reverse complement of a portion of the nucleic acid sequence of one or more of the primers.

Blocking region oligonucleotide

Some embodiments of the methods described herein relate to the use of blocking region oligonucleotides (also referred to herein as "blocking regions"). One problem when testing RPA in some cases is the inability to distinguish between true positive amplification ("positive") and false positive amplification ("NTC"). Some RPA primer sets showed NTCs that amplified as fast as positives, and sometimes the signal amplitude from NTCs was also greater than that from positives. This may be due to the low temperature nature of RPA, which may allow more primer-primer interactions to occur. Most researchers have only used probes to solve this problem because they only show signals from positives. A solution to this problem that is compatible with the sensors described herein is the use of blocking region oligonucleotides.

In some embodiments, the blocking region comprises a short oligonucleotide sequence having the following exemplified features:

(1) reverse complementation of the primer for RPA, starting from the 3' end. In other words, the 5 'terminal base of the blocking region hybridizes to the 3' terminal base of the primer. This may render the primer considered partially double stranded.

(2) The length ranges from 8 nucleotides up to 20 or more. It was found that the exact length may depend on the primer, but lengths approaching 15 nucleotides are generally best.

(3) Chemically modified at the 3' end to prevent extension by polymerase. For example, they may be phosphorylated. Although sufficient for our purposes, phosphorylation may not be suitable for reactions comprising exonucleases (e.g., exo + polymerase, or reactions using probes for detection), as exonucleases may remove phosphate groups from oligonucleotides. However, in these cases, other polymerase blocking (e.g., 3-carbon spacer) modifications that are exonuclease resistant are widely available.

Overview of exemplary enhanced conductivity changes

Some embodiments of the methods, systems, and compositions provided herein include enhancing the change in solution conductivity resulting from nucleic acid amplification. In some embodiments, chelation of pyrophosphate ("PPi") produced by nucleic acid amplification may be used to enhance the change in solution conductivity as the amplification reaction continues. Without being bound by any one theory, the change in conductivity that may occur during nucleic acid amplification may be based on the precipitation of magnesium cations and PPi ions from solution. Some embodiments of the methods provided herein may include increasing the change in conductivity by changing the equilibrium, which additionally results in the precipitation of magnesium cations and PPi ions. In some embodiments, this is achieved by the addition of molecules that compete for PPi with magnesium cations. In some such embodiments, compounds with high ion mobility are provided that will result in a high contribution to the net solution conductivity. Thus, removal of the compound from the solution by precipitation of the compound with PPi produces a significant change in the conductivity of the solution. In some embodiments of the methods provided herein, compounds/complexes that can bind PPi as amplification continues and cause a change in solution conductivity and/or an enhanced change in solution conductivity include Cd ²⁺-cyclen-coumarin, Zn with bis (2-pyridylmethyl) amine (DPA) units²⁺Complex, DPA-2Zn²⁺Phenate, acridine-DPA-Zn²⁺、DPA-Zn²⁺-pyrene and aza crown-Cu²⁺A complex compound. See, e.g., Kim S.K.et al, (2008) Accounts of Chemical Research 42:23-31, and Lee D-H, et al, (2007) Bull. Korean chem.Soc.29: 497-; credo G.M.et al, (2011) analysis 137: 1351-.

Some embodiments includeCompounds such as 2 amino-6-mercapto-7-methylpurine ribonucleoside (MESG). Use of MESG in a kit for detection of pyrophosphate, e.g.Pyrophosphate assay kit (ThermoFischer Scientific) in which MESG is converted by Purine Nucleoside Phosphorylase (PNP) to ribose 1-phosphate and 2 amino-6-mercapto-7 methylpurine in the presence of inorganic phosphate. Enzymatic conversion of MESG shifted the absorbance maximum from 330nm to 360 nm. PNP catalyzes the conversion of pyrophosphate to two equivalents of phosphate. The phosphate was then consumed by the MESG/PNP reaction and detected by the increase in absorbance at 360 nm. Additional sensitivity was obtained by amplifying one molecule of pyrophosphate into two molecules of phosphate. Another kit includes the PIPER pyrophosphate assay kit (ThermoFischer Scientific).

In some embodiments, the change that enhances the conductivity of the solution resulting from nucleic acid amplification comprises a compound that binds to the amplified DNA. In some such embodiments, as amplification continues, charge-carrying species bind to the increased amount of amplified DNA, causing a net decrease in solution conductivity. In some embodiments, the charge carrying substance may include positively charged molecules (e.g., ethidium bromide, crystal violet, SYBR) that are commonly used as DNA/RNA colorants/dyes that bind to nucleic acids by electrostatic attraction. The binding of these small charged molecular species to large low mobility amplification products can reduce the conductivity of the solution by effectively reducing the charge mobility of the dye molecules. It should be noted that although such electrostatic attraction is a mechanism by which DNA is often stained for gel electrophoresis, the molecules bound to the amplicon need not be compounds that are traditionally used as DNA stains. Since these molecules are utilized for their function as charge carriers (contributors to solution conductivity) and their ability to bind to amplicons, they do not need to have any DNA staining properties. In some embodiments, the substance that carries a charge comprises alizarin red S. For example, alizarin red S can interact with the amplified DNA molecules and voltammetrically change the behavior of the amplified DNA, thereby enhancing detection of the amplified DNA by the systems or devices described herein.

Some embodiments include the use of an antibody or aptamer attached to a nanoparticle. In some such embodiments, the presence of the target antigen causes aggregation of the antibody and a change in the conductivity of the solution. Without being bound by any one theory, the effective conductivity of colloidal nanosuspensions in liquids may exhibit a complex dependence on Electric Double Layer (EDL) characteristics, volume fraction, ion concentration, and other physicochemical properties. See, e.g., Angayarkanni SA, et al, Journal of Nanofluids,3:17-25, which is expressly incorporated herein in its entirety by this reference. Antibody-conjugated nanoparticles are well known in the art. See, e.g., Arruebo M.et al, Journal of Nanomaterials 2009 Article ID 439389 and Zawrah MF., et al, HBRC Journal 2014.12.001, each of which is expressly incorporated herein by reference in its entirety. Examples of nanoparticles for use in the methods provided herein include γ -Al₂O₃、SiO₂、TiO₂And alpha-Al₂O₃See, for example, Abdelhalim, MAK., et al, International Journal of the Physical Sciences,6: 5487-. The use of antibodies attached to the nanoparticles can also enhance the signal generated at the surface by performing measurements using Electrochemical Impedance Spectroscopy (EIS). See, for example, Lu j., et al, Anal chem.84: 327-.

Some embodiments of the methods, systems, and compositions provided herein include the use of antibodies or aptamers linked to enzymes. In some embodiments, the enzymatic activity causes a change in the conductivity of the solution. In some such embodiments, the change in conductivity is detected by transferring a charge to a substrate in contact with the assay component.

Overview of exemplary viral targets

Some embodiments of the methods, systems, and compositions provided herein include the detection of certain viruses and viral targets. The viral target may include viral nucleic acids, viral proteins, and/or viral active products (e.g., enzymes or activities thereof). Examples of viral proteins detected using the methods and apparatus provided herein include viral capsid proteins, viral structural proteins, viral glycoproteins, viral membrane fusion proteins, viral proteases, or viral polymerases. Viral nucleic acid sequences (RNA and/or DNA) corresponding to at least a portion of the genes encoding the above viral proteins are also detected using the methods and apparatus described herein. The nucleotide sequences of these targets are readily available from public databases. Primers for isothermal amplification are readily designed from the nucleic acid sequence of the desired viral target. Antibodies and aptamers to the proteins of these viruses are also readily obtained commercially and/or by techniques well known in the art. Examples of viruses that are detected using the methods, systems, and compositions provided herein include DNA viruses (e.g., double-stranded DNA viruses and single-stranded viruses), RNA viruses (e.g., double-stranded RNA viruses, single-stranded (+) RNA viruses, and single-stranded (-) RNA viruses), and retroviruses (e.g., single-stranded retrorna viruses and double-stranded retrodna viruses). Viruses detected using this technique include animal viruses (e.g., human viruses, livestock viruses) or plant viruses. Examples of human viruses that are detected using the methods, systems, and compositions provided herein include those listed in table 2 below, which also provides exemplary nucleotide sequences from which primers for amplification can be readily designed.

TABLE 2

Overview of exemplary bacterial targets

Some embodiments of the methods, systems, and compositions provided herein include the detection of certain bacteria and bacterial targets. Bacterial targets include bacterial nucleic acids, bacterial proteins, and/or bacterial activity products (e.g., toxins and enzyme activities). Nucleotide sequences indicative of certain bacteria are readily available from public databases. Primers for isothermal amplification are readily designed from the nucleic acid sequences of these bacterial targets. Antibodies and aptamers to certain bacterial proteins are readily obtained commercially and/or by techniques well known in the art. Examples of bacteria detected using the methods, systems, and compositions provided herein include gram negative bacteria or gram positive bacteria. Examples of bacteria detected using the methods, systems, and compositions provided herein include: pseudomonas aeruginosa, Pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas acidovorans (Pseudomonas acidovorans), Pseudomonas alcaligenes (Pseudomonas alcaligenes), Pseudomonas putida (Pseudomonas putida), Stenotrophomonas maltophilia (Stenotrophora maltholii), Burkholderia cepacia (Burkholderia cepacia), Aeromonas hydrophila (Aeromonas hydrophila), Escherichia coli (Escherichia coli), Citrobacter freundii (Citrobacter freundii), Salmonella typhimurium, Salmonella typhi (Salmonella typhimurium), Salmonella paratyphi (Salmonella paratyphi), Salmonella enteritidis (Salmonella enterica), Shigella dysenteriae (Shigella dysenteriae), Salmonella aerogenes (Salmonella aerogenesis), Salmonella choleraesula, Salmonella enterica), Salmonella enterica (Escherichia coli (Klebsiella pneumoniae), Salmonella enterica (Escherichia coli), Salmonella aerogenes), Shigella (Escherichia coli), Shigella acidovorax lactis (Klebsiella pneumoniae), Escherichia coli (Klebsiella pneumoniae (Escherichia coli), Escherichia coli (Escherichia coli) Francisella tularensis (Francisella tularensis), Morganella morganii (Morganella morganii), Proteus mirabilis, Proteus vulgaris (Proteus vulgaris), Alkaligenes proliferis (Providence alcalifaciens), Providence Providencia (Providence rettgeri), Providence Providencia (Providence stuartii), Acinetobacter baumannii (Acinetobacter baumannii), Acinetobacter calcoaceticus (Acinetobacter calcoaceticus), Acinetobacter haemolyticus (Acinetobacter halyelyticus), Yersinia enterocolitica (Yersinia entorolytica), Yersinia pestis (Yersinia pestis), Pseudobulbus tuberculosis (Yersinia vernospora), Yersinia parahaemolytica (Bordetella), Bordetella parahaemolytica (Bordetella parahaemolytica), Bordetella pertussis Haemophilus (Bordetella), Bordetella parahaemophilus (Bordetella), Bordetella pertussis Haemophilus (Bordetella), Bordetella Haemophilus (Bordetella Haemophilus), Bordetella Haemophilus, Bordetella Haemophilus pestis, Bordetella Haemophilus, Bordetella, haemophilus parahaemolyticus (Haemophilus parahaemolyticus), Haemophilus ducreyi (Haemophilus ducreyi), Pasteurella multocida (Pasteurella multocida), Pasteurella haemolyticus (Pasteurella haemolytica), Moraxella catarrhalis (Branhamella catarrhalis), Helicobacter pylori (Helicobacter pylori), Campylobacter foetidus (Campylobacter fetalis), Campylobacter jejuni (Campylobacter jejuni), Campylobacter coli (Campylobacter coli), Borrelia burgdorferi (Borrelia burgdorferi), Vibrio cholerae (Vibrio cholerae), Vibrio parahaemolyticus (Vibrio parahaemolyticus), Legionella pneumophila (Legionlla), Salmonella parahaemolyticus (Legionnella), Salmonella parahaemolyticus (Salmonella), Salmonella choleraesuis serotype meningitidis (Salmonella), Salmonella cholera viridans (Salmonella cholerae), Salmonella cholera viridans (Salmonella cholera), Salmonella cholera meningitidis (Salmonella cholerae), Salmonella cholera viridans (Salmonella), Salmonella cholera, and Salmonella cholera, or Salmonella cholera, Bacteroides vulgatus (Bacteroides vulgatus), Bacteroides ovatus (Bacteroides ovatus), Bacteroides thetaiotaomicron (Bacteroides thetaiotaomicron), Bacteroides monoformans (Bacteroides uniflora), Bacteroides exuberans (Bacteroides eggerthii), Bacteroides visceral (Bacteroides sp.), Clostridium difficile (Clostridium difficile), Mycobacterium tuberculosis (Mycobacterium tuberculosis), Mycobacterium avium (Mycobacterium avium), Mycobacterium intracellulare (Mycobacterium intracellularis), Mycobacterium leprae (Mycobacterium intracellularis), Corynebacterium diphtheriae (Corynebacterium diphtheriae), Streptococcus pneumoniae, Streptococcus agalactiae (Streptococcus pneumoniae), Staphylococcus aureus (Staphylococcus aureus), Staphylococcus aureus (Staphylococcus epidermidis), Staphylococcus aureus (Staphylococcus aureus) Staphylococcus haemolyticus (Staphylococcus haemolyticus), Staphylococcus hominis (Staphylococcus hominis) and/or Staphylococcus saccharolyticus (Staphylococcus saccharolyticus). Further examples include bacillus anthracis (b. anthracris), bacillus sphaericus (b. globigii), Brucella (Brucella), erwinia herbicola (e. herbicola), or francisella tularensis.

Overview of exemplary antigen targets

Some embodiments of the methods, systems, and compositions provided herein include the detection of certain antigen targets. The antigen is detected using an aptamer, or an antibody, binding fragment thereof, linked to a primer configured for amplification, e.g., isothermal amplification. Antibodies and aptamers to certain antigens are readily obtained commercially and/or by techniques well known in the art. As used herein, "antigen" includes a compound or composition that is specifically bound by an antibody, binding fragment thereof, or aptamer. Examples of antigens that can be detected using the methods, systems, and compositions provided herein include proteins, polypeptides, nucleic acids, and small molecules (e.g., pharmaceutical compounds). Further examples of analytes include toxins such as ricin, abrin, botulinum toxin, or staphylococcal enterotoxin B.

Overview of exemplary parasite targets

Some embodiments of the methods, systems, and compositions provided herein include detection of certain parasite targets. Parasite targets include parasite nucleic acids, parasite proteins, and/or parasite activity products (e.g., toxins and/or enzymes, or enzyme activities). Nucleotide sequences indicative of certain parasites are readily available from public databases. Primers for isothermal amplification are readily designed from the nucleic acid sequences of such parasite targets. Antibodies and aptamers to certain parasite proteins are readily obtained commercially and/or by techniques well known in the art. Examples of parasites that can be detected using the methods, systems and compositions provided herein include certain endoparasites, such as protozoan organisms such as Acanthamoeba (Acanthamoeba spp.), Babesia (Babesia spp.), Babesia divergens (B.divergens), Babesia bovis (B.bigemina), Babesia equina (B.eq.), Babesia microti (B.micfti), Babesia dunnii (B.dunniani), Balamhia malabarilla (Balamutia mangrillii), Bahasa coli (Balanidium coli), Blastomyces granulosus (Blastocystis spp.), Cryptosporidium (Cryptosporidium spp.), Toxosporidium sp., Sporospora (Cryptosporidium parvum), Trichloropsidium sp), Trichloropsis (Plasmodium falciparum, Sporida, Plasmodium falciparum, etc.), Plasmodium falciparum, Plasmodium falciparum, and the like, Plasmodium vivax (Plasmodium vivax), Plasmodium ovale classical subspecies (Plasmodium ovale cutis), Plasmodium ovale variant subspecies (Plasmodium ovale walikeri), Plasmodium malariae (Plasmodium malariae), Plasmodium knowlesi (Plasmodium knowlesi), nosema sp (Rhinosporidium seeber), bovine-human Sarcocystis (Sarcocystis boviminis), porcine-human Sarcocystis (Sarcocystis suis), Toxoplasma gondii (Toxoplasma gondii), Trichomonas vaginalis (Trichomonas vaginalis), Trypanosoma brucei (Trypanosoma brucei) or Trypanosoma cruzi (Trypanosoma cruzi); certain helminthic organisms such as short-tipped tapeworm (berthiella multicontata), slabby tapeworm (berthiella studeri), tapeworm (Cestoda), Taenia multiceps (Taenia multiceps), tricholobus latifolium (diphylodotrichum latum), Echinococcus granulosus (Echinococcus grandis), Echinococcus polyacticus (Echinococcus multilocularis), Echinococcus volvulus (e.gevolli), Echinococcus oligomicus (e.oligarthrius), Echinococcus microcarpus (hymolepis nana), hymenotheca minitans (hymolepis diminus), Echinococcus ohormis (spirometaria), Echinococcus bovis tapeworm (spirometricius), Taenia tenuis (spirometaria), Taenia tenuis (Taenia tenuis), or Taenia solium (taenii); certain trematode organisms, such as Clonorchis sinensis (Clonorchis sinensis); the mammalian species may be selected from the group consisting of Clostridia mustards (Clorocystis virverini), Dipterocarpa lanceolatum (Dicrooelium dendriticum), Echinostomus spinosus (Echinostoma echinatum), Fasciola hepatica (Fasciola hepatica), Fasciola giganteum (Fasciola gigantis), Kaempferia bracteata (Fasciola bunki), Microfluus spinifera (Gthostoma spinosus), Microfluus rigidus (Gnathus spiniferum), Microfluus transversus (Metagonis yogawarsii), Microchoides (Methochis conjuncus), Microfluus catarrhalis (Opisthia virveris), Microschistosoma felis feldiana (Opistus schucheri), Schistolonicera sinensis (Clonospora Schistosoma fasciata), Paragonia paragonicus (Paragonicus), Paragonia paragonia paragonicus, Paragonia paragonia, Paragonia paragonia (Paragonia), Paragonia paragonia, Paragonia (Paragonia) and Paragonia paragonia, Paragonia (Paragonia) and Paragonia, Paragonia (Paragonia) A, Paragonia, and Paragonia, and Paragonia (Paragonia, or Paragonia (Paragonia, and Paragonia, or Paragonia (Paragonia, or Paragonia (Paragonia, or Paragoni, Mei male schistosome (Schistosoma mekongi), Schistosoma sp, Trichiza longifolia or Schistosoma (Schistosoma sp.); certain nematode organisms, for example, Ancylostoma duodenale (Ancylostoma duodenale), Arctostoma americanum (Necator americanus), Costa punctata (Angostridianus costalis), Heterophyllus anisum (Anisakis), Ancylostoma Ascaris (Ascaris sp.), ascaridoides (Ascaris lumbricoides), Cocaris fuliginosus (Baylis procyconis), Trichosporoides (Brugia malayanus), Trichosporoides (Brugia malayanyi), Trichosporoides (Brugia timer), Trichostrongylus rensis (Dicotyphyceae renalis), Trichostrongylus madillis (Drynaudis medius), Trichostrongylus fasciatus (Drynaudis medius), Trichostrongylus vermicularis (Enteroides), Trichostrongylus griseus manorigi (Enteri), Trichostrongylus destructor (Kluyveris), Trichostrongylus fascicularis (Kluyveria), Trichostrongylus striatum, Trichostrongylus fascicularis (Toxoides), Trichostrongylus fascicularia (Toxoides), Trichostrongylus caris (Toxoides), Trichostrongylus fascicularia), Trichostrongylus caris (Toxoides), Trichostrongylus caris (Toxoides), Trichostrongylus fascicularia), Trichostrongylus caris (Toxoides), Trichostrongylus fascicularia), Trichostrongylus carina), Trichostrongylus caris (Toxoides), Trichostrongylus carina), Trichostrongylus caris (Toxoides), Trichostrongylus caris (Toxoides), Trichostrongylus caris), Trichostrongylus kayas (Toxoides), Trichostrongylus caris), Trichostrongylus carina), Trichostrongylus kayas (Toxoides), Trichostrongylus kayas (Toxoides), Trichostrongylus kayas), Strongia roseus), Trichostrongylus carina), Trichostrongylus kayas), Trichostrongylus carina), and Sarcophi (Toxoides), Trichostrongylus carina), Trichostrongylus kayas), Trichostrongylus carina), Strongia lactis), and Sarcophi (Toxoides), Strongia lactis), Strongia roseus), Strongia lactis, Trichinella spiralis, Trichinella britannii, Trichinella briti, Trichinella neliono, Trichinella nelsonii, Trichinella nativa, Trichinella, Trichinella (Trichuris vulpis), Trichinella (Trichuris vulpis) or Wuchereria bambusi (Wuchereria bancrofti); other parasites, such as Echinococcus protothecoides (Archiaceae), Echinococcus moniliforme (Moniliformis moniliformes), Oesophaga serrata (Linguatula sericata), Musca racemosa (Oestroidea), Calliphoridae (Calliphoridae), Sarcophagae (Sarcophagidae), Cochlomyia spiralis (Cochliomyia hominivorax; Calliporidae), Tocophaga penetrans (Tunga pendans), and Sciaconidae (Cimicidae): warm-blooded bed bugs (Cimex lectularius) or human skin flies (Dermatobia hominis). Further examples of parasites include ectoparasites, such as human lice (Pediculus humanus), body lice (Pediculus humanus coproris), pubic lice (Pthirus pubis), follicular Demodex (Demodex folliculorum), sebaceous Demodex brevices, canine Demodex (Demodex cantis), Sarcoptes (Sarcoptes scabies), or Arachnida such as Trombiculidae (Trombiculidae), or fleas (Pulex irliteans), or Arachnida such as Hydraceae (Ixodidae) and/or Cryptoridae (Argasidaceae).

Overview of exemplary microRNA targets

Some embodiments of the methods, systems, and compositions provided herein include detection of certain microrna (mirna) targets. mirnas include small, non-coding RNA molecules that play a role in RNA silencing or post-transcriptional regulation of gene expression. Some mirnas are associated with dysregulation in various human diseases caused by abnormal epigenetic patterns, including abnormal DNA methylation and histone modification patterns. For example, the presence or absence of certain mirnas in a sample from a subject is indicative of a disease or disease state. Primers for detecting mirnas and for isothermal amplification are easily designed from the nucleotide sequences of mirnas. The nucleotide sequences of mirnas are readily available from public databases. Examples of miRNA targets detected using the methods, systems, and compositions provided herein include: hsa-miR-1, hsa-miR-1-2, hsa-miR-100, hsa-miR-100-1, hsa-miR-100-2, hsa-miR-101, hsa-miR-101-1, hsa-miR-101a, hsa-miR-101b-2, hsa-miR-102, hsa-miR-103, hsa-miR-103-1, hsa-miR-103-2, hsa-miR-104, hsa-miR-105, hsa-miR-106a, bsa-miR-106a-1, hsa-miR-106b, hsa-miR-106b-1, hsa-miR-107, hsa-miR-10a, hsa-miR-10b, hsa-miR-122, hsa-miR-122a, hsa-miR-123, hsa-miR-124a, hsa-miR-124a-1, hsa-miR-124a-2, hsa-miR-124a-3, hsa-miR-125a, hsa-miR-125a-5p, hsa-miR-125b, hsa-miR-125b-1, hsa-miR-125b-2, hsa-miR-126, hsa-miR-126-5p, hsa-miR-127, hsa-miR-128a, hsa-miR-128b, hsa-miR-129, hsa-miR-129-1, hsa-miR-129-2, hsa-miR-130, hsa-miR-130a, hsa-miR-130a-1, hsa-miR-130b, hsa-miR-130b-1, hsa-miR-132, hsa-miR-133a, hsa-miR-133b, hsa-miR-134, hsa-miR-135a, hsa-miR-135b, hsa-miR-136, hsa-miR-137, hsa-miR-138, hsa-miR-138-1, hsa-miR-138-2, hsa-miR-139, hsa-miR-139-5p, hsa-miR-140, miR-140-3p, hsa-miR-141, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-143, hsa-miR-144, hsa-miR-145, hsa-miR-146a, hsa-miR-146b, hsa-miR-147, hsa-miR-148a, hsa-miR-148b, hsa-miR-149, hsa-miR-15, hsa-miR-150, hsa-miR-151, hsa-miR-151-5p, hsa-miR-152, hsa-miR-153, hsa-miR-154, hsa-miR-155, hsa-miR-15a, hsa-miR-15a-2, hsa-miR-15b, hsa-miR-16, hsa-miR-16-1, hsa-miR-16-2, hsa-miR-16a, hsa-miR-164, hsa-miR-170, hsa-miR-172a-2, hsa-miR-17, hsa-miR-17-3p, hsa-miR-l7-5p, hsa-miR-l7-92, hsa-miR-18, hsa-miR-18a, hsa-miR-18b, hsa-miR-181a, hsa-miR-181a-1, hsa-miR-181a-2, hsa-miR-18lb, hsa-miR-181b-1, hsa-miR-181b-2, hsa-miR-181c, hsa-miR-181d, hsa-miR-182, hsa-miR-183, hsa-miR-184, hsa-miR-185, hsa-miR-186, hsa-miR-187, hsa-miR-188, hsa-miR-189, hsa-miR-190, hsa-miR-191, hsa-miR-192, hsa-miR-192-1, hsa-miR-192-2, hsa-miR-192-3, hsa-miR-193a, hsa-miR-193b, hsa-miR-194, hsa-miR-195, hsa-miR-196a, hsa-miR-196a-2, hsa-miR-196b, hsa-miR-197, hsa-miR-198, hsa-miR-199a, hsa-199 a-1, hsa-miR-199a-1-5p, hsa-miR-199a-2, hsa-miR-199a-2-5p, hsa-miR-199a-3p, hsa-miR-199b, hsa-miR-199b-5p, hsa-miR-19a, hsa-miR-19b, hsa-miR-19b-1, hsa-miR-19b-2, hsa-miR-200a, hsa-miR-200b, hsa-miR-200c, hsa-miR-202, hsa-miR-203, hsa-miR-204, hsa-miR-205, hsa-miR-206, hsa-miR-207, hsa-miR-208, hsa-miR-208a, hsa-miR-20a, hsa-miR-20b, hsa-miR-21, hsa-miR-22, hsa-miR-210, hsa-miR-211, hsa-miR-212, hsa-miR-213, hsa-miR-214, hsa-miR-215, hsa-miR-216, hsa-miR-217, hsa-miR-218, hsa-miR-218-2, hsa-miR-219, hsa-miR-219-1, hsa-miR-22, hsa-miR-220, hsa-miR-221, hsa-miR-222, hsa-miR-223, hsa-miR-224, hsa-miR-23a, hsa-miR-23b, hsa-miR-24, hsa-miR-24-1, hsa-miR-24-2, hsa-miR-25, hsa-miR-26a, hsa-miR-26a-1, hsa-miR-26a-2, hsa-miR-26b, hsa-miR-27a, hsa-miR-27b, hsa-miR-28, hsa-miR-296, hsa-miR-298, hsa-miR-299-3p, hsa-miR-299-5p, hsa-miR-29a, hsa-miR-29a-2, hsa-miR-29b, hsa-miR-29b-l, hsa-miR-29b-2, hsa-miR-29c, hsa-miR-301, hsa-miR-302, hsa-miR-302a, hsa-miR-302b, hsa-miR-302c, hsa-miR-302c, hsa-miR-302d, hsa-miR-30a, hsa-miR-30a-3p, hsa-miR-30a-5p, hsa-miR-30b, hsa-miR-30c, hsa-miR-30c-1, hsa-miR-30d, hsa-miR-30e, hsa-miR-30e-5p, hsa-miR-31, hsa-miR-31a, hsa-miR-32, hsa-miR-32, hsa-miR-320, hsa-miR-320-2, hsa-miR-320a, hsa-miR-322 and hsa-miR-323, hsa-miR-324-3p, hsa-miR-324-5p, hsa-miR-325, hsa-miR-326, hsa-miR-328, hsa-miR-328-1, hsa-miR-33, hsa-miR-330, hsa-miR-331, hsa-miR-335, hsa-miR-337, hsa-miR-337-3p, hsa-miR-338, hsa-miR-338-5p, hsa-miR-339, hsa-miR-339-5p, hsa-miR-34a, hsa-miR-340, hsa-miR-340, hsa-miR-341, hsa-miR-342, hsa-miR-342-3p, hsa-miR-345, hsa-miR-346, hsa-miR-347, hsa-miR-34a, hsa-miR-34b, hsa-miR-34c, hsa-miR-351, hsa-miR-352, hsa-miR-361, hsa-miR-362, hsa-miR-363, hsa-miR-355, hsa-miR-365, hsa-miR-367, hsa-miR-368, hsa-miR-369-5p, hsa-miR-370, hsa-miR-371, hsa-miR-372, hsa-miR-373, hsa-miR-374, hsa-miR-375, hsa-miR-376a, hsa-miR-376b, hsa-miR-377, hsa-miR-378, hsa-miR-378, hsa-miR-379, hsa-miR-381, hsa-miR-382, hsa-miR-383, hsa-miR-409-3p, hsa-miR-419, hsa-miR-422a, hsa-miR-422b, hsa-miR-423, hsa-miR-424, hsa-miR-429, hsa-miR-431, hsa-miR-432, hsa-miR-433, hsa-miR-449a, hsa-miR-451, 484-miR-452, hsa-miR-451, hsa-miR-452, hsa-miR-452, hsa-miR-483, hsa-miR-483-3p, hsa-miR-484, hsa-miR-485-5p, hsa-miR-485-3p, hsa-miR-486, hsa-miR-487b, hsa-miR-489, hsa-miR-491, hsa-miR-491-5p, hsa-miR-492, hsa-miR-493-3p, hsa-miR-493-5p, hsa-miR-494, hsa-miR-495, hsa-miR-497, hsa-miR-498, hsa-miR-499, hsa-miR-5, hsa-miR-500, hsa-miR-501, hsa-miR-503, hsa-miR-508, hsa-miR-509, hsa-miR-510, hsa-miR-511, hsa-512-5 p, hsa-miR-513, hsa-miR-513-1, hsa-miR-513-2, hsa-miR-515-3p, hsa-miR-516-5p, hsa-miR-516-3p, hsa-miR-518b, hsa-miR-519a, hsa-miR-519d, hsa-miR-520a, hsa-miR-520c, hsa-miR-521, hsa-miR-532-5p, hsa-miR-539, hsa-miR-542-3p, hsa-miR-542-5p, hsa-miR-550, hsa-miR-551a, hsa-miR-561, hsa-5637, hsa-miR-565, hsa-miR-572, hsa-miR-582, hsa-miR-584, hsa-miR-594, hsa-miR-595, hsa-miR-598, hsa-miR-599, hsa-miR-600, hsa-miR-601, hsa-miR-602, hsa-miR-605, hsa-miR-608, hsa-miR-611, hsa-miR-612, hsa-miR-614, hsa-miR-615, hsa-miR-615-3p, hsa-miR-622, hsa-miR-627, hsa-miR-628, hsa-miR-635, hsa-miR-637, hsa-miR-638, hsa-miR-642, hsa-miR-648, hsa-miR-652, hsa-miR-657, hsa-miR-658, hsa-miR-659, hsa-miR-661, hsa-miR-662, hsa-miR-663, hsa-miR-664, hsa-miR-7, hsa-miR-7-1, hsa-miR-7-2, hsa-miR-7-3, hsa-miR-708, hsa-miR-765, hsa-miR-769-3p, hsa-miR-802, hsa-miR-885-3p, hsa-miR-9, hsa-miR-9-1, hsa-miR-9-3, hsa-miR-9-3p, hsa-miR-92, hsa-miR-92-1, hsa-miR-92-2, hsa-miR-9-2, hsa-miR-92, hsa-miR-92a, hsa-miR-93, hsa-miR-95, hsa-miR-96, hsa-miR-98, hsa-miR-99a, and/or hsa-miR-99 b.

Overview of exemplary agricultural analytes

Some embodiments of the methods, systems, and compositions provided herein include the detection of certain agricultural analytes. Agricultural analytes include nucleic acids, proteins, or small molecules. Nucleotide sequences indicative of certain agricultural analytes are readily available from public databases. Primers for isothermal amplification are readily designed from the nucleic acid sequences of such agricultural analytes. Antibodies and aptamers to proteins of certain agricultural analytes are readily obtained commercially and/or by techniques well known in the art.

Some embodiments of the methods and apparatus provided herein are used to identify organisms or products of organisms in meat products, fish products, or yeast products (such as beer, wine, or bread). In some embodiments, species-specific antibodies or aptamers, or species-specific primers are used to identify the presence of certain organisms in a food product.

Some embodiments of the methods, systems, and compositions provided herein include detection of a pesticide. In some embodiments, the pesticide is detected in a sample (e.g., a soil sample or a food sample). Examples of pesticides that can be detected using the apparatus and methods described herein include herbicides, insecticides, or fungicides. Examples of herbicides include 2, 4-dichlorophenoxyacetic acid (2,4-D), atrazine, glyphosate, 2-methyl-4-chloropropionic acid, dicamba, paraquat, glufosinate, metam, dazomet, dithiopyr, pendimethalin, EPTC, trifluralin, flazasulfuron, metsulfuron-methyl, diuron, aclonifen, trifluoromethoxyfen, acifluorfen, mesotrione, sulcotrione or nitisinone. Examples of pesticides tested using the apparatus and methods described herein include organic chlorides, organophosphates, carbamates, pyrethroids, neonicotinoids and ryanoids. Examples of fungicides that can be detected using the apparatus and methods described herein include carbendazim, diethofencarb, azoxystrobin, metalaxyl-M, streptomycin, oxytetracycline, chlorothalonil, tebuconazole, zineb, mancozeb, tebuconazole, myclobutanil, triadimefon, fenbuconazole, deoxynivalenol or mancozeb.

Overview of exemplary biomarkers

Some embodiments of the methods, systems, and compositions provided herein include the detection of certain biomarkers of certain disorders. Biomarkers can include nucleic acids, proteins, protein fragments, and antigens. Some biomarkers may include a target provided herein. Exemplary disorders include cancers such as breast cancer, colorectal cancer, gastric cancer, gastrointestinal stromal tumors, leukemias and lymphomas, lung cancer, melanoma, brain cancer, and pancreatic cancer. Some embodiments may include detecting the presence or absence of a biomarker, or the level of a biomarker, in a sample. Biomarkers can indicate the presence, absence, or stage of certain disorders. Exemplary biomarkers include estrogen receptor, progesterone receptor, HER-2/neu, EGFR, KRAS, UGT1A1, c-KIT, CD20, CD30, FIP1L1-PDGFR α, PDGFR, Philadelphia chromosome (BCR/ABL), PML/RAR- α, TPMT, UGT1A1, EML4/ALK, BRAF, and elevated levels of certain amino acids (e.g., leucine, isoleucine, and valine).

Overview of exemplary methods for amplifying and detecting target nucleic acids

Some embodiments include a method 4100 of amplifying and detecting a target nucleic acid. An example of method 4100 is depicted in FIG. 41. In some embodiments, method 4100 comprises providing, preferably in a single container, a Recombinase Polymerase Amplification (RPA) reagent solution comprising a target nucleic acid, primers each complementary to a region of the target nucleic acid, a buffer, dntps, a recombinase, a single-stranded DNA binding protein (SSB), and a strand-displacing DNA polymerase 4110; combining, preferably in the single container, the RPA reagent solution with a second reagent solution configured for a second isothermal amplification reaction without the use of a recombinase to produce an amplification reaction solution 4120; performing RPA and optionally the second isothermal amplification in the amplification reaction solution, preferably in the single vessel, to produce amplified target nucleic acid 4130; and detecting the presence of amplified target nucleic acid by measuring modulation of an electrical signal (e.g., impedance) in the amplification reaction solution when the amplification reaction is subjected to an electric field, as compared to a control 4140. Some embodiments include performing helical RPA using, for example, paired primers, wherein the forward and reverse primer sequences are reverse complementary to each other at their 5 'ends and their 3' end sequences are complementary to the target sequence.

Some embodiments of method 4100 include providing RPA reagent solution 4110. In some embodiments, the RPA reagent solution comprises reagents compatible with the RPA described herein. In some embodiments, the RPA solution comprises a target nucleic acid, primers each complementary to a region of the target nucleic acid, a buffer, dntps, a recombinase, an SSB, and/or a strand-displacing DNA polymerase. In some embodiments, providing the RPA reagent solution comprises providing the RPA reagent in a single container. In some embodiments, the RPA agent is provided in more than one container.

Some embodiments of method 4100 include combining an RPA reagent solution with a second reagent solution configured for a second isothermal amplification reaction without the use of a recombinase to produce amplification reaction solution 4120. In some embodiments, the RPA reagent solution is combined with the second reagent solution in the single container.

Some embodiments of method 4100 include performing RPA 4130 in the amplification reaction solution. Examples of RPA reaction solutions are described herein. Some embodiments include performing the second isothermal amplification. In some embodiments, RPA and the second isothermal amplification are performed in the single vessel. In some embodiments, performing RPA and optionally a second isothermal amplification produces an amplified target nucleic acid.

Some embodiments of method 4100 include the use of the apparatus described herein. For example, the devices described herein can be used to detect amplified nucleic acid targets. Some embodiments of method 4100 include detecting the presence of amplified target nucleic acids in an amplification reaction solution by measuring modulation of an electrical signal, e.g., when subjecting the amplification reaction to an electric field 4140. In some embodiments, the electrical signal comprises an impedance. In some embodiments, the electrical signal is compared to a control.

In some embodiments of method 4100, the primers and/or amplification reaction solution do not comprise a label or dye. In some embodiments, amplification and detection are performed in the absence of detection reagents (e.g., dyes, clouding agents, fluorophores, double-stranded nucleic acid intercalators, sequencing indexes, and/or nanoparticles).

Some embodiments include a method 4200 of amplifying and detecting a target nucleic acid. An example of method 4200 is depicted in FIG. 42. In some embodiments, method 4200 includes providing, preferably in a single container, a Recombinase Polymerase Amplification (RPA) reagent solution comprising a target nucleic acid, primers each complementary to a region of the target nucleic acid, a buffer, dntps, a recombinase, a single-stranded DNA binding protein (SSB), and a strand-displacing DNA polymerase 4210; performing RPA of the RPA reagent solution to produce an amplified target nucleic acid 4220; combining, preferably in the single vessel, the amplified target nucleic acid with a second reagent solution configured for a second isothermal amplification reaction without the use of a recombinase to produce a second amplification reaction solution 4230; performing a second isothermal amplification of the second amplification reaction solution to produce further amplified target nucleic acid 4240; the presence of amplified target nucleic acid is detected 4250 by measuring the modulation of an electrical signal (e.g., impedance) in the amplification reaction solution when the amplification reaction is subjected to an electric field, as compared to a control.

Some embodiments of method 4200 include providing Recombinase Polymerase Amplification (RPA) reagent solution 4210. In some embodiments, the RPA reagent solution comprises reagents compatible with the RPA described herein. In some embodiments, the reagent solution comprises a target nucleic acid, primers each complementary to a region of the target nucleic acid, a buffer, dntps, a recombinase, a single-stranded DNA binding protein (SSB), and/or a strand-displacing DNA polymerase. In some embodiments, the reagents are in a single container.

Some embodiments include performing RPA of the RPA reagent solution to produce amplified target nucleic acid 4220. Examples of RPAs are described herein.

Some embodiments include combining, preferably in the single vessel, the amplified target nucleic acid with a second reagent solution configured for a second isothermal amplification reaction without the use of a recombinase to produce a second amplification reaction solution 4230. Examples of isothermal amplification reactions without the use of a recombinase are provided herein.

Some embodiments include performing a second isothermal amplification of the second amplification reaction solution to produce further amplified target nucleic acid 4240.

Some embodiments of method 4200 include the use of an apparatus as described herein. For example, the devices described herein can be used to detect amplified nucleic acid targets. Some embodiments of method 4200 include detecting the presence 4250 of amplified target nucleic acid by measuring modulation of an electrical signal in an amplification reaction solution, e.g., when subjecting the amplification reaction to an electric field. In some embodiments, the electrical signal comprises an impedance. In some embodiments, the electrical signal is compared to a control.

In some embodiments of method 4200, the primers and/or amplification reaction solution do not comprise a label or dye. In some embodiments, amplification and detection are performed in the absence of detection reagents (e.g., dyes, clouding agents, fluorophores, double-stranded nucleic acid intercalators, sequencing indexes, and/or nanoparticles).

Some embodiments include a method 4300 of amplifying and detecting a target nucleic acid. An example of a method 4300 is depicted in FIG. 43. In some embodiments, the method 4300 comprises subjecting the target nucleic acid to Recombinase Polymerase Amplification (RPA) and loop-mediated isothermal amplification (LAMP) in a single vessel to produce amplified target nucleic acid, preferably without isolating or purifying the amplified target nucleic acid between the RPA amplification and the LAMP amplification, e.g., 4310 in a single vessel; and detecting the presence of amplified target nucleic acid by measuring a modulation of an electrical signal (e.g., impedance) in the amplification reaction solution when the amplification reaction is subjected to the electric field, as compared to a control 4320.

Some embodiments of the method 4300 include performing Recombinase Polymerase Amplification (RPA) and loop-mediated isothermal amplification (LAMP) on a target nucleic acid in a single vessel to produce an amplified target nucleic acid, preferably without isolating or purifying the amplified target nucleic acid between the RPA amplification and the LAMP amplification, e.g., 4310 in a single vessel. Some embodiments include detecting the presence of amplified target nucleic acid by measuring a modulation of an electrical signal (e.g., impedance) in the amplification reaction solution when subjecting the amplification reaction to an electric field, as compared to a control 4320.

In some embodiments of method 4300, the primers in the RPA and/or LAMP amplification do not comprise a label or dye. In some embodiments, RPA and/or LAMP amplification and detection are performed in the absence of detection reagents (e.g., dyes, clouding agents, fluorophores, double-stranded nucleic acid intercalators, sequencing indexes, and/or nanoparticles).

In some embodiments of the methods described herein, the detection of amplified target nucleic acids is performed in a device comprising a test well. In some embodiments, the test well includes an excitation electrode and a sensor electrode. In some embodiments, the detecting further comprises applying an excitation signal from the reader device to the excitation electrode. In some embodiments, the detecting further comprises sensing a signal from the test well using the excitation electrode. In some embodiments, the signal is representative of the impedance of the amplification reaction solution. In some embodiments, the detecting further comprises transmitting the signal to a reader device, wherein the reader device analyzes the signal. In some embodiments, the detection of the amplified target nucleic acid is performed in a device comprising a test well comprising an excitation electrode and a sensor electrode, and wherein the detecting further comprises: applying an excitation signal from a reader device to an excitation electrode; sensing a signal from the test well using the excitation electrode, wherein the signal is representative of an impedance of the amplification reaction solution; and transmitting the signal to a reader device, wherein the reader device analyzes the signal.

In some embodiments of the methods described herein, the amplifying and detecting steps are performed in the presence of about 0.25mM or less of Dithiothreitol (DTT), or in the absence of DTT.

In some embodiments of the methods described herein, the RPA comprises a leading strand RPA (lsrpa), a synchronous leading and trailing strand synthesis, or a nested RPA. In some embodiments, the second isothermal amplification comprises self-sustained sequence replication reaction (3SR), 90-I, BAD Amp, cross-primer amplification (CPA), isothermal index amplification reaction (EXPAR), isothermal chimeric primer-primed nucleic acid amplification (ICAN), Isothermal Multiple Displacement Amplification (IMDA), ligation-mediated SDA, multiple displacement amplification, polymerase helix reaction (PSR), restriction cascade index amplification (RCEA), smart amplification program (SMAP2), Single Primer Isothermal Amplification (SPIA), transcription-based amplification system (TAS), transcription-mediated amplification (TMA), Ligase Chain Reaction (LCR), and/or multiple cross-displacement amplification (MCDA), rolling circle Replication (RCA), nickase amplification reaction (NEAR), or nucleic acid sequence-based amplification (NASBA).

In some embodiments of the methods described herein, the second isothermal amplification comprises loop-mediated isothermal amplification (LAMP). In some embodiments, the amplification reaction solution comprises LAMP-compatible primer oligonucleotides. In some embodiments, the amplification reaction solution comprises a LAMP-compatible FIP, BIP, LF, or LB primer oligonucleotide, and a RPA-compatible primer. In some embodiments, the amplification reaction solution comprises a LAMP-compatible FIP, BIP, LF, LB, F3, or B3 primer oligonucleotide.

In some embodiments of the methods described herein, the RPA and the second isothermal amplification are performed at the same or substantially the same temperature. In some embodiments, the RPA or second isothermal amplification is performed at 25 ℃, 30 ℃, 37 ℃, 40 ℃, 45 ℃, 50 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, or at a temperature within a range defined by any two of the aforementioned temperatures. In some embodiments, the RPA is performed at a lower or higher temperature than the second isothermal amplification. In some embodiments, the RPA is performed at 37 ℃, about 37 ℃, 40 ℃, or about 40 ℃, and the second isothermal amplification is performed at 60 ℃, about 60 ℃, 65 ℃, or about 65 ℃.

In some embodiments of the methods described herein, providing the RPA reagent solution, combining the RPA reagent solution with a second reagent solution, and/or performing RPA in the amplification solution is performed at 5224 and/or 5228 of fig. 52D or 5718 and/or 5740 of any of fig. 57A-58D. In some embodiments, performing RPA in an RPA reagent solution to produce amplified target nucleic acids is performed at 5224 and/or 5228 of fig. 52D or at 5718 and/or 5740 of any of fig. 57A-58D, combining the amplified target nucleic acids with a second reagent solution configured for a second isothermal amplification reaction, and/or performing a second isothermal amplification of the second amplification reaction solution to produce further amplified target nucleic acids. In some embodiments, RPA and LAMP are performed on target nucleic acids in a single vessel to produce amplified target nucleic acids at 5224 and/or 5228 of fig. 52D or 5718 and/or 5740 of any of fig. 57A-58D.

Examples

Example 1 detection of fC4D before/after LAMP amplification in PDMS

The LAMP reaction mixture was prepared according to the standard protocol of NEB, using the 5' untranslated region of the genome of Haemophilus influenzae as a target. The mixture was aliquoted into pre-amplification vials (-control) and post-amplification vials (+ control). The pre-amplification vials were heat inactivated at 85 ℃ for 20 minutes to prevent amplification. The post amplification vials were amplified at 63 ℃ for 60 minutes. Aliquots from each vial were loaded sequentially (alternating between two vials at room temperature) onto PDMS/glass chip v.1.1 while real-time data collection was performed. FIG. 24 is a graph depicting sensor voltage over time.

Example 2 Pre/post amplification detection of fC4D in PDMS with whole blood

Reaction mixtures were prepared with 0%, 1% and 5% whole blood (v/v) using the 5' untranslated region of the genome of Haemophilus influenzae as a target. The mixture was aliquoted into pre-amplification vials (-control) and post-amplification vials (+ control). The pre-amplification vials were heat inactivated at 85 ℃ for 20 minutes to prevent amplification. The post amplification vials were amplified at 63 ℃ for 60 minutes. Aliquots from each vial were loaded sequentially (alternating between two vials at room temperature) onto PDMS/glass chip v.1.1 while real-time data collection was performed. Fig. 25, 26, and 27 are graphs depicting pre-amplification (-control) and post-amplification (+ control) sensor voltage versus time for 0%, 1%, and 5% whole blood, respectively.

Example 3 filtration of LAMP Pre/post amplification

Samples were prepared as described in example 1. Prior to measurement, all samples (one removed as a control) were spin filtered using a 50kD filter. Aliquots from each vial were loaded sequentially (alternating between two vials at room temperature) onto PDMS/glass chip v.1.1 while real-time data collection was performed. Filtration improves the S/N and conductivity change. FIGS. 28 and 29 are graphs depicting sensor voltage before amplification (-control) and after amplification (+ control) with 0% whole blood versus time for unfiltered and filtered samples, respectively.

Example 41 conductivity detection of copy of k-1M target

The reaction mixture was prepared using the 5' untranslated region of the haemophilus influenzae genome as a target. Using fC⁴And D, detecting by using an instrument. Data were repeatedly averaged for 3. Figure 30 depicts a graph of time as a function of target loading with error bars showing standard deviation. The no template negative control showed no signal at 60 minutes heating.

Example 5 pre/post amplification detection of fC4D in PDMS with whole blood

The reaction mixture was prepared with 0% or 1% whole blood (v/v) using the 5' untranslated region of the genome of Haemophilus influenzae as a target. The mixture was aliquoted into pre-amplification vials (-control) and post-amplification vials (+ control). The pre-amplification vials were heat inactivated at 85 ℃ for 20 minutes to prevent amplification. The post amplification vials were amplified at 63 ℃ for 60 minutes. Aliquots from each vial were loaded sequentially (alternating between two vials at room temperature) onto PDMS/glass chip v.1.1 while real-time data collection was performed. FIG. 31 depicts a plot of the conductivity of various samples from pre-amplification vial (-control) and post-amplification vial (+ control).

Example 6 detection of hepatitis B surface antigen Using MAIA

Biotinylated polyclonal antibody capture probes (anti-HBsAg) were conjugated to streptavidin-functionalized 1 micron magnetic microspheres (Dynal T1). Chimeric detection complexes were synthesized by conjugating biotinylated polyclonal capture probes (anti-HBsAg) to streptavidin and streptavidin-antibody complexes to biotinylated DNA targets. Antibody-functionalized beads capture HBs antigen from solution. HBs antigen is detected by binding of the chimera Ab-DNA complex and subsequent amplification of the DNA template portion of the chimera complex. Figure 32 depicts binding between an antibody conjugated to a nucleic acid and an antigen. FIG. 33 depicts a graph showing detection of hepatitis B surface antigen.

Example 7 detection with Low Ionic Strength buffer

Commercial amplification solutions and T10 amplification solutions were prepared using the reagents listed in table 2 and table 3, respectively. Commercial amplification solutions are commonly used in general amplification reactions. The T10 amplification solution had reduced Tris-HCl content and was absent ammonium sulfate. 400 μ L of each solution was prepared and about 15 μ L of each solution was loaded into a different channel of the cartridge. The solution was heated to 63.0 ℃. Data was collected using a data collection plate.

Fig. 34 depicts the results. The T10 amplification buffer provided at least a 30% higher signal than that provided by commercial amplification solutions.

TABLE 2

TABLE 3

Reagent	1 × concentration (mM)	10 Xconcentration (mM)	FW	10mL 10 Xadded mg
					Tris-HCl	2	20	157.6	31.52
KCl	50	500	74.55	372.75
					MgSO₄	2	20	246.48	49.30
Tween 20	0.10％	1％	100％	0.1mL
					DI water				9.9mL

EXAMPLE 8 impedance characteristics of fluid cells

The channels of the fluidic cartridge depicted in fig. 17A were filled with 1288mS/cm reference buffer and the excitation frequency was swept from less than about 100Hz to greater than about 1MHz and the impedance ("| Z |") or arg Z was measured as a function of frequency. The results are shown in fig. 35, which depicts the variation of | Z | or arg Z with frequency.

Example 9 amplification of nucleic acids containing HCV sequences

Samples containing nucleic acids comprising Hepatitis C Virus (HCV) sequences were amplified in a series of experiments by LAMP under various conditions, and the threshold cycle (C) was determined_t) Values are along with Standard Deviation (SD) and Relative Standard Deviation (RSD)%. The nucleic acid includes: a synthetic nucleic acid comprising an HCV sequence; synthetic RNA comprising HCV sequences. All reactions contained 5% tween-20. For an experiment containing about one million copies of a synthetic nucleic acid comprising an HCV sequence, the average Ct is 856s, SD is 15s, and RSD is 1.72%.

Amplification of plasma samples containing synthetic RNA comprising HCV sequences by LAMP under various conditions including: untreated, treated by heating before addition of synthetic RNA, treated by heating after addition of synthetic RNA, and added 100mM DTT. Each reaction contains about 25k copies of the nucleic acid. Table 4 summarizes the results.

TABLE 4

The addition of 100mM DTT or heating the treated plasma prior to the addition of synthetic RNA improves amplification as shown by Relative Standard Deviation (RSD) compared to untreated samples. Addition of DTT or heating of the treated plasma prior to addition of synthetic RNA also resulted in faster amplification (approximately 50s faster) compared to untreated samples (P ═ 0.03 and P ═ 0.002, respectively).

HCV-containing plasma samples (SeraCare, Milford MA) were amplified by LAMP under various conditions including: the plasma was heat treated, 100mM DTT added, SDS and/or DTT added. Table 5 summarizes the results.

TABLE 5

As shown by RSD values, heating the plasma or adding DTT improved the amplification results compared to untreated plasma. The addition of 0.05% SDS or 0.1% SDS decreased the reproducibility and speed of amplification compared to untreated, heat treated, or DTT added plasma.

Example 10 amplification of clinical samples containing HCV

Clinical plasma samples containing HCV were amplified by LAMP with different concentrations of DTT. A WarmStart LAMP reaction premix (New England Biolabs) was used to prepare four samples. The samples included: 5% plasma containing about-20 k copies of HCV/reactive (SeraCare, Milford MA), 50U/reactive murine RNase inhibitor, with varying concentrations of Tween and DTT. Samples containing synthetic nucleic acids comprising HCV sequences (1M copies/rxn) were tested with 1% and 5% tween 20. A No Target Control (NTC) was also tested. LAMP was performed on an Applied Biosystems QuantStaudio 3(QS3) system at 67 ℃ for 60 min. Fluorescence was measured once per minute to assess reaction progress. The results are summarized in table 6.

TABLE 6

As shown by RSD values, samples containing 5% tween had improved amplification compared to samples containing 1% tween. Similar studies were performed to further alter the tween concentration in the reaction tubes. The results are summarized in table 7.

TABLE 7

Higher concentrations of emetic and DTT reaction volumes have better reproducibility of the amplification results of HCV samples, in particular, fewer extreme outliers, fewer failed amplifications, and lower amplified repeat RSD values in repeated reactions. At 5mM DTT and 10mM DTT, there were no replicates that were not amplified for any concentration of Tween. Similarly, there were no failed replicates or extreme outliers at 4% and 5% spit temperatures, except for low DTT concentrations (1mM and below).

Example 11 amplification of targets with cartridges

A series of three experiments were performed using a cartridge substantially similar to that described in figure 2, with six wells, each with a ring electrode. Each hole is associated with a measured channel. The sample includes a target nucleic acid comprising a sequence from haemophilus influenzae (Hinf) or Hepatitis B Virus (HBV). Samples were amplified by LAMP and changes in impedance were measured.

Wells were prepared by pre-heating the cassette to 72 ℃ for 20 min, filling each well with 25 μ L of "no template and primer control" (NTPC) buffer, covering the buffer with mineral oil, heating the cassette to 72 ℃ for 20 min, removing air bubbles from the wells, and cooling the cassette at room temperature for 10 min. The sample is injected into the bottom of the pre-filled wells and the cassette is placed at 67 ℃ or 76.5 ℃ for LAMP of a particular experiment. The frequency used for the Hinf study was 60 kHz. The samples and corresponding wells/channels for each cartridge are listed in table 8. The target sequences and primers are listed in table 9. The reaction components are listed in table 10.

TABLE 8

Hole/channel	Sample (I)
		1	Synthesis of HBV
2	Synthesis of HBV
		3	Synthesis of HBV
4	NTPC
		5	Hinf
6	Hinf

TABLE 9

Watch 10

Data for LAMP performed on the cassette at 65 ℃ are shown in fig. 36A and 36B. Figure 36A is a graph of the out-of-phase portion of the attenuated excitation signal sensed in the test well of the cartridge of figure 2, where the x-axis is time, the line represents LAMP on a sample of NTPC, and is labeled with Hinf and an example of synthesizing HBV. FIG. 36B is a graph of the in-phase portion of the attenuated excitation signal sensed in the test well of the cartridge of FIG. 2, with lines representing synthetic HBV (channels 1-3), NTPC (channel 4), and Hinf (channels 5-6). The sample containing the synthetic HBV was not amplified on the cassette at 65 ℃. The labeled Hinf samples show exemplary signal cliffs indicative of positive samples.

Data for LAMP performed on the cassette at 67 ℃ are shown in fig. 36C and 36D. Figure 36C is a graph of the out-of-phase portion of the attenuated excitation signal sensed in the test well of the cartridge of figure 2, where the x-axis is time, and the line represents LAMP on a sample of NTPC, and is labeled with Hinf and an example of synthesizing HBV. FIG. 36D is a graph of the in-phase portion of the attenuated excitation signal sensed in the test well of the cartridge of FIG. 2, with lines representing synthetic HBV (channels 1-3), NTPC (channel 4), and Hinf (channels 5-6). Samples containing synthetic HBV were amplified on the cassette at 67 ℃ for approximately 49 minutes. The labeled Hinf samples show exemplary signal cliffs indicative of positive samples.

Data for LAMP performed on the cassette at 67 ℃ are shown in fig. 36E and 36F. Figure 36E is a graph of the out-of-phase portion of the attenuated excitation signal sensed in the test well of the cartridge of figure 2, where the x-axis is time, the line represents LAMP on a sample of NTPC, and is labeled with Hinf and an example of synthesizing HBV. FIG. 36F is a graph of the in-phase portion of the attenuated excitation signal sensed in the test well of the cartridge of FIG. 2, with lines representing synthetic HBV (channels 1-3), NTPC (channel 4), and Hinf (channels 5-6). Samples containing synthetic HBV were amplified on the cassette at 67 ℃ for approximately 46 minutes.

Samples were also tested by LAMP at 67 ℃ using QS 3. Table 11 lists the average Ct values for samples containing Hinf or synthetic HBV. The data in the figure is shown in seconds(s).

TABLE 11

Sample (target concentration)	Average Ct(s)	SD(s)	RSD(％)
				Hinf PC (1M copy)	1704.5	10.4	0.6
HBV Synt (10B copy)	380.4	5.5	1.5

Example 12RPA reaction premix and protocol

The following are examples of base reaction premixes for the RPA reaction: 50mM Tris-acetate, pH 7.8; 5.5% polyethylene glycol-35 k; 5% trehalose; 100mM potassium acetate; 6 μ g T4 UvsX recombinase; 2.5 μ g T4 UvsY; 6 μ g T4 gp32 SSB; 2.5 μ g creatine kinase; 5mM Dithiothreitol (DTT); 50mM creatine phosphate (from disodium salt); 1U polymerase from Staphylococcus aureus (Sau polymerase) or Bacillus subtilis (Bsu polymerase). In some embodiments, the base reaction premix is used in a 25 μ L reaction. Additional disclosure related to RPA and base reaction premixes is provided in U.S. patent No. 9,057,097B 2. The entire contents of U.S. patent No. 9,057,097B2 are incorporated herein by reference. In some embodiments, the amplification reaction solution or RPA reagent solution comprises a base reaction premix or one or more components of a base reaction premix.

The following are examples of the RPA protocol:

(1) all RPA reagents (buffers, enzymes, primers, etc.) except the target DNA/RNA and magnesium acetate (the enzyme used in the activation reaction) were mixed together,

(2) the reaction mixture was pipetted into a separate 25 μ L PCR tube,

(3) pipetting the mixture of target and activator into the cap of each tube,

(4) the lid was closed, inverted several times to mix, and the tube was then centrifuged briefly,

(5) the tube is placed into a thermal cycler,

(6) the RPA reaction was run at 40 ℃ and data was acquired every minute.

Some embodiments of the methods provided herein include performing RPA and/or another isothermal amplification, such as LAMP. According to some embodiments, the basic reaction premix and/or the RPA protocol described in example 12, or certain aspects thereof, is used to perform RPA and/or LAMP or another isothermal amplification.

Example 13 RPA with blocking region

Initial attempts at RPA without optical probes (i.e., only forward and reverse primers + reporter dye embedded in dsDNA) found that the curve from NTC substantially or completely overlapped the curve from the positive sample. This is the case for each primer set and target. This is assumed to be due to the relatively low RPA reaction temperature that makes polymerase extendible interactions between primers at their 3' ends feasible. Therefore, it was tested whether the blocking oligonucleotides could alleviate this problem by putting the 3' ends of the primers in duplex until they completely annealed to their target sequence. 3 repeats of the target (100 million copies/reaction Synt H inf) (SEQ ID NO:08) and 3 repeats of the NTC reaction were prepared using a twist Amp Liquid Basic Master Mix (Basic reaction premix described in example 12) and with the following combinations of blocking region oligonucleotides, at concentrations in parentheses: positive controls (no blocking region), F1_ B10+ R3_ B10 (2400 nM and 4800nM each), F1_ B12+ R3_ B12 (2400 nM and 4800nM each), F1_ B15+ R3_ B15 (2400 nM and 4800nM each), F1_ B10+ R3_ B15 (4800 nM each), F1_ B15+ R3_ B10 (4800 nM each).

The RPA experiment was run on a QuantStaudio 5 thermal cycler (Thermo Fisher Scientific) at 65 ℃ for 60 minutes, and fluorescence was recorded every minute to assess the progress of the reaction. The samples are named in the following format: (P or N _ Length _ F-Length R _ concentration), therefore, a positive with two positives of 10@2400nM would be P _10-10_2400, while an NTC of F1_ B15+ R3_ B10 would be N _15-10_ 4800.

The results are shown in table 12. The oligonucleotide sequences are included in table 13.

TABLE 12

Watch 13

As shown in table 12, the samples with F1_ B15+ R3_ B15 (4800 nM each) worked well; positive and negative samples without blocking regions were separated by approximately 1 minute in Ct, while the P-15-4800 and N-15-4800 samples were separated by an amplification time of approximately 40 minutes. These results indicate that, according to some embodiments, inclusion of a blocking region oligonucleotide in the RPA reaction solution enhances RPA or another amplification reaction by increasing specificity. The inventors next used a tenfold dilution from 100 million copies per reaction down to 0 copies of the target per reaction to evaluate the dynamic range of our assay with this blocking region combination. The results are shown in table 14.

TABLE 14

As shown in table 14, the template was detected as low as 10 copies per reaction, but it appeared in the NTC range at 10 copies per reaction. NTC was successfully inhibited by the blocking zone. These results indicate that, according to some embodiments, inclusion of a blocking region oligonucleotide in the RPA reaction solution enhances RPA or another amplification reaction by increasing specificity.

Example 14 isothermal amplification with Low level or in the absence of DTT (e.g., LAMP)

Another problem encountered in some cases is the difficulty of including Dithiothreitol (DTT) in commercial RPA kits. DTT is a reducing agent whose thiol group has a strong affinity for gold. Some embodiments of the apparatus and methods used herein relate to electrodes comprising gold. For example, some embodiments of the electrodes of the cartridge comprise gold. In some embodiments, some concentrations of DTT prevent detection on the cartridge or with the devices described herein. For example, it was found that in some cases concentrations of DTT above-1 mM prevented detection on the cartridge, but amounts of DTT of about 0.5mM were acceptable.

DTT was found to have no detrimental effect on the optical detection method using the thermal cycler, indicating that DTT does interfere with the cartridge's electrical detection method, not the amplification reaction itself. To avoid this problem, in some embodiments, a lower concentration of DTT (e.g., 0.25mM) may be substituted where a concentration of 5mM or 1mM-10mM DTT is typically used without the detection methods or devices described herein. In other words, if a reaction premix having a higher amount (e.g., 5mM) is described, in some embodiments a similar reaction premix may be prepared, including, for example, 0.25mM DTT may alternatively be used. For example, some embodiments include a base reaction premix (e.g., the base reaction premix in example 12), but with about 0.25mM DTT instead of 5mM DTT.

Experiments were performed to determine whether in some cases the reducing agents DTT and tris (2-carboxyethyl) phosphine (TCEP) reacted with the cartridge electrode in a manner that did not properly detect the amplification signal. If one or both of these reducing agents are shown to not affect signal transduction, they can be used in a cartridge assay that both lyses viruses and mitigates the negative effects of nasal fluid sample testing. For this experiment, the sample consisted of a Haemophilus influenzae target and LAMP primers (SEQ ID NO: 08-SEQ ID NO: 13) with 5mM DTT or TCEP in the LAMP reaction solution. The samples were loaded directly into the preheated cassettes and run at 65 ℃ for 60 minutes and were electrically tested. The same samples were also run on a QS3 thermal cycler to optically assess the effect of DTT/TCEP on the reaction. Ct values from the amplification curves are shown in table 15.

Watch 15

The results of the cassette test are shown in fig. 44. The experimental results show that the Haemophilus influenzae sample added with 5mM TCEP was amplified normally in a thermocycler, indicating that it does not interfere with LAMP chemistry. It also produces a normal amplification signal when the sample is run in the cartridge with electrical detection. With these results, we should expect that TCEP is compatible with isothermal amplification assays (e.g., LAMP) in a cassette according to some embodiments, and that isothermal amplification assays (e.g., LAMP) can be performed in the absence of DTT. In some embodiments of the methods described herein, the amplification reaction solution comprises TCEP. In some embodiments, the TCEP in the amplification reaction solution is 5mM, about 5mM, 1mM-10mM, or 3mM-7 mM.

The results of the experiment also show that a sample of haemophilus influenzae with 5mM DTT was amplified normally in a thermocycler, indicating that DTT does not interfere with LAMP chemistry. When Haemophilus influenzae with a 5mM DTT sample was run in the cassette with the electrical test, no amplification was detected. Because the DTT sample was amplified in the thermal cycler, there is a high probability that there was amplification in the cassette, but the signal was not properly reported due to possible corrosion of the electrodes. This result may be due to the affinity of the thiol group of DTT for gold.

Example 15 isothermal amplification with or without calcium in amplification reaction solution (e.g., LAMP or RPA)

Experiments were performed to test with and without CaCl₂The inventors found in previous experiments that H Inf was detected with low dNTPs (1.8 mM in this experiment) and was signal-enhanced. An experiment was performed according to the procedure using the base reaction premix in example 12, but the modification included changing the dNTP concentration to 1.8mM and CaCl₂The amounts of (c) differ as follows.

Inf LAMP primers F3 and B3(SEQ ID NO: 23 and SEQ ID NO: 25) + CaCl-free₂+ synthetic H.Inf (SEQ ID NO: 08), 1M copy/rxn (LAMP F3+ B3 for short)

Inf LAMP primers F3 and B3+0.9mM CaCl ₂+ synthetic H.Inf, 1M copy/rxn (LAMP F3+ B3 and 0.9mM CaCl₂)

·H Inf_180430_RPA F1(SEQ ID NO:14)+R1(SEQ ID NO:27:) + no CaCl₂+ synthetic H.Inf, 1M copy/rxn (RPA F1+ R1 for short)

·H Inf_180430_RPA F1+R1+0.9mM CaCl₂+ synthetic H.Inf, 1M copy/rxn (RPA F1+ R1 and 0.9mM CaCl₂)

LAMP F3+ B3 and CaCl-free₂NTC (negative temperature coefficient)

LAMP F3+ B3 and 0.9mM CaCl₂NTC (negative temperature coefficient)

RPA F1+ R1 and CaCl-free₂NTC (negative temperature coefficient)

RPA F1+ R1 and 0.9mM CaCl₂NTC (negative temperature coefficient)

Absence of CaCl₂NPC of

·0.9mM CaCl₂NPC of

Amplification was performed on 6 replicates of each group. The results are shown in fig. 45A-45E. FIG. 45A includes the following amplification curves: LAMP F3+ B3, without CaCl₂And with 0.9mM CaCl₂Synthetic H Inf is 1M copy/rxn, and NTC. Fig. 45B shows the following melting curves: LAMP F3+ B3, without CaCl₂And with 0.9mM CaCl₂Synthetic H Inf is 1M copy/rxn, and NTC. Fig. 45C includes the following amplification curves: RPA F1+ R1 without CaCl₂And with 0.9mM CaCl₂Synthetic H Inf is 1M copy/rxn, and NTC. Fig. 45D includes the following melting curves: RPAF1+ R1 without CaCl₂And with 0.9mM CaCl₂Synthetic H Inf is 1M copy/rxn, and NTC. No Primer Control (NPC) lacked amplification. Ct values are shown in fig. 45E. The positive control was distinguished from NTC. Without CaCl₂Positive control(s) amplified at 24.5 min without CaCl ₂NTC of (3) was amplified at 40.0 minutes. With 0.9mM CaCl₂Slightly delayed and amplified at 31.3 min, with 0.9mM CaCl₂NTC of (3) was amplified at 42.1 minutes.

Similar experiments were performed using the cassettes described herein for detection of amplification products. Fig. 46A shows the RPA curve for the sample without calcium. No amplification signal was detected in these RPA reactions without calcium. (note: the initial impedance drop of the reaction is normal.) FIG. 46B shows the RPA curve for the sample with 0.9mM calcium. The signal is clearly visible at approximately 5 min. These results indicate that the detection methods using the cartridges according to some embodiments are compatible with isothermal amplification assays (e.g., RPA), and that isothermal amplification (e.g., RPA) can work at higher levels of DTT (e.g., about 5 mM). In some embodiments, calcium (e.g., CaCl) is included in the amplification reaction solution or reagent solution (e.g., RPA reagent solution)₂E.g., 0.5mM-1.5mM or about 0.9mM CaCl₂) Or calcium ions, and calcium may allow the amplification reaction to proceed at higher DTT levels.

In another example, the RPA reaction is performed similarly as in this example, but in the absence of DTT or at low levels of DTT (e.g., 0.1mM-0.5mM, 0.5mM-1.0mM, or about 0.25 mM). The RPA reaction is carried out in and detected by a cartridge detection system as described herein. In such instances (in the absence of DTT or at low levels of DTT), no calcium or calcium ions are required to allow the RPA reaction to proceed and can be detected in a cartridge detection system.

Example 16 protocol for RPA as Pre-amplification (RAMP) before Another amplification (e.g., LAMP)

In some embodiments of the methods described herein, RPA is performed as a pre-amplification followed by LAMP in the same reaction vessel. In some embodiments of preamplification prior to RPA being used as LAMP, LAMP primers include FIP/BIP and LF/LB. In some embodiments, the outer LAMP primers (F3/B3) may be replaced by RPA primers as long as they produce amplicons of the desired length (i.e., the LAMP-specific primers must be spatially nested within the RPA primers along the target sequence). In some embodiments of RAMP, RPA primers are used in the RPA phase, followed by the addition of FIP/BIP and LF/LB prior to the LAMP phase. The addition of all six LAMP primers (effectively with RPA primer + LAMP F3/B3) also works, but in some embodiments is not necessary.

The following is an exemplary protocol for performing RPA followed by LAMP as a pre-amplification:

(1) the base reaction premix of example 12 was used, except that:

WarmStart Bst 2.0LAMP polymerase from New England Biolabs, including 8U. Note that this was not activated at the RPA reaction temperature of 40 ℃.

Increase the dNTP concentration to a total of 5.6mM (or 1.8mM of each dNTP)

Reduction of the amount of magnesium activator from 13+ mM to 8mM, which are commonly used in RPA

(2) Step 2-step 6 are run as in RPA. Step 6 is run as a pre-amplification for the desired length of time.

(3) After pre-amplification, the tubes were opened and pipetted into the LAMP primers (FIP/BIP and LF/LB only; RPA primers can replace F3/B3)

(4) The LAMP phase of the reaction was run at 65 ℃.

Example 17 RPA as Pre-amplification (RAMP) before Another amplification (e.g., LAMP)

In experiments where RPA reagent solutions (but including 8U of NEB's WarmStart Bst 2.0 and 5.6mM dNTPs instead of 1.8mM, and varying amounts of magnesium) were prepared according to the base reaction premix of example 12, the RPA stage contained only the RPA primer, and then LAMP FIP/BIP and LF/LB were added after the RPA stage was completed.

These experiments were performed with the aim of adding NEB WarmStart Bst 2.0 to the RPA stage as a step towards the one-pot (one-pot) RAMP reaction, since all components except the LAMP primers and additional dntps are contained in the RPA stage. The total volume of the RPA stage was 23. mu.L, and 2. mu.L of LAMP primer and dNTP were added after the RPA run.

A LAMP reagent solution is provided comprising 1 μ L of the hnf LAMP primer and 1 μ L of a 95mM dNTP mix. After the RPA reaction run, 2 μ L of this mixture was added to each 23 μ L of RPA reaction. This volume can be added to the lid or side of the RPA reaction tube and then inverted and spun down, similar to the RPA activation step. The LAMP stage was run in the same band used for the RPA stage, as the LAMP reagent solution was added directly to the tube, rather than removing the RPA reaction and adding to a new tube. The LAMP primer sequences are shown in table 17. The RPA primers were H Inf RPA F1(SEQ ID NO: 14) and H Inf RPA R3(SEQ ID NO: 28: ). The experiment was run on a QS3 instrument. The target is synthetic H Inf (10)⁶Copy/reaction).

TABLE 17

Fig. 47A and 47B depict RPA stage data (RPA pre-amplification stage before LAMP primer addition). FIG. 47A is an amplification plot of RPA positive controls with 8mM, 10mM, and 12mM Mg without addition of Bst 2.0. FIG. 47B is an amplification plot of an RPA positive control with WarmStart Bst 2.0 at 8mM, 10mM, and 12mM Mg and NEB.

Fig. 48A-48F depict LAMP stage amplification data (after LAMP primer addition). FIG. 48A includes H Inf 10 with 8mM Mg⁶Amplification profiles of LAMP phase of c/rxn and NTC. FIG. 48B includes H Inf 10 with 8mM Mg⁶Melting profiles of LAMP phase of c/rxn and NTC. FIG. 48C includes H Inf 10 with 10mM Mg⁶Amplification profiles of LAMP phase of c/rxn and NTC. FIG. 48D includes H Inf 10 with 10mM Mg⁶Melting profiles of LAMP phase of c/rxn and NTC. FIG. 48E includes H Inf 10 with 12mM Mg⁶Amplification profiles of LAMP phase of c/rxn and NTC. FIG. 48F includes H Inf 10 with 12mM Mg⁶Melting profiles of LAMP phase of c/rxn and NTC.

The data in fig. 47A-48F indicate that the RAMP method was successful at all magnesium concentrations used. Thus, some embodiments include methods comprising RAMP or RPA as a pre-amplification reaction prior to another amplification (e.g., LAMP).

Example 18 two-pot RPA on cassette and Another amplification (e.g., LAMP)

Experiments were performed to test serial dilutions with H Inf in cassettes (10)⁶An⁰Copies/reaction) to determine sensitivity, specificity and limit of detection in the cassette.

RPA was performed in a similar manner to RPA in example 17 (using the modified base reaction premix of example 12), except that the LAMP primer was not added to the RPA reaction at the end of the RPA stage, and the reaction was performed on a cassette. The RPA phase was run on the cassette at 40 ℃ for 15 minutes. No data is collected during the RPA phase. A 2.5 μ L aliquot was transferred from the RPA reaction to the LAMP reaction premix. The LAMP phase was run on the cassette at 65 ℃ for 60 minutes. In fact, the product of the RPA reaction is used as a template in LAMP.

Since the RPA product is diluted 10 x when transferred to the LAMP reaction, the target concentration in the RPA stage is 10 times higher than that in the LAMP stage. The target concentrations listed in the data areRefers to the final concentration after dilution. Thus, the initial RPA target concentration was 10 per reaction⁷、10⁶、10⁵、10⁴、10³、10²And 10¹Copies (c/rxn). The reason for 10 x dilution from RPA stage to LAMP stage is that the RPA reaction mixture contains 5mM DTT, which in some cases may be incompatible with the electrical detection sensor. By diluting it by 10 ×, the final DTT concentration becomes 0.5mM, which enables the cartridge to detect an amplification signal.

The data are depicted in fig. 49A-49D. FIG. 49A includes H Inf 10 at 1000Hz⁶c/rxn and 10⁵c/rxn amplification data. FIG. 49B includes H Inf 10 at 1000Hz⁴c/rxn and 10³c/rxn amplification data. FIG. 49C includes H Inf 10 at 1000Hz²c/rxn and 10¹c/rxn amplification data. FIG. 49D includes H Inf 10 at 1000Hz⁰Amplification data of c/rxn and NTC c/rxn.

In addition to having 10⁴c/rxn and 10³Several repeats on the c/rxn cassette, almost all of which observed amplification. Overall, Ct is slower than that seen in the thermocycler; 10⁶c/rxn appears almost immediately in the LAMP phase, whereas 10⁶Appearing in the box for approximately 10 minutes. These data indicate that LAMP can be performed on RPA reaction products as templates, according to some embodiments.

Example 19 one-pot RPA with an alternative amplification (e.g., LAMP)

Experiments were performed to determine whether RPA and LAMP could be performed together in one reaction mixture (including a blocking region in the reaction solution). Using a twist Amp Liquid Basic Master Mix, an amplification reaction solution was provided comprising Bst 2.0, 5.6mM dNTP and 10mM Mg in MM²⁺. The experiment included 3 replicates of Synt H inf and 3 replicates of NTC with the following conditions:

1. A one-pot method: FIP/BIP @ 0.32. mu.M each, no blocking region

2. A one-pot method: FIP/BIP @ 0.32. mu.M each, 0.1 × (0.032. mu.M each) LAMP Block

3. A one-pot method: FIP/BIP @ 0.32. mu.M each, 1 × (0.32. mu.M each) LAMP blocking region

4. A one-pot method: FIP/BIP @ 0.32. mu.M each, 2 × (0.64. mu.M each) LAMP blocking region

5. A one-pot method: FIP/BIP @ 0.32. mu.M each, 5 × (1.6. mu.M each) LAMP blocking region

The experiment was run on QS3 for 15 minutes at 40 ℃ and then 45 minutes at 65 ℃. Fluorescence was measured every minute to assess the progress of the reaction. The RPA primers used in each reaction were HINf-180430 RPA-F1 (SEQ ID NO:14) and H Inf-180430 RPA-R3 (SEQ ID NO:28), in each case 480 nM. The LAMP primers used in each reaction were H Inf 180430_ FIP (SEQ ID NO:22) and H Inf 180430_ BIP (SEQ ID NO: 21). The primer F3/B3 or LF/LB is not included, and only H Inf 180430_ FIP and H Inf 180430_ BIP are included. The blocking region sequences are shown in table 18. The blocking regions are designed to bind directly to the LAMP primers to prevent them from functioning during the RPA phase.

Watch 18

The data are depicted in fig. 50A-50C. As shown in fig. 50A, inclusion of the LAMP blocking region resulted in faster amplification, indicating that the blocking region prevented the LAMP primer from inhibiting RPA. Fig. 50B includes a melting profile for the one-pot method: FIP/BIP @ 0.32. mu.M each, without blocking region. Fig. 50C contains a melting profile for the one-pot method: FIP/BIP @ 0.32. mu.M each, 0.1 × (0.032. mu.M each) LAMP blocking region. Melting curves indicate that different species are generated in the presence of the blocking region. These experiments show that, according to some embodiments, blocking regions may be used, for example, as part of an amplification reaction solution to improve specificity of nucleic acid amplification.

Implementation System and terminology

Embodiments disclosed herein provide systems, methods, and devices for the detection of the presence and/or amount of a target analyte. Those skilled in the art will recognize that these embodiments can be implemented in hardware or a combination of hardware and software and/or firmware.

The signal processing and reader device control functions described herein may be stored as one or more instructions on a processor-readable medium or a computer-readable medium. The term "computer-readable medium" refers to any available medium that can be accessed by a computer or processor. By way of example, and not limitation, such media can comprise RAM, ROM, EEPROM, flash memory, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store desired program code in the form of instructions or data structures and that can be accessed by a computer. It should be noted that computer-readable media may be tangible (transitory) and non-transitory. The term "computer program product" refers to a computing device or processor in combination with code or instructions (e.g., a "program") that may be executed, processed, or computed by the computing device or processor. As used herein, the term "code" may refer to software, instructions, code or data that is executable by a computing device or processor.

The various illustrative logical blocks and modules described in connection with the embodiments disclosed herein may be implemented or performed with a machine, such as a general purpose processor, a Digital Signal Processor (DSP), an Application Specific Integrated Circuit (ASIC), a Field Programmable Gate Array (FPGA) or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. A general purpose processor may be a microprocessor, but in the alternative, the processor may be a controller, microcontroller, combination thereof, or the like. A processor may also be implemented as a combination of computing devices, e.g., a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration. Although the description herein is primarily with respect to digital technology, the processor may also primarily include analog components. For example, any of the signal processing algorithms described herein can be implemented in analog circuitry. The computing environment may include any type of computer system, including but not limited to microprocessor-based computer systems, mainframe computers, digital signal processors, portable computing devices, personal organizers, device controllers, and computing engines in appliances, to name a few.

The methods disclosed herein comprise one or more steps or actions for achieving the described method. The method steps and/or actions may be interchanged with one another without departing from the scope of the claims. In other words, unless a specific order of steps or actions is required for proper operation of the method being described, the order and/or use of specific steps and/or actions may be modified without departing from the scope of the claims.

The term "comprising" as used herein is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

The above description discloses some methods and materials of the present invention. The present invention is susceptible to modifications in method and materials, as well as variations in manufacturing methods and apparatus. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed herein, but that the invention will include all modifications and alternative embodiments falling within the true scope and spirit of the present invention.

All references cited herein (including but not limited to published and unpublished applications, patents, and references) are hereby incorporated by reference in their entirety and made a part of this specification. If publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Sequence listing

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<223> RPA blocking region

<400> 20

cttttggatt aatttcttgt t 21

<210> 21

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> LAMP primer

<400> 21

acttctttac caaaggcatc atttaatttg cgtttgttga attctggg 48

<210> 22

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> LAMP primer

<400> 22

ttttactgat gatatgggta catctgggct cgaagaatga gaagttttgt 50

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> LAMP primer

<400> 23

tggtacgcca atacattcaa 20

<210> 24

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> LAMP primer

<400> 24

cgccataact tcatcttagc acc 23

<210> 25

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> LAMP blocking region

<400> 25

cccagaattc aacaa 15

<210> 26

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> LAMP blocking region

<400> 26

acaaaacttc tcattc 16

Claims

1. A method of amplifying and detecting a target nucleic acid, the method comprising:

providing, preferably in a single container, a Recombinase Polymerase Amplification (RPA) reagent solution comprising a target nucleic acid, primers each complementary to a region of the target nucleic acid, a buffer, dntps, a recombinase, a single-stranded DNA binding protein (SSB), and a strand-displacing DNA polymerase;

combining, preferably in the single container, the RPA reagent solution with a second reagent solution configured for a second isothermal amplification reaction without the use of a recombinase to produce an amplification reaction solution;

performing RPA in the amplification reaction solution, and optionally the second isothermal amplification, preferably in the single vessel, to produce amplified target nucleic acid;

Optionally, helical RPA is performed using paired primers, the forward primer sequence and the reverse primer sequence being reverse complementary to each other at their 5 'ends and their 3' end sequence being complementary to said target sequence, and

detecting the presence of the amplified target nucleic acid by measuring the modulation of an electrical signal, e.g., impedance, in the amplification reaction solution when subjecting the amplification reaction to an electric field, as compared to a control.

2. The method of claim 1, wherein the detection of the amplified target nucleic acid is performed in a device comprising a test well comprising an excitation electrode and a sensor electrode, and wherein the detecting further comprises:

applying an excitation signal from a reader device to the excitation electrode;

sensing a signal from a test well using an excitation electrode, wherein the signal is representative of an impedance of the amplification reaction solution; and

transmitting the signal to the reader device, wherein the reader device analyzes the signal.

3. The method of claim 1 or 2, wherein the recombinase comprises UvsX or RecA.

4. The method of any one of claims 1-3, wherein the SSB comprises T4 bacteriophage gp32 or E.

5. The method of any one of claims 1-4, wherein the strand-displacing DNA polymerase comprises Bst DNA polymerase large fragment, Bst 2.0 polymerase, Bst 3.0 polymerase, Gsp polymerase, Sau polymerase, Bsu DNA polymerase large fragment, Deep Vent_RDNA polymerase, Deep Vent_R(exo-) DNA polymerase, Klenow fragment (3'→ 5' exo-), DNA polymerase I Large (Klenow) fragment, phi29 DNA polymerase, Vent_RDNA polymerase or Vent_R(exo-) DNA polymerase.

6. The method of any one of claims 1-5, wherein said RPA reagent solution further comprises a reagent selected from the group consisting of: tris-acetate, polyethylene glycol, trehalose, potassium acetate, creatine kinase, creatine phosphate, adenosine triphosphate, recombinase-loaded proteins, such as uvsY, or wherein the RPA reagent solution or the amplification reaction solution does not contain a primer with a detectable label or dye.

7. The method of any one of claims 1 to 6, wherein the amplification reaction solution comprises DTT at a concentration of 0.5mM to 1mM, 0.25mM to 0.5mM, about 0.25mM, 0mM to 0.25mM, or 0 mM.

8. The method according to any one of claims 1 to 7, wherein the amplification reaction solution comprises DTT at a concentration of 1mM-10mM, about 5mM, or 5mM, and calcium chloride at a concentration of 5mM-15mM, about 0.9mM, 5mM-15mM, Ca ²⁺Ca of about 0.9mM²⁺Or 0.9mM Ca²⁺。

9. The method of any one of claims 1 to 8, wherein the amplification reaction solution comprises magnesium or magnesium ions at a concentration of 1mM to 20mM, 1mM to 5mM, 5mM to 10mM, 7mM to 9mM, 8mM, about 8mM, 10mM to 20mM, about 13mM, or 13 mM.

10. The method of any one of claims 1-9, wherein the amplification reaction solution comprises a dNTP mixture at a concentration of 1mM-10mM, 1mM-3mM, about 1.8mM, 5mM-6mM, about 5.6mM, or 5.6 mM.

11. The method of any one of claims 1-10, wherein the amplification reaction solution further comprises a blocking region oligonucleotide comprising a nucleic acid sequence that is reverse complementary to a portion of the nucleic acid sequence of one or more of the primers.

12. The method of any one of claims 1-11, wherein the RPA comprises a leading strand RPA (lsrpa), a synchronous leading and trailing strand synthesis, or a nested RPA.

13. The method of any one of claims 1-12, wherein the second isothermal amplification comprises self-sustained sequence replication reaction (3SR), 90-I, BAD Amp, cross-primer amplification (CPA), isothermal index amplification reaction (EXPAR), isothermal chimeric primer-primed nucleic acid amplification (ICAN), Isothermal Multiple Displacement Amplification (IMDA), ligation-mediated SDA, multiple displacement amplification, polymerase helix reaction (PSR), restriction cascade index amplification (RCEA), smart amplification program (SMAP2), Single Primer Isothermal Amplification (SPIA), transcription-based amplification system (TAS), transcription-mediated amplification (TMA), Ligase Chain Reaction (LCR) or multiple cross-displacement amplification (MCDA), rolling circle Replication (RCA), nickase amplification reaction (NEAR), or nucleic acid sequence-based amplification (NASBA).

14. The method of any one of claims 1-13, wherein said RPA and said second isothermal amplification are performed at the same or substantially the same temperature.

15. The method of claim 14, wherein the RPA or the second isothermal amplification is performed at 25 ℃, 30 ℃, 37 ℃, 40 ℃, 45 ℃, 50 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, or at a temperature within a range defined by any two of the aforementioned temperatures.

16. The method of any one of claims 1-15, wherein said RPA is performed at a lower or higher temperature than said second isothermal amplification.

17. The method of claim 16, wherein the RPA is performed at 37 ℃, about 37 ℃, 40 ℃, or about 40 ℃, and the second isothermal amplification is performed at 60 ℃, about 60 ℃, 65 ℃, or about 65 ℃.

18. The method of any one of claims 1-17, wherein the second isothermal amplification comprises loop-mediated isothermal amplification (LAMP).

19. The method of claim 18, wherein the amplification reaction solution comprises LAMP-compatible primer oligonucleotides.

20. The method of claim 19, wherein the amplification reaction solution comprises a FIP, BIP, LF or LB primer oligonucleotide compatible with LAMP and a primer compatible with RPA.

21. The method of claim 19, wherein the amplification reaction solution comprises a LAMP-compatible FIP, BIP, LF, LB, F3, or B3 primer oligonucleotide.

22. The method of any one of claims 1-21, wherein the primer does not comprise a label or dye.

23. The method of any one of claims 1-22, wherein the amplifying and detecting are performed in the absence of a detection reagent, such as a dye, a clouding agent, a fluorophore, a double-stranded nucleic acid intercalator, a sequencing index, or a nanoparticle.

24. A method of amplifying and detecting a target nucleic acid, the method comprising:

preferably, providing in a single container a Recombinase Polymerase Amplification (RPA) reagent solution comprising a target nucleic acid, primers each complementary to a region of the target nucleic acid, a buffer, dntps, a recombinase, a single-stranded DNA binding protein (SSB), and a strand-displacing DNA polymerase;

performing RPA in an RPA reagent solution to produce amplified target nucleic acids;

combining, preferably in the single vessel, the amplified target nucleic acid with a second reagent solution configured for a second isothermal amplification reaction without the use of a recombinase to produce a second amplification reaction solution;

Performing a second isothermal amplification of the second amplification reaction solution to produce further amplified target nucleic acids; and

the presence of amplified target nucleic acids is detected by measuring the modulation of an electrical signal, e.g., impedance, in the amplification reaction solution when the amplification reaction is subjected to an electric field, as compared to a control.

25. The method of claim 24, wherein combining the amplified target nucleic acid with a second reagent solution configured for a second isothermal amplification reaction comprises: adding the second reagent solution to the RPA reagent solution after performing RPA to produce the amplified target nucleic acid.

26. The method of claim 24, wherein combining the amplified target nucleic acid with a second reagent solution configured for a second isothermal amplification reaction comprises: adding the RPA reagent solution or a portion of the RPA reagent solution to the second reagent solution after performing RPA to produce an amplified target nucleic acid.

27. The method of any one of claims 24-26, wherein the recombinase enzyme comprises UvsX or RecA.

28. The method of any of claims 24-27, wherein the SSB comprises gp32 or escherichia coli SSB.

29. The method of any one of claims 24-28, wherein the strand-displacing DNA polymerase comprises Bst DNA polymerase large fragment, Bst 2.0 polymerase, Bst 3.0 polymerase, Gsp polymerase, Sau polymerase, Bsu DNA polymerase large fragment, Deep Vent_RDNA polymerase, Deep Vent_R(exo-) DNA polymerase, Klenow fragment (3'→ 5' exo-), DNA polymerase I Large (Klenow) fragment, phi29 DNA polymerase, Vent_RDNA polymerase or Vent_R(exo-) DNA polymerase.

30. The method of any one of claims 24-29, wherein said RPA reagent solution further comprises a reagent selected from the group consisting of: tris-acetate, polyethylene glycol, trehalose, potassium acetate, creatine kinase, creatine phosphate, adenosine triphosphate, recombinase-loaded proteins, such as uvsY, or wherein the RPA reagent solution or the amplification reaction solution does not contain a primer with a detectable label or dye.

31. The method of any one of claims 24-30, wherein the amplification reaction solution comprises DTT at a concentration of 0.5mM-1mM, 0.25mM-0.5mM, about 0.25mM, 0mM-0.25mM, or 0 mM.

32. The method of any one of claims 24-31, wherein the amplification reaction solution comprises DTT at a concentration of 1mM-10mM, about 5mM or 5mM, and calcium chloride at a concentration of 5mM-15mM, calcium chloride at a concentration of about 0.9mM, calcium chloride at a concentration of 0.9mM, Ca at a concentration of 5mM-15mM ²⁺Ca of about 0.9mM²⁺Or 0.9mM Ca²⁺。

33. The method of any one of claims 24-32, wherein the amplification reaction solution comprises magnesium or magnesium ions at a concentration of 1mM-20mM, 1mM-5mM, 5mM-10mM, 7mM-9mM, 8mM, about 8mM, 10mM-20mM, about 13mM, or 13 mM.

34. The method of any one of claims 24-33, wherein the amplification reaction solution comprises a dNTP mixture at a concentration of 1mM-10mM, 1mM-3mM, about 1.8mM, 5mM-6mM, about 5.6mM, or 5.6 mM.

35. The method of any one of claims 24-34, wherein the amplification reaction solution further comprises a blocking region oligonucleotide comprising a nucleic acid sequence that is reverse complementary to a portion of the nucleic acid sequence of one or more of the primers.

36. The method of any one of claims 24-35, wherein the RPA comprises a leading strand RPA (lsrpa), a synchronous leading and trailing strand synthesis, or a nested RPA.

37. The method of any one of claims 24-36, wherein the second isothermal amplification comprises self-sustained sequence replication reaction (3SR), 90-I, BAD Amp, cross-primer amplification (CPA), isothermal index amplification reaction (EXPAR), isothermal chimeric primer-primed nucleic acid amplification (ICAN), Isothermal Multiple Displacement Amplification (IMDA), ligation-mediated SDA, multiple displacement amplification, polymerase helix reaction (PSR), restriction cascade index amplification (RCEA), smart amplification program (SMAP2), Single Primer Isothermal Amplification (SPIA), transcription-based amplification system (TAS), transcription-mediated amplification (TMA), Ligase Chain Reaction (LCR) or multiple cross-displacement amplification (MCDA), rolling circle Replication (RCA), nickase amplification reaction (NEAR), or nucleic acid sequence-based amplification (NASBA).

38. The method of any one of claims 24-37, wherein said RPA and said second isothermal amplification are performed at the same or substantially the same temperature.

39. The method of claim 38, wherein said RPA or said second isothermal amplification is performed at 25 ℃, 30 ℃, 37 ℃, 40 ℃, 45 ℃, 50 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, or at a temperature within a range defined by any two of the foregoing temperatures.

40. The method of any one of claims 24-39, wherein said RPA is conducted at a lower or higher temperature than said second isothermal amplification.

41. The method of claim 40, wherein said RPA is performed at 37 ℃, about 37 ℃, 40 ℃, or about 40 ℃, and said second isothermal amplification is performed at 60 ℃, about 60 ℃, 65 ℃, or about 65 ℃.

42. The method of any one of claims 24-41, wherein the second isothermal amplification comprises loop-mediated isothermal amplification (LAMP).

43. The method of claim 42, wherein the amplification reaction solution comprises LAMP-compatible primer oligonucleotides.

44. The method of claim 43, wherein the amplification reaction solution comprises LAMP-compatible FIP, BIP, LF or LB primer oligonucleotides, and RPA-compatible primers.

45. The method of claim 43, wherein the amplification reaction solution comprises a LAMP-compatible FIP, BIP, LF, LB, F3, or B3 primer oligonucleotide.

46. The method of any one of claims 24-45, wherein the primer does not comprise a label or dye.

47. The method of any one of claims 24-46, wherein the amplifying and detecting are performed in the absence of a detection reagent, such as a dye, a clouding agent, a fluorophore, a double-stranded nucleic acid intercalator, a sequencing index, or a nanoparticle.

48. A method of amplifying and detecting a target nucleic acid, the method comprising:

performing Recombinase Polymerase Amplification (RPA) and loop-mediated isothermal amplification (LAMP) on a target nucleic acid in a single vessel to produce an amplified target nucleic acid, preferably without separating or purifying the amplified target nucleic acid between the RPA amplification and LAMP amplification, e.g., in a single vessel; and

49. The method of claim 48, wherein the RPA and LAMP amplification are performed at the same or substantially the same temperature.

50. The method of claim 49, wherein the RPA or LAMP amplification is performed at 25 ℃, 30 ℃, 37 ℃, 40 ℃, 45 ℃, 50 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, or at a temperature within a range defined by any two of the aforementioned temperatures.

51. The method of any one of claims 48-50, wherein the RPA is performed at a lower or higher temperature than the LAMP amplification.

52. The method of claim 48, wherein the RPA is performed at 37 ℃, about 37 ℃, 40 ℃, or about 40 ℃, and the LAMP amplification is performed at 60 ℃, about 60 ℃, 65 ℃, or about 65 ℃.

53. The method of any one of claims 48-52, wherein the primers in the RPA or LAMP amplification do not comprise a label or dye.

54. The method of any one of claims 48-53, wherein the RPA and LAMP amplification and the detection are performed in the absence of detection reagents, such as dyes, clouding agents, fluorophores, double-stranded nucleic acid intercalators, sequencing indexes, or nanoparticles.

55. The method of any one of claims 1-54, wherein detection of the amplified target nucleic acids is performed in a device comprising a test well comprising an excitation electrode and a sensor electrode, and wherein the detecting further comprises:

Applying an excitation signal from a reader device to the excitation electrode;

sensing a signal from the test well using the excitation electrode, wherein the signal is representative of an impedance of the amplification reaction solution; and