RU2772116C2 - Methods, systems and devices for detecting analytes - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: system is described for detecting a target agent, containing: an analytical cartridge including a test cell containing an exciting electrode and a sensor electrode, wherein the test cell is made with the possibility of accommodating a sample containing the target agent subjected to the amplification process, wherein the specified target agent contains nucleic acid; and a reading device including: an area made with the possibility of receiving the analytical cartridge, a heater located in such a way that to heat the used analytical cartridge inside a cavity, memory storing at least machine-readable storage instructions, and a processor. In this case, the processor is configured by the specified instructions in such a way that at least: to lead to heating of the analytical cartridge by the heater to a set temperature to perform the amplification process inside the test cell; to supply excitation current to the exciting electrode for at least part of a time of the amplification process, to receive a signal from the sensor electrode, corresponding to excitation current after its attenuation due to the interaction with at least the sample inside the test cell, to decompose the specified signal to an active resistance component and a reactive resistance component, to analyze the reactive resistance component to determine a signal drop relatively to the time and on a certain frequency of the specified signal indicating a positive sample containing the target agent for at least part of a time of the amplification process, and, in response to the determination of the signal drop, to output a positive test result; or, in response to the determination of absence of the signal drop, to output a negative test result. A device and a method for studying a sample for a target agent are also described.

EFFECT: invention provides a low-cost device that is undemanding to resources, provides quick and stable sensor detection and identification; such a device can use microfluidics, biochemistry and electronics to detect one or more targets simultaneously in the field, as well as near the place or in the place of provision of medical services.

62 cl, 36 dwg, 12 tbl, 11 ex

Description

RELATED APPLICATIONS

[0001] The present application claims priority over U.S. Provisional Application No. 62/398959, filed Sept. 23, 2016, entitled: "METHODS AND COMPOSITIONS FOR DETECTING VIRAL TARGETS", provisional U.S. Patent Application No. 62/399047, filed September 23, 2016, entitled: "METHODS AND COMPOSITIONS FOR DETECTING BACTERIAL TARGETS", U.S. Provisional Application No. 62/398925, filed on September 23, 2016, having the title: “METHODS AND COMPOSITIONS FOR DETECTING ANTIGENS” (“METHODS AND COMPOSITIONS FOR DETECTING ANTIGENS”), US Provisional Application No. 62/398913, filed on September 23, 2016, having the title: “ METHODS AND COMPOSITIONS FOR DETECTING PARASITES, Provisional Application for US Patent No. 62/398955, filed on September 23, 2016, titled: "METHODS AND COMPOSITIONS FOR DETECTING PARASITES". METHODS AND COMPOSITIONS FOR DETECTING MICRORNA TARGETS” and Provisional Application for US Patent No. 62/398965, filed September 23, 2016, titled: “METHODS AND COMPOSITIONS FOR DETECTION OF AGRICULTURAL ANALYTES” (“ METHODS AND COMPOSITIONS FOR DETECTING AGRICULTURAL ANALYTES"), each of which is hereby incorporated by reference in its entirety.

LINK TO SEQUENCE LISTING

[0002] This application is filed with a sequence listing in electronic format. This sequence listing is represented by a file named ALVEO010WOSEQ created on September 13, 2017, which is approximately 4 Kb in size. Information from the sequence listing in electronic format is incorporated herein in its entirety by reference.

FIELD OF TECHNOLOGY

[0003] The present invention relates generally to systems, methods and devices for diagnosing, recognizing and/or identifying pathogens, genomic materials, proteins and/or other small molecules or biomarkers. In some embodiments, the resource-saving, low-cost device provides fast and robust recognition and identification. Such a device may use microfluidics, biochemistry, and electronics to detect one or more targets simultaneously in the field and at or near a healthcare facility.

BACKGROUND OF THE INVENTION

[0004] Pathogens in a sample can be identified by detecting specific genomic material (DNA or RNA). In addition to pathogen detection, many other biomarkers are available for research, including molecules that provide early cancer detection, vital prenatal information, or a better understanding of a patient's microbiome. In standard nucleic acid testing ("NAT"), the genomic material in a sample can first be copied exponentially using a molecular amplification process known as polymerase chain reaction ("PCR") until there is enough DNA available to measurements. 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 using PCR.

BRIEF DESCRIPTION OF THE INVENTION

[0005] Some embodiments include a system for detecting a target agent (target) comprising: an assay cartridge comprising a test cell containing an excitation electrode and a sensor electrode, said test cell being configured to contain a sample containing the target agent being subjected to the amplification process; and a reader including: an area configured to receive an assay cartridge, a heater positioned to heat said assay cartridge in use within the cavity, a memory storing at least machine-readable storage instructions, and a processor configured with said instructions. so as to at least: cause the assay cartridge to be heated by the heater to a predetermined temperature to perform the amplification process within the test cell; apply excitation current to the excitation electrode for at least part of the time of the amplification process, receive a signal from the sensor electrode corresponding to the excitation current after it decays due to interaction with at least the sample inside the test cell, decompose the specified signal into a component of active resistance and a component reactance, analyze the reactance component to determine the presence of a signal drop indicative of a positive sample containing the target agent for at least part of the time of the amplification process, and in response to determining the occurrence of a signal drop, output a positive test result; or, in response to the determination of the absence of a signal edge, output a negative test result.

[0006] In some embodiments, said assay cartridge further comprises: a sample entry area configured to receive a sample; and a flow channel fluidly connecting the sample injection area to the test cell.

[0007] In some embodiments, said assay cartridge also includes a sealed chamber containing liquid constituents of the amplification process located in an area of the assay cartridge having an opening leading into the flow channel, wherein the sample entry area is located between said opening and the test cell along the flow channel; and a pneumatic fluid conduit fluidly connecting the pneumatic interface to an area of the assay cartridge, wherein dried components of the amplification process are placed in said test cell.

[0008] In some embodiments, said reader includes a pneumatic system configured to apply pressure through a pneumatic interface, a processor further configured by said instructions to at least: drive a motor coupled to an actuator located in such a way as to break through the sealed chamber and result in the ingress of liquid constituents into the area of the analytical cartridge; activate the pneumatic system, causing the liquid components to enter the flow channel and transfer the sample from the sample injection area to the test cell.

[0009] In some embodiments, said assay cartridge further comprises a mixing chamber located between the sample injection area and the test cell along the flow channel, configured to mix the liquid constituents and the sample into a substantially uniformly mixed test fluid.

[0010] In some embodiments, said assay cartridge comprises a first electrode interface, including a first pad leading to a drive electrode and a second pad leading to a sensor electrode.

[0011] In some embodiments, said reader comprises a second electrode interface configured to couple to the first electrode interface, wherein the assay cartridge enters the area of the reader.

[0012] In some embodiments, said reader further comprises a voltage source configured to generate a drive current, and in that the second electrode interface includes: a third pad positioned to interface with the first pad coupled to the voltage source; and a fourth pad, the location of which provides interfacing with the second pad associated with the memory.

[0013] In some embodiments, to decompose said signal into a resistance component and a reactance component, said processor is further configured by said instructions to at least: measure a signal at a frequency greater than its Nyquist frequency, wherein said signal matches the impedance of the specified sample; decompose said signal into an in-phase component and an out-of-phase component; and calculating a resistance component based on the common mode component, and calculating a reactance component based on the out of phase component.

[0014] In some embodiments, to analyze the reactance component, said processor is further configured with said instructions to evaluate at least predetermined expected edge characteristics from the memory.

[0015] In some embodiments, said predetermined expected signal edge characteristics stored in memory include a window of time over the course of the amplification process when said signal edge is predicted to occur.

[0016] In some embodiments, said predetermined expected edge characteristics stored in the memory include a threshold change in the value of the reactance component.

[0017] In some embodiments, said predetermined expected ramp characteristics stored in memory include a threshold slope of the reactance component curve, wherein the reactance component curve corresponds to the reactance component values measured over at least a portion of the time the amplification process is running.

[0018] In some embodiments, said amplification process includes bringing the processed sample into contact with a capture probe. In some embodiments, said capture probe is selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, and a nucleic acid. In some embodiments, said capture probe contains a detectable nucleic acid. In some embodiments, said detectable nucleic acid is amplified.

[0019] In some embodiments, said amplification process includes isothermal amplification. In some embodiments, said amplification process comprises an isothermal amplification reaction selected from the group consisting of a self-sustaining sequence replication reaction (3SR); 90-I; detection by amplification using beacons (BAD Amp); cross-priming amplification (CPA); exponential isothermal amplification reactions (EXPAR); isothermal chimeric primer initiated nucleic acid amplification (ICAN); isothermal amplification with multiple displacement (IMDA); SDA ligation mediated (strand exclusion amplification); multiple displacement amplifications; polymerase helical reaction (PSR); exponential restriction cascade amplification (RCEA); smart amplification process (SMAP2); single primer amplification reactions (SPIA); transcription-based amplification systems (TAS); transcription-mediated amplification (TMA); ligase chain reaction (LCR); and cross-displacement amplification (MCDA). In some embodiments, said amplification process includes loop isothermal amplification (LAMP).

[0020] In some embodiments, the target temperature for performing the amplification process inside the test cell is greater than 30°C. In some embodiments, the specified target temperature for performing the amplification process within the test cell is greater than 37°C. In some embodiments, the specified target temperature for performing the amplification process inside the test cell is above 60°C. In some embodiments, the target temperature for performing the amplification process within the test cell is in the range of 60°C to 70°C.

[0021] In some embodiments, said liquid constituents of the amplification process comprise a component selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, a nucleic acid such as a primer, a buffer, and an enzyme such as a polymerase.

[0022] In some embodiments, said dried amplification process components comprise a component selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, a nucleic acid such as a primer, a buffer, and an enzyme such as a polymerase.

[0023] Some embodiments include a device for testing a sample for a target agent, comprising: a sample input area configured to receive a sample containing the target agent; a test cell comprising an excitation electrode and a sensor electrode, said test cell being configured to: contain a sample during the amplification process, apply current to the sample during the amplification process using the excitation electrode, and receive, using a sensing electrode, a signal corresponding to the current after its attenuation due to interaction with at least the sample inside the test cell; and a flow channel fluidly connecting the sample injection area to the test cell.

[0024] Some embodiments also include a sealed chamber containing the liquid components of the amplification process, located in the region of the device with an opening leading to the flow channel, and the sample injection area is located between the specified hole and the test cell along the flow channel; and dried components of the amplification process, placed in a test cell.

[0025] Some embodiments also include a tip configured to break through the sealed chamber and cause liquid constituents to enter the specified area; and a pneumatic fluid conduit fluidly connecting the pneumatic interface device to the area of the device, configured to apply pressure to the specified area, which leads to the ingress of liquid components into the flow channel and transfers the sample received from the sample injection area to the test cell.

[0026] Some embodiments also include a mixing chamber located between the sample injection area and the test cell along the flow channel, said mixing chamber being configured to mix the liquid constituents and the sample into a substantially uniformly mixed test fluid.

[0027] In some embodiments, said assay cartridge comprises a first electrode interface device, including a first pad leading to a drive electrode and a second pad leading to a sensor electrode.

[0028] Some embodiments also include a circuit board including a drive electrode and a sensor electrode, wherein the sample entry area and at least a portion of the flow channel form a single piece of liquid impervious material, and the circuit board is attached to the portion of liquid impervious material.

[0029] Some embodiments also include a lid located over the liquid-impervious material and a circuit board, containing an opening located above the sample entry area, and a cap configured to seal said opening with the possibility of subsequent opening.

[0030] In some embodiments, the sides of the test cell form a circular opening in the liquid-impermeable material, and the bottom of the test cell is formed by a circuit board.

[0031] In some embodiments, said excitation electrode and said sensor electrode are located at the bottom of the test cell, away from the sides of the test cell.

[0032] In some embodiments, said drive electrode and said sensor electrode are configured to be substantially flush with the underlying circuit board layer.

[0033] Some embodiments also include a vent configured to vent gas from the test cell, said vent being covered by a liquid-tight gas permeable filter.

[0034] In some embodiments, said excitation electrode includes a circular electrode located in the center of the cell, and wherein said sensor electrode comprises an annular electrode disposed concentrically around said excitation electrode.

[0035] In some embodiments, said annular electrode is separated from said circular electrode by a gap approximately equal to the radius of the annular electrode.

[0036] In some embodiments, said annular electrode is separated from said circular electrode by a gap that is at least twice the radius of the annular electrode.

[0037] In some embodiments, said excitation electrode comprises a first semicircular electrode, and wherein said sensor electrode comprises a second semicircular electrode separated by a gap from the first semicircular electrode, wherein the rectilinear portions of said first and second semicircular electrodes face each other through the gap.

[0038] In some embodiments, said excitation electrode comprises a first line electrode, and wherein said sensor electrode comprises a second line electrode separated by a gap from the first line electrode.

[0039] In some embodiments, said excitation electrode comprises a first square electrode, and wherein said sensor electrode comprises a second square electrode separated by a gap from the first linear electrode.

[0040] In some embodiments, said amplification process includes bringing the processed sample into contact with a capture probe. In some embodiments, said capture probe is selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, and a nucleic acid. In some embodiments, said capture probe contains a detectable nucleic acid. In some embodiments, said detectable nucleic acid is amplified.

[0041] In some embodiments, said amplification process includes isothermal amplification. In some embodiments, said amplification process comprises an isothermal amplification reaction selected from the group consisting of a self-sustaining sequence replication reaction (3SR); 90-I; (BAD Amp); cross-priming amplification (CPA); exponential isothermal amplification reactions (EXPAR); isothermal chimeric primer initiated nucleic acid amplification (ICAN); isothermal amplification with multiple displacement (IMDA); mediated by SDA ligation; multiple displacement amplifications; polymerase helical reaction (PSR); exponential restriction cascade amplification (RCEA); smart amplification process (SMAP2); single primer amplification reactions (SPIA); transcription-based amplification systems (TAS); transcription-mediated amplification (TMA); ligase chain reaction (LCR); and cross-displacement amplification (MCDA). In some embodiments, said amplification process includes loop isothermal amplification (LAMP).

[0042] In some embodiments, said test cell is configured to heat the sample to a temperature in excess of 30°C. In some embodiments, said test cell is configured to heat the sample to a temperature in excess of 37°C. In some embodiments, said test cell is configured to heat the sample to a temperature in excess of 60°C. In some embodiments, said test cell is configured to heat the sample to a temperature in the range of 60°C to 70°C.

[0043] In some embodiments, said liquid constituents of the amplification process comprise a component selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, a nucleic acid such as a primer, a buffer, and an enzyme such as a polymerase.

[0044] In some embodiments, said dried amplification process components comprise a component selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, a nucleic acid such as a primer, a buffer, and an enzyme such as a polymerase.

[0045] Some embodiments include a non-volatile computer-readable medium that stores instructions that, when executed by a reader configured to receive an assay cartridge containing a sample containing a target agent and a test cell, causes said reader to perform operations including : supplying a drive current to a drive electrode located inside the test cell for at least part of the time of the amplification process occurring inside the test cell; receiving a signal from a sensor electrode located inside the test cell, said signal corresponding to the excitation current after it decays due to interaction with at least the sample being amplified inside the test cell; decomposing said signal into an active resistance component and a reactance component; analysis of the reactance component to determine the presence of a signal drop indicative of a positive sample containing the target agent for at least a portion of the time of the amplification process; and, in response to determining the occurrence of a signal drop, outputting a positive test result; or, in response to determining the absence of a signal edge, outputting a negative test result.

[0046] In some embodiments, these steps further include causing the heater to heat the assay cartridge to a predetermined temperature to perform the amplification process.

[0047] In some embodiments, these operations further include transmitting a positive test result or a negative test result over the network.

[0048] In some embodiments, said decomposition operations further comprise: measuring a signal at a frequency greater than its Nyquist frequency, where said signal corresponds to the impedance of said sample; decomposing said signal into an in-phase component and an out-of-phase component; calculating a resistance component based on the common mode component; and calculating a reactance component based on the out of phase component.

[0049] In some embodiments, these steps for analyzing the reactance component further include estimating predetermined expected edge characteristics from the memory.

[0050] In some embodiments, said predetermined expected signal edge characteristics stored in memory include a window of time over the course of the amplification process when said signal edge is predicted to occur.

[0051] In some embodiments, said predetermined expected edge characteristics stored in the memory include a threshold change in the value of a reactance component.

[0052] In some embodiments, said predetermined expected ramp characteristics stored in memory include a threshold slope of the reactance component curve, wherein the reactance component curve corresponds to the reactance component values measured for at least a portion of the time the amplification process is running.

[0053] In some embodiments, these steps further include activating a pneumatic system to pressurize the flow path of the assay cartridge, which causes the sample to mix with the amplification process components in the assay cartridge and enter the test cell.

[0054] In some embodiments, these steps further include activating a motor to push the drive into the blister pack of the assay cartridge, which causes the liquid constituents of the amplification process to be released into the flow channel.

[0055] In some embodiments, said amplification process includes bringing the processed sample into contact with a capture probe. In some embodiments, said capture probe is selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, and a nucleic acid. In some embodiments, said capture probe contains a detectable nucleic acid. In some embodiments, said detectable nucleic acid is amplified.

[0056] In some embodiments, said amplification process includes isothermal amplification. In some embodiments, said amplification process comprises an isothermal amplification reaction selected from the group consisting of a self-sustaining sequence replication reaction (3SR); 90-I; BAD Amp; cross-priming amplification (CPA); exponential isothermal amplification reactions (EXPAR); isothermal chimeric primer initiated nucleic acid amplification (ICAN); isothermal amplification with multiple displacement (IMDA); mediated by SDA ligation; multiple displacement amplifications; polymerase helical reaction (PSR); exponential restriction cascade amplification (RCEA); smart amplification process (SMAP2); single primer amplification reactions (SPIA); transcription-based amplification systems (TAS); transcription-mediated amplification (TMA); ligase chain reaction (LCR); and cross-displacement amplification (MCDA). In some embodiments, said amplification process includes loop isothermal amplification (LAMP).

[0057] In some embodiments, the specified target temperature for performing the amplification process is greater than 30°C. In some embodiments, the specified target temperature for performing the amplification process is greater than 37°C. In some embodiments, the specified target temperature for performing the amplification process is greater than 60°C. In some embodiments, said target temperature for performing the amplification process is in the range of 60°C to 70°C.

[0058] In some embodiments, said liquid constituents of the amplification process comprise a component selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, a nucleic acid such as a primer, a buffer, and an enzyme such as a polymerase.

[0059] In some embodiments, said amplification process further comprises dried moieties selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, a nucleic acid such as a primer, a buffer, and an enzyme such as a polymerase.

[0060] Some embodiments include a method for detecting a target agent, said method comprising: providing a cartridge containing a test cell including a drive electrode and a sensor electrode; introducing a sample containing the target agent into said cartridge; inserting the cartridge into the reader; conducting amplification of the target agent contained in the sample inside the test cell; applying an excitation signal from the reader to the excitation electrode; sensory detection, using an excitation electrode, of a signal from the test cell corresponding to the impedance of the sample being amplified; signal transmission to the reader; and detecting the target agent based on the reader's analysis of the reactive portion of the impedance.

[0061] Some embodiments also include supplying a sample to the sample input area of the cartridge; breaking through the sealed chamber within the cartridge to release the liquid constituents of the amplification process into the flow channel of the cartridge; and allowing the liquid constituents and the sample to flow along the flow channel into the test cell, thereby mixing the liquid constituents and the sample to obtain a test fluid.

[0062] Some embodiments also include moisturizing the dried components of the amplification process contained within the test cell being examined by the fluid.

[0063] Some embodiments also include expelling gas entrained in the test fluid through a vent that is in fluid communication with the test cell.

[0064] Some implementations also include analyzing the reactive portion of the signal to identify a signal edge that indicates a positive test result.

[0065] Some implementations also include identifying a signal edge based on the portion of the curve generated from the reactive portion that differs in either one or both of more than a threshold value change and temporal location within a given time window in the process. amplification.

[0066] In some embodiments, said amplification process includes bringing the processed sample into contact with a capture probe. In some embodiments, said capture probe is selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, and a nucleic acid. In some embodiments, said capture probe contains a detectable nucleic acid. In some embodiments, said detectable nucleic acid is amplified.

[0067] In some embodiments, said amplification includes isothermal amplification. In some embodiments, said amplification comprises an isothermal amplification reaction selected from the group consisting of a self-sustaining sequence replication reaction (3SR); 90-I; BAD Amp; cross-priming amplification (CPA); exponential isothermal amplification reactions (EXPAR); isothermal chimeric primer initiated nucleic acid amplification (ICAN); isothermal amplification with multiple displacement (IMDA); mediated by SDA ligation; multiple displacement amplifications; polymerase helical reaction (PSR); exponential restriction cascade amplification (RCEA); smart amplification process (SMAP2); single primer amplification reactions (SPIA); transcription-based amplification systems (TAS); transcription-mediated amplification (TMA); ligase chain reaction (LCR); and cross-displacement amplification (MCDA). In some embodiments, said amplification includes loop isothermal amplification (LAMP).

[0068] In some embodiments, carrying out amplification of the target agent includes heating the sample to a temperature greater than 30°C. In some embodiments, carrying out amplification of the target agent includes heating the sample to a temperature greater than 37°C. In some embodiments, carrying out amplification of the target agent includes heating the sample to a temperature in excess of 60°C. In some embodiments, carrying out amplification of the target agent includes heating the sample to a temperature in the range of 60°C to 70°C.

[0069] In some embodiments, said liquid constituents of the amplification process comprise a component selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, a nucleic acid such as a primer, a buffer, and an enzyme such as a polymerase.

[0070] In some embodiments, said dried amplification process components comprise a component selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, a nucleic acid such as a primer, a buffer, and an enzyme such as a polymerase.

[0071] Some embodiments include a method for detecting a target agent, said method comprising: providing a device comprising an excitation electrode and a sensor electrode; introducing a sample containing the target agent into said device; processing the sample within said device; and target detection by measuring an electrical property of the treated sample.

[0072] In some embodiments, said electrical property is selected from the group consisting of complex admittance, impedance, conductivity, resistivity, resistance, and dielectric constant.

[0073] In some embodiments, said electrical property is a complex admittance.

[0074] In some embodiments, detection includes applying a drive signal to a drive electrode.

[0075] In some embodiments, said drive signal includes alternating current.

[0076] In some embodiments, said drive signal includes direct current.

[0077] In some embodiments, said drive signal includes a voltage and frequency sweep.

[0078] In some embodiments, the detection includes measuring the induced current at the sensor electrode.

[0079] In some embodiments, said electrical property is measured over a period of time.

[0080] In some embodiments, at least one electrode is passivated.

[0081] In some embodiments, said electrode is passivated with a dielectric material.

[0082] In some embodiments, said electrode is passivated with titanium oxide.

[0083] In some embodiments, detection includes bringing the processed sample into contact with a capture probe. In some embodiments, said capture probe comprises a magnetic bead.

[0084] In some embodiments, said capture probe is selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, and a nucleic acid. In some embodiments, said capture probe contains a detectable nucleic acid. In some embodiments, detection comprises amplification of said detectable nucleic acid.

[0085] In some embodiments, said amplification includes isothermal amplification. In some embodiments, said amplification includes loop isothermal amplification (LAMP). In some embodiments, said processed sample contains a low ionic strength solution.

[0086] In some embodiments, ammonium sulfate is absent from said treated sample.

[0087] In some embodiments, detection includes bringing the sample into contact with an agent to enhance changes in the conductivity of a solution containing the sample. In some embodiments, said agent binds to an inorganic pyrophosphate. In some embodiments, said agent is selected from the group consisting of Cd2+-cyclen-coumarin; a Zn2+ complex with a bis(2-pyridylmethyl)amine (DPA) unit; DPA-2Zn2+-phenoxide; acridine-DPA-Zn2+; DPA-Zn2+-pyrene; and the azacrown-Cu2+ complex. In some embodiments, said agent comprises a 2-amino-6-mercapto-7-methylpurine ribonucleoside.

[0088] Some embodiments include a method for detecting a target agent using a frequency-dependent capacitively coupled non-contact conductivity detection device, said method comprising: introducing a sample containing the target agent into said device; processing the sample within said device; and target detection by analyzing the frequency-dependent capacitively coupled non-contact conductivity of the sample.

[0089] In some embodiments, processing includes a step selected from the group consisting of enriching the sample with a target agent, removing non-target material from the sample, lysing the cells, precipitating proteins, and adding a preservative.

[0090] In some embodiments, detection includes bringing the processed sample into contact with a capture probe. In some embodiments, said capture probe is selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, and a nucleic acid. In some embodiments, said capture probe contains a detectable nucleic acid. In some embodiments, detection comprises amplification of said detectable nucleic acid.

[0091] In some embodiments, said amplification includes isothermal amplification. In some embodiments, said amplification comprises an isothermal amplification reaction selected from the group consisting of a self-sustaining sequence replication reaction (3SR); 90-I; BAD Amp; cross-priming amplification (CPA); exponential isothermal amplification reactions (EXPAR); isothermal chimeric primer initiated nucleic acid amplification (ICAN); isothermal amplification with multiple displacement (IMDA); mediated by SDA ligation; multiple displacement amplifications; polymerase helical reaction (PSR); exponential restriction cascade amplification (RCEA); smart amplification process (SMAP2); single primer amplification reactions (SPIA); transcription-based amplification systems (TAS); transcription-mediated amplification (TMA); ligase chain reaction (LCR); and cross-displacement amplification (MCDA). In some embodiments, said amplification includes loop isothermal amplification (LAMP).

[0092] In some embodiments, detection includes bringing the sample into contact with an agent to enhance changes in conductivity of a solution containing the sample. In some embodiments, said agent binds to an inorganic pyrophosphate. In some embodiments, said agent is selected from the group consisting of Cd2+-cyclen-coumarin; a Zn2+ complex with a bis(2-pyridylmethyl)amine (DPA) unit; DPA-2Zn2+-phenoxide; acridine-DPA-Zn2+; DPA-Zn2+-pyrene; and the azacrown-Cu2+ complex. In some embodiments, said agent comprises a 2-amino-6-mercapto-7-methylpurine ribonucleoside.

[0093] In some embodiments, AC is involved in said detection.

[0094] In some embodiments, high frequency alternating current is involved in said detection.

[0095] In some embodiments, DC is involved in said detection.

[0096] Some embodiments include a device for detecting a target agent in a sample, comprising: a chamber capable of containing a liquid sample; a channel with at least one side wall in fluid communication with said chamber and containing one or more nucleic acid amplification reagents; a heater capable of heating said channel; the first electrode in contact with the side wall; a second electrode in contact with the side wall and located at a distance from the first electrode along the channel; and a circuit electrically connected to the first and second electrodes, capable of supplying a current to said first electrode and detecting a current signal received by said second electrode indicative of a target agent.

[0097] In some embodiments, said current is DC.

[0098] In some embodiments, said current is AC.

[0099] In some embodiments, said heater is capable of heating a liquid sample to at least 30°C. In some embodiments, said heater is capable of heating a liquid sample to at least 37°C. In some embodiments, said heater is capable of heating a liquid sample to at least 60°C. In some embodiments, said heater is capable of heating a liquid sample to a temperature in the range of 60°C to 70°C.

[0100] In some embodiments, said channel is formed in the dielectric substrate and the heater is located adjacent to the channel.

[0101] In some embodiments, said device is configured to be electronically and mechanically coupled to a companion device.

[0102] According to some embodiments, said companion device is a consumer product, including a processor, memory, user graphic display.

[0103] In some embodiments, said associated device is selected from the group consisting of a smartphone, tablet device, laptop, and smart watch.

[0104] In some embodiments, said one or more nucleic acid amplification reagents comprise a primer and a polymerase.

[0105] In some embodiments, the one or more nucleic acid amplification reagents comprise an agent to enhance changes in the conductivity of the solution containing the sample. In some embodiments, said agent binds to an inorganic pyrophosphate. In some embodiments, said agent is selected from the group consisting of Cd2+-cyclen-coumarin; a Zn2+ complex with a bis(2-pyridylmethyl)amine (DPA) unit; DPA-2Zn2+-phenoxide; acridine-DPA-Zn2+; DPA-Zn2+-pyrene; and the azacrown-Cu2+ complex. In some embodiments, said agent comprises a 2-amino-6-mercapto-7-methylpurine ribonucleoside.

[0106] Some of the embodiments described above include a device, a durable computer-readable medium, or a method, characterized in that the target agent is selected from the group consisting of a viral nucleic acid, a viral capsid protein, a viral structural protein, a viral glycoprotein, a viral membrane fusion protein, viral protease and viral polymerase.

[0107] In some embodiments, the virus contains a target agent.

[0108] In some embodiments, said virus is selected from the group consisting of double-stranded DNA virus, single-stranded DNA virus, double-stranded RNA virus, single-stranded (+) RNA virus, single-stranded (-) RNA virus, single-stranded reverse transcribed RNA virus and double-stranded reverse transcribed DNA virus.

[0109] In some embodiments, said virus is selected from the group consisting of adeno-associated virus, Aichi virus, Australian bat lyssavirus, VK polyomavirus, Bann virus, Barma forest virus, Bunyamwera virus, La Crosse bunyavirus, American hare bunyavirus, marmoset herpesvirus, Chandipur virus, Chikungunya virus, Cosavirus A, Vaccinia virus, Coxsackie virus, Crimean-Congo hemorrhagic fever virus, Dengue fever virus, Dhori virus, Dagbe virus, Duvenhage virus, Oriental equine encephalitis virus, Ebolavirus, Echovirus, Encephalomyocarditis virus, Epstein virus -Barr, European bat Lyssavirus, GBV-C/Hepatitis G, Hantaan virus, Hendra virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Hepatitis delta virus, Equine pox virus, Human adenovirus, Astrovirus human, human coronavirus, human cytomegalovirus, human enterovirus 68, 70, herpesvir human ca 1, human herpesvirus 2, human herpesvirus 6, human herpesvirus 7, human herpesvirus 8, human immunodeficiency virus, human papillomavirus 1, human papillomavirus 2, human papillomavirus 16, 18, human parainfluenza, human parvovirus B19, human respiratory syncytial virus , human rhinovirus, human SARS coronavirus, human spumaretrovirus, human T-lymphotropic virus, human torovirus, influenza A virus, influenza B virus, influenza C virus, Isfahan virus, polyomavirus JC, Japanese encephalitis virus, Junin arenavirus, polyomavirus KI, polyomavirus Kunjin , Lagos Island Bat Virus, Lake Victoria Marburg Virus, Langat Virus, Lassa Virus, Lordsdale Virus, Twirl Virus, Lymphocytic Choriomeningitis Virus, Machupo Virus, Mayaro Virus, Middle East Respiratory Syndrome Coronavirus (MERS), Measles Virus, Mengo Encephaloamyocarditis Virus, Polyomavirus Merkel cells, Mokola virus, contagio virus Molluscum Virus, Monkeypox Virus, Mumps Virus, Murray Valley Encephalitis Virus, New York Virus, Nipah Virus, Norwalk Virus, O'Nyong Nyong Virus, Pustular Dermatitis Contagious Virus, Oropush Virus, Pichinde Virus, Poliovirus, Punta Phlebovirus Toro, Puumala virus, Rabies virus, Rift Valley fever virus, Rosavirus A, Ross river virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, Sagiyama virus, Salivirus A, Sicilian phlebotomic fever virus serotype, Sapporo virus, Forest virus Semliki virus, Seoul virus, Monkey foam virus, Monkey virus 5, Sindbis virus, Southampton virus, St. Louis encephalitis virus, Powassan tick-borne encephalitis virus, TTV hepatitis virus, Tuscan fever virus, Uukuniemi virus, vaccinia virus, varicella-zoster virus, natural virus smallpox, Venezuelan equine encephalitis virus, vesicular stomatitis virus, western equine encephalitis virus, WU polyomavirus, Zapa virus Nile Bottom, Yab Monkey Tumor Virus, Yab-like Disease Virus, Yellow Fever Virus and Zika Virus.

[0110] Some of the embodiments described above include a device, durable computer-readable medium, or method, characterized in that the target agent is selected from the group consisting of a bacterial nucleic acid, a bacterial protein, and a bacterial toxin.

[0111] In some embodiments, the bacterium contains the target agent.

[0112] In some embodiments, said bacterium is selected from the group consisting of a Gram-positive bacterium or a Gram-negative bacterium.

[0113] In some embodiments, said bacterium is selected from the group consisting of Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli, Salmonybacter phimurtyphi, , Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia, Providencia alcalifaciens stuartii, Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilu s influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Branhamella catarrhalis, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes , Neisseria gonorrhoeae, Neisseria meningitidis, Kingella, Moraxella, Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis, группы гомологии Bacteroides 3452A, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, Bacteroides splanchnicus, Clostridium difficile, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium leprae, Corynebacterium diphtheriae, Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faec ium, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus hyicus subspecies hyicus, Staphylococcus haemolyticus, Staphylococcus hominis and Staphylococcus saccharolyticus.

[0114] Some of the embodiments described above include a device, a durable computer-readable medium, or a method, characterized in that the target agent is selected from the group consisting of a protein, a polypeptide, a nucleic acid, a small molecule, and a pharmaceutical compound.

[0115] Some of the embodiments described above include a device, durable computer-readable medium, or method, characterized in that the parasite contains a target agent.

[0116] In some embodiments, said parasite is selected from the group consisting of an endoparasite and an ectoparasite.

[0117] In some embodiments, said parasite is selected from the group consisting of protozoa, helminths, trematodes, and roundworms.

[0118] In some embodiments, said endoparasite is selected from the group consisting of (spp.) Acanthamoeba, Babesia spp., B. divergens, B. bigemina, B. equi, B. microfti, B. duncani, Balamuthia mandrillaris, Balantidium coli, Blastocystis spp., Cryptosporidium spp., Cyclospora cayetanensis, Dientamoeba fragilis, Entamoeba histolytica, Giardia lamblia, Isospora belli, Leishmania spp. knowlesi, Rhinosporidium seeberi, Sarcocystis bovihominis, Sarcocystis suihominis, Toxoplasma gondii, Trichomonas vaginalis, Trypanosoma brucei, Trypanosoma cruzi, Bertiella mucronata, Bertiella studeri, tapeworms (Cestoda), Taenia multiceps, Dilobothrium latum, Echinococcularis locus, multivocus. , E. oligarthrus, Hymenolepis nana, Hymenolepis diminuta, Spirometra erinaceieuropaei, Taenia saginata, Taenia solium, Clonorchis sinensis; Clonorchis viverrini, Dicrocoelium dendriticum, Echinostoma echinatum, Fasciola hepatica, Fasciola gigantica, Fasciolopsis buski, Gnathostoma spinigerum, Gnathostoma hispidum, Metagonimus yokogawai, Metorchis conjunctus, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Paragonimus westermani, Paragonimus africanus, Paragonimus caliensis, Paragonimus kellicotti , Paragonimus skrjabini; Paragonimus uterobilateralis, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni and Schistosoma intercalatum, Schistosoma mekongi, Schistosoma sp., Trichobilharzia regenti, Schistosomatidae, Ancylostoma duodenale, Necator americanus, Angiostrongylus costaricensis, Anisakis, Ascaris sp. , Brugia timori, Dioctophyme renale, Dracunculus medinensis, Enterobius vermicularis, Enterobius gregorii, Halicephalobus gingivalis, causative agent of loiasis, Mansonella streptocerca, Onchocerca volvulus, Strongyloides stercoralis, Thelazia californiensis, Thelazia callipaeda, Toxocara canis, Toxocarais, Trichinella spiralto, Trichinella brichinto nelsoni, Trichinella nativa, Trichuris trichiura, Trichuris vulpis, Wuchereria bancrofti, Archiacanthocephala, Moniliformis moniliformis, Linguatula serrata, Oestroidea, Calliphoridae, Sarcophagidae, Cochliomyia hominivorax (family Calliphoridae), Tunga penetrans, Cimicidae : Cimex lectularius and Dermatobia hominis.

[0119] In some embodiments, said parasite is an ectoparasite selected from the group consisting of Pediculus humanus, Pediculus humanus corporis, Pthirus pubis, Demodex folliculorum, Demodex brevis, Demodex canis, Sarcoptes scabiei, Trombiculidae ticks, Pulex irritans, ixodid ticks ( Ixodidae) and argas mites (Argasidae).

[0120] Some of the embodiments described above include a device, a durable computer-readable medium, or a method, characterized in that the microRNA contains a target agent.

[0121] In some embodiments, said miRNA is mammalian. In some embodiments, said miRNA is human-derived.

[0122] In some embodiments, said miRNA is selected from the group consisting of 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, hsa-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, hsa-miR-140-3p, hsa-miR-141, hsa-miR-14 2-3p, 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-17-5p, hsa-miR-17-92, hsa-miR-18, hsa-miR-18a, hsa-miR-18b, hsa-miR-181a, hsa-miR-181a-1, hsa-miR- 181a-2, hsa-miR-181b, 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-miR-199a-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-299-3р, hsa-miR-299-5p, hsa-miR-29a, hsa-miR-29a-2, hsa-miR-29b, hsa-miR-29b-1, 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-30е, 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, 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, hs a-miR-429, hsa-miR-431, hsa-miR-432, hsa-miR-433, hsa-miR-449a, hsa-miR-451, hsa-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-miR-512-5p, 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-miR-563, 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-654, 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-9-3р, 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-99b.

[0123] Some of the embodiments described above include a device, a durable computer-readable medium, or a method, characterized in that the agricultural analyte contains the target agent.

[0124] In some embodiments, said agricultural analyte is indicative of the source of the food product. In some embodiments, said agricultural analyte is indicative of an animal source of the food product. In some embodiments, said agricultural analyte indicates the genus of the animal source. In some embodiments, said agricultural analyte is indicative of a plant source of the food product. In some embodiments, said agricultural analyte indicates the genus of the plant source.

[0125] In some embodiments, said agricultural analyte is a pesticide. In some embodiments, said agricultural analyte is a pesticide selected from the group consisting of a herbicide, an insecticide, and a fungicide. In some embodiments, said agricultural analyte is a herbicide selected from the group consisting of 2,4-dichlorophenoxyacetic acid (2,4-D), atrazine, glyphosate, mecoprop, dicamba, paraquat, glufosinate, metam sodium, dazomet, ditopyr, pendimethalin, EPTC, trifluralin, flazasulfuron, metsulfuron-methyl, diuron, nitrofen, nitrofluorfen, acifluorfen, mesotrione, sulcotrione, and nitisinone. In some embodiments, said agricultural analyte is an insecticide selected from the group consisting of an organochlorine compound, an organophosphorous compound, a carbamate, a pyrethroid, a neonicontinoid, and a ryanoid. In some embodiments, said agricultural analyte is a fungicide selected from the group consisting of carbendazim, diethofencarb, azoxystrobin, metalaxyl, metalaxyl-m, streptomycin, oxytetracycline, chlorothalonil, tebuconazole, cineb, mancozeb, tebuconazole, myclobutanil, triadimefon, fenbuconazole, deoxynivalenol and mancozeb.

[0126] Some of the embodiments described above include a device, a durable computer-readable medium, or a method, characterized in that the biomarker of the disorder contains a target agent. In some embodiments, said disorder is cancer. In some embodiments, said cancer is selected from breast cancer, colorectal cancer, gastric cancer, gastrointestinal stromal tumors, leukemias and lymphomas, lung cancer, melanomas, brain cancer, and pancreatic cancer. In some embodiments, said biomarker is selected from estrogen receptor, progesterone receptor, HER-2/neu, rEGFR, KRAS, UGT1A1, C-KIT, CD20, CD30, FIP1L1-rTRF-alpha, rTRF, Philadelphia chromosome (BCR/ABL), PML/RAR-alpha, TPMT, UGT1A1, EML4/ALK, BRAF and elevated levels of certain amino acids such as leucine, isoleucine and valine.

[0127] Some of the embodiments described above include an apparatus, a durable computer-readable medium, or a method, characterized in that said sample originates from a bird.

[0128] Some of the embodiments described above include a device, a durable computer-readable medium, or a method, characterized in that said sample is from a mammal.

[0129] Some of the embodiments described above include a device, a durable computer-readable medium, or a method, characterized in that said sample originates from a human.

[0130] Some of the embodiments described above include a device, durable computer-readable medium, or method, characterized in that said sample is selected from the group consisting of blood, serum, plasma, urine, saliva, ascitic fluid, cerebrospinal fluid, semen, lung fluid lavage, sputum, mucus, mucosal secretions, liquid media containing cells or nucleic acids, solid media containing cells or nucleic acids, and tissues.

[0131] Some of the embodiments described above include an apparatus, a durable computer-readable medium, or a method, characterized in that said sample is obtained by performing a step selected from finger prick, heel prick, venipuncture, adult nasal aspirate, pediatric nasal aspirate , washing the nasopharynx, taking aspirate from the nasopharynx, smear, collecting the contents of the large intestine in a container, tissue biopsy and lavage.

[0132] Some of the embodiments described above include an apparatus, a durable computer-readable medium, or a method, characterized in that said sample is a plant sample.

[0133] Some of the embodiments described above include an apparatus, durable computer-readable medium, or method, characterized in that said sample is an environmental sample.

[0134] Some of the embodiments described above include an apparatus, durable computer-readable medium, or method, characterized in that said sample is a soil sample or a water sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0135] In FIG. 1A-1D depict an example of a target detection cartridge.

[0136] FIG. 2 shows another example of a target detection cartridge.

[0137] FIG. 3A and 3B show another example of a target detection cartridge.

[0138] In FIG. 4A-4G depict various examples of electrodes that may be used in the test cell of the cartridges of FIG. 1A-3B or in the test cell or channel of another suitable target detection cartridge as described herein.

[0139] FIG. 5A shows the first electrode, or excitation electrode; and a second electrode, or signal electrode, which may be spaced apart within the test cell of the cartridges of FIG. 1A-3B, or in the test cell or channel of another suitable target detection cartridge as described herein.

[0140] In FIG. 5B shows an example of a signal that can be extracted from the signal electrode of FIG. 5A.

[0141] In FIG. 5C shows the resistance component and the reactive component extracted from the signal shown in FIG. 5B generated from the positive test example.

[0142] In FIG. 5D shows the resistance component and the reactive component extracted from the signals shown in FIG. 5B, from test examples for positive and negative controls.

[0143] In FIG. 5E shows the resistance component and the reactive component extracted from the signal shown in FIG. 5B generated from another example of a positive test.

[0144] In FIG. 6 is a schematic block diagram of an example reader that can be used with the cartridges described herein.

[0145] In FIG. 7A is a flow chart of an example of the operation of a reader during a test as described in this document.

[0146] FIG. 7B is a flow chart of an example process for analyzing test data for target detection as described herein.

[0147] In FIG. 8 is a diagram of an immunoassay with amplification.

[0148] In FIG. 9 is a diagram of an immunoassay with bead-based amplification.

[0149] In FIG. 10 is a diagram of an immunoassay with magnetic bead amplification.

[0150] In FIG. 11 shows the first electrode, or excitation electrode; and a second electrode, or signal electrode, which may be spaced apart along the channel.

[0151] In FIG. 12 is a graph showing that the impedance signal depends on the drive frequency and changes after the LAMP reaction occurs in the channel, where the inequality on the left can determine the frequency domain.

[0152] In FIG. 13 is a graph showing that in both extreme regions the impedance is capacitive and out of phase (approaching 90°) with the drive voltage.

[0153] FIG. 14 is a graph showing the measured sample chip impedance versus drive frequency.

[0154] In FIG. 15 is a graph showing the response of a synchronous detector as a function of dimensionless conductivity.

[0155] In FIG. 16 is a graph showing the results of the model showing agreement with detector output over a wide range of conductivities and for given frequency steps.

[0156] In FIG. 17A and in FIG. 17B depicts an embodiment of a detection system that can be used to detect the presence or absence of a particular nucleic acid and/or a particular nucleotide in a sample. In FIG. 17A is a plan view of this system, and FIG. 17B is a cross-sectional elevational view of this system.

[0157] FIG. 18 is a process flow chart illustrating an embodiment of a target detection device.

[0158] FIG. 19 is a process flow chart illustrating an embodiment of a target detection device.

[0159] FIG. 20 shows an example of a fluid cartridge.

[0160] In FIG. 21 is a plan view of an example of the fluid cartridge of FIG. 20.

[0161] In FIG. 22 shows an example of an electrode configuration.

[0162] In FIG. 23 shows an example of a channel.

[0163] FIG. 24 is a graph showing sensor voltage over time before amplification (- control) and after amplification (+ control).

[0164] FIG. 25 is a graph showing sensor voltage over time before amplification (- control) and after amplification (+ control) for 0% whole blood.

[0165] In FIG. 26 is a graph showing sensor voltage over time before amplification (- control) and after amplification (+ control) for 1% whole blood.

[0166] In FIG. 27 is a graph showing sensor voltage over time before amplification (- control) and after amplification (+ control) for 5% whole blood.

[0167] FIG. 28 is a graph showing sensor voltage over time before amplification (- control) and after amplification (+ control) for 0% whole blood from an unfiltered sample.

[0168] FIG. 29 is a graph showing sensor voltage over time before amplification (- control) and after amplification (+ control) for 0% whole blood from a filtered sample.

[0169] FIG. 30 is a plot of time versus target load; error bars show the standard deviation.

[0170] In FIG. 31 is a graph of conductivity for various samples from the pre-amplification vessel (- control) and the post-amplification vessel (+ control).

[0171] In FIG. 32 is a diagram of an immunoassay with magnetic bead amplification for the detection of hepatitis B virus antigen (HBsAg).

[0172] In FIG. 33 is a graph illustrating the detection of HBsAg.

[0173] In FIG. 34 is a graph illustrating the detection of HBsAg with low ion buffer (T10).

[0174] FIG. 35 is a graph illustrating the impedance characteristics of a fluid cartridge.

[0175] FIG. 36A is a graph of out-of-phase signals for LAMP performed on a cartridge at 65°C.

[0176] FIG. 36B is a plot of in-phase signals for LAMP performed on a cartridge at 65°C.

[0177] FIG. 36C is a graph of out-of-phase signals for LAMP performed on a cartridge at 67°C.

[0178] FIG. 36D is a plot of in-phase signals for LAMP performed on a cartridge at 67°C.

[0179] FIG. 36E is a graph of out-of-phase signals for LAMP performed on a cartridge at 67°C.

[0180] In FIG. 36F is a plot of in-phase signals for LAMP performed on a cartridge at 67°C.

DETAILED DESCRIPTION OF THE INVENTION

[0181] Aspects of the invention described herein relate to the use of amplification and non-contact electrical sensor detection to detect the presence of a target in a sample. Such a diagnostic platform is capable of replacing the complex optical systems and expensive fluorescent labels used for optical detection, and the electrodes and electroactive agents used for existing electrochemical and FET techniques, with conventional electronic components. In some aspects, the amplification may be isothermal. The platform described in this document is inexpensive, reliable, portable and consumes less power than traditional diagnostic systems. In some aspects, said diagnostic platform is small enough to fit in the palm of a user and capable of operating in the field, such as for in-office diagnostics, at home, at a location remote from a medical facility.

[0182] Many commercially available nucleic acid detection platforms employ conventional PCR, respectively requiring temperature cycling, fluorescent labels, and optical detection instrumentation. These factors lead to the emergence of expensive laboratory instruments that use thin vibration-sensitive detectors, expensive fluorescent markers, and leave a significant environmental footprint. This equipment requires operation and frequent calibration by highly qualified personnel.

[0183] These large, bulky platforms make the routine use of conventional nucleic acid testing (NAT) difficult to use in the clinic, and even more so at home. NAT remains an expensive and slow strategy, heavily tied to centralized lab equipment. At the same time, the technology proposed in this document allows avoiding these difficulties.

[0184] A problem in point-of-care ("POC") testing is the potential inhibition of amplification by interferences often found in unpurified and untreated clinical specimens, such as whole blood or mucosal secretions. Minimizing exposure to amplification inhibitors can make it difficult to directly detect target nucleic acids in clinically relevant biological samples.

[0185] Conventional detection strategies are typically based on fluorescent detection techniques. Such techniques can be complex, more expensive and require precision optical systems. However, the present invention generally relies on electrical detection systems. Such electrical detection systems may use microelectronics that consume relatively little power and can be manufactured at lower cost through high volume production. Thus, the electrical detection of genomic material may allow the advances of the computer industry to be transferred to sensory bioanalysis.

[0186] Existing electronic methods for monitoring amplification may require the binding of an electrochemically active label or the selective binding of the amplified material to a surface. However, in clinical use in real conditions, these methods often have disadvantages in the form of a significant response time, biological contamination of the electrode or bonding surfaces, which leads to unsatisfactory signal-to-noise ratios, as well as limitations on the service life and reliability of the device. Potentially providing high sensitivity, the use of electrochemical detection or FET-based detection increases the level of detection complexity. This can lead to more cost and less robust strategies than is typically required for POC and other user applications. Accordingly, the need for additional diagnostic devices is obvious.

[0187] The platform proposed herein is based on measuring the change in electrical conductivity that occurs during amplification of nucleic acids. Briefly, during the biochemical synthesis of DNA from nucleotide triphosphates, the number and mobility of electrically charged molecules change. This, in turn, leads to a change in the conductivity of the solution as the amplification progresses. Said change in the electrical conductivity of the solution can be detected using the detection method of frequency-dependent capacitive-coupled non-contact conductivity ("ƒC ⁴ D").

[0188] In some embodiments, for ƒC ⁴ D, a pair of electrodes located directly adjacent, but not in contact with the fluid in the amplification chamber, is used to measure the electrical properties of the solution. The ability to measure solution properties in this way, without direct contact, avoids the surface contamination problems associated with other electrical measurement methods.

[0189] In some embodiments where ƒC ⁴ D is used, a high frequency AC signal ("AC") is applied to the drive electrode. Said signal is a capacitively coupled signal through the solution where it is detected at the signal electrode. By comparing the drive signal with the signal at the signal electrode, the conductivity of the solution can be determined.

[0190] When using these high resolution finite element models and empirical studies, specific tolerances for technology based on ƒC ⁴ D can achieve optimal detection sensitivity and optimal dynamic range of detection for specific implementations of this platform. Such calculated and empirically determined microfluidic sizing parameters, capacitive coupling characteristics, and applied frequency may allow parameters to be determined that are effective in detecting changes in fluid conductivity. In some embodiments, the parameters corresponding to optimal detection may be interdependent variables. According to the following equation, the measured impedance is a function of solution resistance, capacitance, and applied frequency:

Z = R - (1/pi*f*C)*j

[0191] As the thickness of the passivating layer of the electrode increases, the parasitic capacitance due to this layer increases accordingly. Therefore, the optimal FET frequency for measuring the conductivity of a solution using ƒC ⁴ D can be chosen taking into account the capacitance of the passivation layer.

Overview of Cartridge, Reader and Signal Processing Examples

[0192] In some aspects, the system for detecting a target in a sample includes a removable fluid cartridge that can be paired with an accompanying reader. The user can enter the sample into the cartridge and then insert it into the reader. Said reader is configured to perform test procedures using said cartridge and analyze test data to determine the presence, absence, or amount of a target in a sample. For example, said cartridge may be provided with the required agents, proteins, or other chemical for an amplification process that amplifies a target originally present in a sample. In particular, some cartridges may be provided with the required nucleic acid testing chemical, wherein the genomic material in the sample is exponentially replicated using the molecular amplification process as described herein. The specified cartridge may also include a test cell in which the amplification process takes place, while the test cell refers to a cell, chamber, channel or other geometric shape configured to contain (or essentially contain) the fluid under study and the components of the amplification process. The reader may maintain the desired temperature or other test environment for said cartridge to facilitate the amplification process, and may provide electronic monitoring of the test cell of the cartridge during some or all of the amplification process. Said reader may accordingly collect signal data corresponding to the impedance of the test cell over the course of the amplification process, and may analyze said impedance as described herein to confirm the presence, absence, or amount of the target in the sample. For example, the duration of the amplification process may vary from five minutes to 60 minutes, with some examples ranging from ten minutes to thirty minutes. Preferably, in some embodiments, the amplification products are detected in fluid suspension within the wells, such that there is no attachment or sequestration of the amplification products in the wells, or fixation or binding to the probes that are bound in the wells. In other embodiments, amplification products are detected when they are attached or sequestered in the wells, such as by fixation or binding to probes that are bound in the wells.

[0193] Such systems can advantageously provide target detection that is feasible in a clinical setting, or even at the user's home, without the need to send the sample to a laboratory for amplification and analysis. In a clinical setting, this can ensure that there are no delays in routine nucleic acid testing, thus allowing clinicians to determine a diagnosis within a typical patient visit to the facility. Accordingly, the proposed systems allow clinicians to develop treatment plans for patients during their initial visit to a facility without requiring the clinician to wait hours or even days for test results from the laboratory. For example, when a patient visits a clinic, a nurse or other healthcare professional may take a sample from the patient and begin testing using the described system. This system can provide a test result at the time of the patient's consultation with a doctor or clinician to determine a treatment plan. In particular, when used to diagnose pathologies that progress rapidly, the proposed systems can avoid delays associated with laboratory tests that can negatively affect treatment and patient outcome.

[0194] Another advantage of the proposed systems is that they can be used outside of clinical settings (e.g., in the field, in rural areas without easy access to an organized medical clinic) to detect health conditions such as infectious diseases (e.g., Ebola), respectively allowing suitable personnel to take immediate action to prevent or reduce the spread of a contagious disease. Similarly, the proposed systems can be applied in the field or at the site of a suspected presence of a hazardous contaminant (e.g., anthrax) to quickly determine if a sample contains a hazardous contaminant, respectively, allowing appropriate personnel to take immediate action to prevent or mitigate exposure to the person of the specified polluting agent. In addition, the proposed systems can be used to detect contaminants in blood or plasma supplies, or in the food industry. It should be understood that the proposed systems may provide similar benefits in other scenarios where real-time target detection provides more effective action than delayed detection when the sample is sent to an external laboratory.

[0195] Another advantage of such systems is the use of inexpensive disposable cartridges for single use along with a reusable reader that can be used many times with different cartridges and/or for tests on different targets.

[0196] FIG. 1A-1D depict an example cartridge 100 configured for target detection. As described herein, said target may be a viral target, a bacterial target, an antigenic target, a parasitic target, a microRNA target, or an agricultural analyte. Some embodiments of cartridge 100 may be configured to test a single target, while some embodiments of cartridge 100 may be configured to test multiple targets.

[0197] FIG. 1A depicts a cartridge 100 with a cap 105 over base 125. In use, cap 105 can function to seal the provided sample in cartridge 100, thereby preventing test operators from exposing the sample and preventing any liquid from entering the electronics of the associated reader. Lid 105 may be permanently attached to base 125 or may be removable according to certain embodiments. Lid 105 may be formed from suitable materials such as plastic and may be opaque, as shown, or in other examples, may be translucent or transparent.

[0198] The lid 105 includes an opening 115 located above the sample entry area 120 of the base 125. "Above" herein refers to the opening 115 located above the sample entry area 120 when viewed from top to bottom with the cartridge 100 at right angles to the flat surface of the cover 105 including opening 115. Cap 105 also includes a cap 110 configured to seal aperture 115 with fluid before and after sample is introduced through aperture 115. Cap 110 includes a cylindrical protrusion 111 that plugs aperture 115 while sealing cap 110 with opening 115, a trigger latch 113 configured to facilitate removal by a user of cap 110 from opening 115 while sealing cap 110 with opening 115; and a hinge 112 configured to allow retraction of the cap 110 from the opening 115 and from the sample path while maintaining the attachment of the cap 110 to the cap 105. It should be understood that other forms of the cap 110 can similarly be used to achieve sealing of the opening 115; also, in some embodiments, said hinge 112 and/or trigger lock 113 may be modified or omitted. In the illustrated embodiment, cap 105 and cap 110 are integrally formed from a single piece of material, however, in other embodiments, cap 110 may be a separate structure from cap 105.

[0199] In use, the user opens the cap 110 and introduces a sample potentially containing a target or targets into the specified sample entry area 120 of the base 125 through the opening 115 in the lid. For example, the user may prick a finger and introduce a whole blood sample into the sample entry area 120, such as through a capillary. Cartridge 100 may be configured to accept any one or more of liquid, semi-solid, and solid sample types. After the sample has been introduced, the user may close the cap 110 to seal the opening 115. Preferably, sealing the inlet to the base 125 flow path allows the sample (and other fluids) to move through the base 125 flow path into the test cell. For example, a user may insert a sealed cartridge 100 containing a sample into a reader as described herein, and said reader may activate an optional pneumatic interface to move the sample into the test cell. The flow channel and test cell are described in more detail with reference to FIG. 1B and 1C, and an example reader is described with reference to FIG. 6.

[0200] The lid 105 also includes a notch 130 for exposing the electrode interface 135 of the base 125, described in more detail below. In some embodiments, cover 105 may include a movable shutter or removable shroud to protect electrode interface 135 prior to use.

[0201] In FIG. 1B shows the cartridge 100 of FIG. 1A with the cover removed to show features of base 125. Base 125 may be formed from a fluid impervious material such as cast or milled acrylic or plastic. The base 125 includes a sample entry area 120, a blister pack 140, a pneumatic interface 160, a testing area 170A including test cells 175, and a flow path 150 configured to mix the applied sample with the fluids contained in the blister pack 140, and transferring said mixed liquid to test cells 175. It should be understood that, according to other embodiments, the specific geometries or relative positions of said features may vary.

[0202] The blister pack 140 includes a film, such as a thermoformed plastic, forming a sealed chamber containing fluids for mixing with the applied sample. Said liquids may include amplification reagents, buffer solutions, water, or other required liquid constituents for the testing process. The specific selection and chemical properties of these liquids may be individualized for the particular target or targets for which cartridge 100 is designed to test. Some embodiments of blister pack 140 may further 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 comprising a pneumatic fluid passage 161 leading into the chamber and an opening 141 leading out of the chamber into the flow passage 150. For example, to secure the blister pack 140 a ring of pressure-sensitive adhesive may be applied in situ along the outer edge of one or both surfaces of the blister pack 140.

[0203] In use, a user or reader may mechanically actuate a point (e.g., a needle or other pointed body) to pierce the blister pack 140 and release its liquid contents through the opening 141 into the first segment 151 of the flow channel 150. The point may be included into a cartridge 100, for example, is localized in a chamber containing a blister pack 140, said chamber being fluidly connected to said first flow channel segment 151. As used herein, fluid connection refers to the ability to carry fluids (eg, liquefied gas). According to another embodiment, said user or reader may press the bottom surface of the blister pack 140 (although the bottom surface is not shown in the illustrations, it is opposite the surface visible in Fig. 1B), pushing it up on the point and piercing the blister pack 140. According to in other embodiments, the tip may be omitted and the user or reader may squeeze the blister pack 140 until the pressure of the liquid content causes the blister pack 140 to burst. provide mechanically openable chambers configured to similarly release internally contained liquids into the first segment 151 of the flow channel 150.

[0204] As described above, after sample application, the user seals the cap opening 115, thereby sealing the flow path 150 within the cartridge 100. Pneumatic interface 160 is configured to supply fluid, such as air, to the sealed flow path 150 through the chamber blister pack, facilitating fluid flow in the desired direction along flow path 150 into test cells 175. Pneumatic interface 160 may be an orifice leading to a pneumatic fluid path and fluidly coupled to a pneumatically controlled flow path 161 which, in turn, in turn leads into the blister pack 140 or the chamber containing the blister pack 140 and is fluidly connected to it. In some embodiments, said pneumatic interface 160 may be a compressible one-way valve that forces air from the environment into the pneumatic fluid passage 161 when compressed and draws air from the environment when decompressed. In such embodiments, repeated compression of the pneumatic interface 160 may force the fluid in said cartridge along the flow path.

[0205] The flow channel 150 includes segments 151, 152, 153, 154, 155, and 156, as well as a sample entry area 120, a test cell 175, a test cell inlet path 176, and a test cell outlet path 177. The first segment 151 of the flow channel 150 leads from the blister pack 140 to the sample entry area 120. The second segment 152 of the flow channel 150 leads from the sample entry area 120 to the mixing chamber 153. The mixing chamber 153 is the third segment of the flow channel 150, which is extended compared to the second segment 152 and the fourth segment 154 The fourth segment 154 of the flow channel 150 leads from the mixing chamber 153 to the fifth segment 155 of the flow channel. The fifth segment 155 of the flow channel 150 is formed in the test area 170A. The fifth segment 155 of the flow channel 150 leads simultaneously into the inlet path of the first test cell 176 and into the sixth segments 156 of the flow channel 150. Each of the sixth segments 156 of the flow channel 150 forms an extension of the flow channel 150 between adjacent inlets of the test cells until the last inlet of the test cell 176. test cell path 176 fluidly connects test cell 175 to flow path 150, and may be closed by valve 174, for example, to prevent cross-amplification between test cells. Test cell outlet 177 leads from test cell 175 to outlet 178 which allows gas to exit test cell 175 and out of cartridge 100.

[0206] Uniform or homogeneous mixing of the liquid from the sample loaded blister pack 140 may produce more accurate test results in some embodiments. Accordingly, the mixing chamber 153 is configured to promote uniform mixing of the liquid from the sample loaded blister pack 140, for example, by including curved regions and/or a cross-sectional shape that promotes turbulent rather than laminar flow of liquids within the mixing chamber 153. Turbulent flow is a flow regime in fluid dynamics characterized by chaotic changes in pressure and fluid flow rate. Turbulent flow is different from laminar flow, which occurs when fluid flows in parallel layers, with no breaks between said layers.

[0207] The segments 151, 152, 153, 154 of the flow channel 150 may be completely enclosed within the base material 125, or may include three surfaces formed from the base material 125, where the cover 105 forms the top surface that seals said channels. The segments 155, 156 of the flow channel 150, the test cell inlet path 176, and the test cell outlet path 177 may be completely enclosed within the base material 125, may include three surfaces formed from the base material 125, where the cover 105 forms a top surface that seals these features. , or may include two surfaces formed from base material 125, where circuit board 179 forms the bottom surface of said features and cover 105 forms the top surface of said features.

[0208] FIG. 1C illustrates the direction of flow along the flow channel 150, where circled numbers indicate certain points along the flow channel. The circled numbers are discussed below as examples of steps to advance fluid 180 as it moves through flow channel 150 within cartridge 100, with each step marked with an arrow indicating the direction of fluid flow in that step.

[0209] Before step (1), the user inserts a sample into the sample input area 120. For clarity and simplicity, in FIG. 1C does not label the components labeled with reference numbers in FIG. 1B. Also prior to step (1), the blister pack 140 is ruptured so that its liquid contents are released from the previously sealed chamber.

[0210] In step (1), the air or other fluid flowing out of the pneumatic interface 160 moves in the illustrated direction along the pneumatically operated flow channel 161 towards the ruptured blister pack 140.

[0211] In step (2), the liquid released from the ruptured blister pack 140 (herein referred to as the "master mix") moves through the opening 141 in the direction shown in the illustration to the first segment 151 of the flow channel 150. The master mix continues to flow along the first segment 151 until step (3) when it enters the sample entry area 120 and begins to carry the sample further along the flow channel.

[0212] In step (4), the master mix and sample leave the sample entry area 120 and flow along the second segment 152 of the flow channel 150 in the direction shown in the illustration. The volume of the master mix can be pre-selected to completely or substantially completely wash out the applied sample from the sample entry area 120 and/or to at least fill the test cells 175 and their respective inlet paths 176.

[0213] In step (5), the master mix and sample flow in the direction shown in the illustration to the inlet of the wider third segment 153 of the flow channel 150, and in step (6), the master mix and sample are mixed into a homogeneous solution where the sample is uniformly distributed in the master mix. As described above, the third segment 153 includes curved segments and a flat mixing chamber configured to facilitate mixing of the master mix and sample. In some embodiments, the fluid velocity provided by pneumatic interface 160 may be selected to further assist in said mixing.

[0214] In step (7), the mixed master mix and sample (referred to as "test fluid") leave the mixing chamber 153 and enter the fourth segment 154 of the flow channel 150, which leads to the testing area 170A.

[0215] In step (8), the test fluid moves along the fifth segment 155 of the flow channel 150 in the illustrated direction through the test area 170A towards the test cells 175.

[0216] In step (9), the test fluid reaches the inlet path of the first test cell 176, and its flow is directed along three possible paths, shown by three branches extending from the flow channel arrow of step (9).

[0217] The path in step (10) corresponds to the flow of the test fluid further along the segment 156 of the flow channel 150 into the inlet paths of the next test cells 176. Optionally, the valve 174 in the inlet path of the test cell 176 can be closed, which prevents the flow of the test fluid to step (10).

[0218] The path in step (11) corresponds to optionally flowing the gas portion of the test fluid through valve 174. In some embodiments, said valve 174 may include a liquid-tight gas permeable filter to allow any gas present in the test fluid to be vented through valve 174 before entering test cell 175. In some embodiments, said valve 174 may not necessarily be configured to vent gas.

[0219] The path in step (12) corresponds to the direction of flow of test fluid into test cell 175. In some embodiments, valve 174 may be closed to seal test cell 175 in the event of a predetermined trigger signal. Said trigger signal may occur after a predetermined volume of fluid corresponding to the volume of at least test cell 175 (and additionally inlet and outlet paths 176, 177) has flowed along the path in step (12). Another example of a valve-closing start signal may occur after a predetermined period of time has elapsed, corresponding to the time for which the specified volume of fluid is expected to flow along the path in step (12). In another embodiment, said trigger signal may cause the pneumatic interface device 160 to deactivate, whereupon the fluid may begin to flow back along the depicted paths, causing cross-contamination of the amplification processes taking place in other test cells. In some embodiments, the depicted location of valve 174 may optionally be a gas outlet, optionally covered by a liquid-tight gas-permeable filter, and the described valve may be located along the inlet path of the test cell 176 or along a segment of the flow channel 156.

[0220] The path in step (13) corresponds to the direction of flow of the test fluid or its gas component from the test cell 175 through the outlet path 177. The outlet path 177 may be a channel leading out of the test cell 175, and the test fluid may be pushed into the outlet path 177 is the pressure provided by pneumatic interface 160. In some embodiments, a liquid-tight gas-permeable filter can be installed on test cell interface 175 and exhaust path 177 so that only the gas component of the test fluid flows through exhaust path 177.

[0221] In step (14), gas from the test fluid is vented from cartridge 100 through outlet 178. Outlet 178 may be covered with a liquid-tight gas-permeable filter to allow gas to be vented and prevent liquid from escaping cartridge 100. Preferably, providing and facilitating removing gas from the test fluid can minimize the amount of gas remaining in the test cell, maximize the amount of liquid in the test cell. As discussed below, minimizing the potential formation of gas bubbles in the path between the electrodes can advantageously lead to more reliable signals and more accurate test results.

[0222] Returning to FIG. 1B, test area 170A includes segments 155, 156 of flow path 150, test cells 175, test cell inlet paths 176, test cell outlet paths 177, orifices/valves 176, 178, and circuit board 179. Circuit board 179 includes test cell electrodes 171A, 171B. cells, conductors 172 for carrying current or other electrical signals, and an electrode interface 135. Electrode interface 135 includes pads 173; half of the pads 173 are configured to couple the test cell drive electrode to the reader voltage or current source, and the other half of the pads 173 are configured to electrically couple the test cell signal electrode to the signal sensing test lead. For clarity, in FIG. 1B, only certain recurring features of test area 170A are marked with reference numbers.

[0223] The circuit board 179 may be a printed circuit board, such as a screen-printed or silk-screened printed circuit board with multiple layers. The circuit board 179 may be printed on a flexible plastic substrate or a semiconductor substrate. The circuit board 179 may be formed at least in part from a material different from that of the base 125 and secured to the underside of the base 125, wherein the top region 126 of the base 125 includes segments 155, 156 of the flow channel 150, test cells 175, test inlets cells 176, test cell outlets 178, and openings/valves 176, 178. For example, circuit board 179 may be a multi-layer printed circuit board affixed, fixed, or laminated to the acrylic of overlying region 126. Electrode interface device 135 may extend beyond the edge of the overlying region 126. The test cells 175 may be formed as openings in the material of the overlying region 126 so that the electrodes 171A, 171B of the circuit board 179 are exposed within the cell 175. Accordingly, the electrodes 171A, 171B may be in direct contact with fluid and medium flowing into cell 175. The top surface of the circuit board 179 may be coated with a thick layer of resin to provide a smooth, flat surface on the bottom of the test cells.

[0224] The test wells 175 may contain dried solid components for the testing process, such as primers and proteins. The particular selection of these dried constituents and their chemical properties may be individualized for the particular target or targets for which cartridge 100 is designed to be tested. Test cells 175 may contain the same or different dried constituents. Said dried constituents may be moistened with the liquid that flows into the test cell (eg, the liquid from the blister pack 140 mixed with the applied sample) and, accordingly, activated for the testing procedure. Preferably, introducing the liquid components in the blister pack 140 separately from the dried solids into the test wells 175 allows the cartridge 100 containing the components necessary for the amplification process to be stored until use, while also delaying the initiation of amplification until the sample is applied.

[0225] The test cells 175 are depicted as round cells staggered in two rows starting from the electrode interface 135. The test cells 175 may be generally cylindrical, for example, in the form of circular openings in the material of the upper part of the region 126 , and are bounded by flat surfaces on the top side (eg, the cover 105 or part of the overlying area 126) and the bottom side (eg, the circuit board 179). Each test cell 175 includes two electrodes 171A, 171B, one of which is a drive electrode configured to apply current to the sample in test cell 175 and the other is a signal electrode configured to detect current output from excitation electrode through the liquid sample. In some embodiments, one or more test cells may include a thermistor instead of electrodes to allow monitoring of the temperature of the fluid within the cartridge 100.

[0226] The monitoring of each test cell may be independent of the monitoring of other test cells, and thus each test cell may represent a different test. The depicted electrodes 171A, 171B within each test cell are line electrodes arranged parallel to each other. The depicted arrangement of test cells 175 provides a compact test area 170A with access from flow channel 150 to each test cell 175. Some implementations may include only one test cell, and other implementations may include two or more test cells located in other configurations. Moreover, the shape of the test cells may vary according to other embodiments, and the shapes of the electrodes may correspond to any of the electrodes shown in FIG. 4A-4G.

[0227] In some embodiments, gas bubbles within test cell 175, particularly when located along the current path between electrodes 171A, 171B, may create noise in the signal received by the signal electrode. This noise can reduce the accuracy of test results based on the signal from the signal electrode. The required high quality signal can be obtained when only liquid or a minimum of gas bubbles are present along the current path. As described above, any air originally present in the fluid flowing along the flow channel 150 may be expelled through the outlet 178. In addition, the electrodes 171A, 171B and/or the test cell 175 may be shaped to soften or prevent the formation of bubbles. air or gas in a liquid sample and their accumulation along the electrodes.

[0228] For example, electrodes 171A, 171B are located at the bottom of test cell 175. This may allow any air or gas to rise in the fluid to the top of the test cell and move away from the path between the electrodes. As used herein, the bottom of the test cell refers to the part of the test cell where a heavier liquid descends under gravity, and the top of the test cell refers to the part of the test cell where a lighter gas rises above heavier liquids. In addition, the electrodes 171A, 171B are spaced from the perimeter or edges of the test cell 175, that is, from the location where bubbles typically occur.

[0229] In addition, the electrodes 171A, 171B may be formed from a thin flat layer of material with a minimum height relative to the underlying circuit board layer forming the bottom of the test cell. In some embodiments, the electrodes 171A, 171B may be formed using electrodeposition and lithography to form a thin metal film layer, for example, about 300 nm high. This minimum height can help prevent or reduce the entrapment of air bubbles along the interface between the electrode and the underlying layer. In some embodiments, a layer of conductive material may be applied over all of the electrodes to create a smoother transition between the edge of the electrode and the bottom of the test cell. For example, a thin polyimide layer (eg, about 5 µm high) can be applied over the electrode, or a resin layer can be applied to the circuit board. In a further or alternative embodiment, the electrodes may be located in grooves in the underlying layer having a depth approximately equal to the height of the electrode. These and other suitable methods can provide an electrode that is nearly flat or recessed into the bottom surface of the cell.

[0230] Preferably, the features described above can help maintain the liquid environment of the electrodes 171A, 171B and prevent or reduce the location of gas bubbles along the current path between the electrodes 171A, 171B.

[0231] In FIG. 1D is a graphical plan view of test area 170B of cartridge 100. As in FIG. 1B, in FIG. 1D certain recurring features are marked with reference numbers in only one localization for simplicity and clarity of representation.

[0232] Test area 170B is an alternative implementation of test area 170A, with the difference between the two implementations being the different electrode configurations within test cells 175. An embodiment of test area 170B provides test cells with ring electrodes 171C and 171D. In the case of line electrodes 171A, 171B of test area 170A, either electrode may be a drive electrode or a signal electrode. According to an embodiment of test area 170B, inner electrode 171D is a drive electrode and outer electrode 171C is a signal electrode.

[0233] The inner electrode 171D may be a disc or ring shaped electrode mated to a current lead 172B, which in turn is mated to a mating pad 173 with an electrode 135 that transmits a current (e.g., a predetermined frequency DC current) to the inner electrode 171D from the reader. The inner electrode 171D may be located in the center of the test cell 175. The outer electrode 171C is a semi-circular electrode located concentrically around the inner electrode 171D and separated from the inner electrode 171D by a gap. The semicircle of the outer electrode 171C is open where the conductive terminal connects the inner electrode 171D to the current-carrying conductor 172B. The outer electrode 171C is coupled to the current sense sensor conductor 172V, which in turn is coupled to the current sense sense pad 173 of the electrode interface 135, which transmits the sensed current to the reader.

[0234] The cartridge 100 of FIG. 1A-1D is a stand-alone, easy-to-use amplification-based target testing device, such as nucleic acid testing, characterized in that the genomic material in the sample is exponentially replicated by a molecular amplification process. Preferably, the user only needs to insert the sample and insert the cartridge 100 into the reader to ensure test results, in some embodiments, since the liquid and solid components of the amplification process are placed within the cartridge and automatically mixed with the sample. In some embodiments, one or both of the cartridges or reader may include a heater and a controller configured to control the heater to maintain said cartridge at the desired temperature for amplification. In some embodiments, one or both of the cartridges or reader may include a motor to vibrate or otherwise agitate the cartridge, causing any entrained gas to rise up in the liquid and vent out of the test cells.

[0235] FIG. 2 is a photograph of another example of a cartridge 200 configured for target detection. Cartridge 200 was used to generate some of the test data described herein and represents an alternative configuration for some of the components described in connection with cartridge 100.

[0236] The cartridge 200 includes a printed circuit board layer 205 and an acrylic layer 210 placed on top and attached to a portion of the printed circuit board layer 205 using a pressure-sensitive adhesive. The acrylic layer 210 includes a series of test cells 215A and a series of temperature monitoring cells 215B in the form of circular holes extending through the acrylic layer 210 along its entire height. The printed wiring 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 cell 215A and a thermistor 225 located within each temperature monitoring cell 215B. Each of the electrodes 220 and thermistors 225 is paired with conductors terminating in a series of printed circuit board leads 230. In the illustration, six pins are labeled "SIG" numbered 1-6 for signal electrodes, six pins are labeled "EXC" numbered 1-6 for drive electrodes, and two thermistor pins are labeled RT1 and RT2.

[0237] During some of the tests described herein, the following example protocol was followed. First, the user filled cells 215A with the test fluid and blocked said fluid with mineral oil. The test fluid may not contain control primers, which provides a clear negative control due to the absence of primers that can cause amplification.

[0238] The user then heated the 200 cartridge to 65 degrees Celsius for 10 minutes to expand any trapped air in the test fluid, causing it to rise to the top in the liquid as bubbles. Bubbles formed in the cells 215A at the indicated initial heating.

[0239] In the next step, the user removes bubbles from the surface of the liquid in cells 215A using a pipette or other instrument. As described above, the elimination of air bubbles may contribute to more accurate test results.

[0240] After the elimination of bubbles, the user allowed the cartridge 200 to cool to room temperature. The user then inserted a loop isothermal amplification (LAMP) positive control (PC) into the bottom of each of the test wells 215A, placed the cartridge 200 on the fuser, and began running the LAMP tests. The detected signals from the signal electrodes were analyzed as described herein to identify a positive signal edge.

[0241] FIG. 3A and 3B depict another example of a cartridge 300 configured for target detection. In FIG. 3A is a top, front, and left perspective view of the cartridge 300, and FIG. 3B is a sectional perspective view showing the outline of cells 320 of cartridge 300. Said cartridge 300 represents an alternate configuration of some of the components described in connection with cartridge 100.

[0242] Said cartridge 300 includes a sample entry area 305, a central channel 310, test cells 320, branches 315 fluidly connecting test cells 320 to a central channel 310, electrodes 325A, 325B located within each test cell 320, and an interface device with an electrode 320 including pads mating with conductors that are in turn mated with respective pads of electrodes 325A, 325B and configured to receive or send signals from or to the reader. As shown in FIG. 3B, the bottom surface of the cells 320 may be curved such that each cell is generally hemispherical. The cartridge 300 in the image is open at the top to show the internal components, however, in use, a lid or other top layer may be present to seal the fluid paths of the cartridge 300. The lid may include vents to allow gas to escape from the cartridge 300, for example, contain liquid-tight gas permeable filters as described above with respect to FIGS. 1A-1D.

[0243] The fluid sample introduced into the sample injection area 305 flows down the central channel 310, for example, under pressure from the reader, pumping the specified sample into the cartridge 300 through a mating port above the sample injection area 305. According to some implementation options, such a reader with a set of cartridges, for example, located one above the other, which can ensure that each cartridge receives the same or different samples. The fluid sample may be essentially liquid with dissolved or entrapped gas (eg, air bubbles). Fluid may exit the central channel 310 through the branched channels 315 and flow into the test cells 320. The branched channels 315 may enter the top of the cell and may be tortuous (eg, include a series of small radius turns) to prevent or attenuate fluid backflow. , which can lead to cross-contamination of amplification processes between different cells.

[0244] FIG. 4A-4G depict various examples of electrode configurations that may be used in the test cell of the cartridges of FIG. 1A-3B or in the test cell or channel of another suitable target detection cartridge as described herein. The test cells shown in Fig. 4A-4G are depicted as annular, however, these electrodes may be used in other test cell geometries in other examples. Unless otherwise indicated, the filled circles in FIG. 4A-4G correspond to the contacts between the described electrodes and conductors leading to or from the electrode. "Width" as used herein below refers to dimensions in the horizontal dimension on the page of FIG. 4A-4G, and "height" herein below refers to the dimensions in vertical dimension on the page of FIG. 4A-4G. Although the exemplary electrodes of FIG. 4A-4G are shown in a particular orientation, in other embodiments they may be turned. In addition, the size examples described correspond to certain potential embodiments of the electrode configurations 400A-400G, and their options may have other sizes with the same ratios as in the proposed size examples. The electrodes shown in Fig. 4A-4G may be made from suitable materials, including platinum, gold, steel, or tin. In experimental testing, tin and platinum functioned in a similar way and were suitable for certain test conditions and targets.

[0245] FIG. 4A shows a first configuration of electrode 400A, characterized in that each of the first and second electrodes 405A, 405B is formed with a semicircular perimeter. The straight edge of the first electrode 405A is adjacent to the straight edge of the second electrode 405B and separated by a gap along the width measurement in configuration 400A. The specified gap is greater than the radius of the semicircular electrodes. Accordingly, the first and second electrodes 405A, 405B with semicircular perimeters are mirror-symmetrical. In one example of the first configuration of electrode 400A, the gap between the most closely spaced portions of said first and second electrodes 405A, 405B is approximately 26.369 mm, the height (along the straight edge) of each of the electrodes 405A, 405B is approximately 25.399 mm, and the radius of the semicircle of each of the electrodes 405A 405B is approximately 12.703 mm.

[0246] FIG. 4B shows a second electrode configuration 400B. Similar to the first electrode configuration 400A, each of the first and second electrodes 410A, 410B in the second electrode configuration 400B is formed with a semicircular perimeter and their semicircles are mirror-symmetrical with their straight edges facing each other. The first and second electrodes 410A, 410B in the second electrode configuration 400B may be the same size as the first and second electrodes 405A, 405B in the first configuration 400A. In the second electrode configuration 400B, the gap along the width measurement in the configuration 400B between the first and second electrodes 410A, 410B is smaller than in the first configuration 400A, and said gap is smaller than the radius of the semicircle of the electrodes 410A, 410B. In one example, in the second configuration of electrode 400B, the gap between the most closely spaced parts of the first and second electrodes 410A, 410B is approximately 10.158 mm, the height (along the straight edge) of each of the electrodes 410A, 410B is approximately 25.399 mm, and the radius of the semicircle of each of the electrodes 410A , 410V is approximately 12.703 mm.

[0247] FIG. 4C shows a third electrode configuration 400C with first and second line electrodes 415A, 415B separated by a gap along the width measurement in configuration 400C, where the gap is approximately equal to the height of the electrodes 415A, 415B. The width of the electrodes 415A, 415B is approximately one half to one third of the height of the electrodes. In one example of the third electrode configuration 400C, the gap between the most closely spaced portions of the first and second electrodes 415A, 415B is approximately 25.399 mm, the height of each of the electrodes 415A, 415B is also approximately 25.399 mm, and the width of each of the electrodes 415A, 415B is approximately 10.158 mm. . The ends of said first and second electrodes 415A, 415B may be rounded, such as having a radius of about 5.078 mm.

[0248] FIG. 4D shows a fourth electrode configuration 400D with first and second rectangular electrodes 420A, 420B separated by a gap along the width measurement in configuration 400D, where said gap is approximately equal to the width of electrodes 420A, 420B. In one example of the fourth electrode configuration 400D, the gap between the most closely spaced portions of said first and second electrodes 420A, 420B is approximately 20.325 mm, the height of each of the electrodes 420A, 420B is also approximately 23.496 mm, and the width of each of the electrodes 420A, 420B is approximately 17.777 mm.

[0249] FIG. 4E shows a fifth electrode configuration 400E with first and second line electrodes 425A, 425B separated by a gap along the width measurement in configuration 400E, where said gap is approximately equal to the height of electrodes 425A, 425B. The fifth electrode configuration 400E is similar to the third electrode configuration 400C, with the width of the electrodes 425A, 425B reduced to about half to two-thirds of the width of the electrodes 415A, 415B at the same height. In one example of the fifth electrode configuration 400E, the gap between the most closely spaced portions of said first and second electrodes 425A, 425B is approximately 25.399 mm, the height of each of the electrodes 425A, 425B is also approximately 25.399 mm, and the width of each of the electrodes 425A, 425B is approximately 5.078 mm. The ends of said first and second electrodes 425A, 425B may be rounded, such as having a radius of about 2.542 mm.

[0250] FIG. 4F shows a sixth electrode configuration 400F with concentric ring electrodes 430A, 430B. The sixth electrode configuration 400F is the configuration shown for the test cells 175 of FIG. 1D. The inner electrode 430B may be a disk or annular electrode and may be located in the center of the test cell. The outer electrode 430A may be a semi-circular electrode located 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 semicircle of outer electrode 430A is open where a conductive terminal connects inner electrode 430B to the current-carrying conductor. In one example of the sixth configuration of electrode 400F, the gap between the inner edge of the annular first electrode 430A and the outer perimeter of the circular second electrode 430B is approximately 11.430 mm, the radius of the circular second electrode 430B is approximately 17.777 mm, and the ring thickness of the annular first electrode 430A is approximately 5.080 mm. The ends of the first electrode 430A may be rounded, such as having a radius of about 2.555 mm, and the gap between the open ends of the ring of the first electrode 435A may be about 28.886 mm, tip to tip.

[0251] FIG. 4G shows a seventh electrode configuration 400G with concentric ring electrodes 435A, 435B. Similar to the embodiment of FIG. 4F, inner electrode 435B may be a disc or ring shaped electrode with the same radius as inner electrode 430B and may be located at the center of the test cell. The outer electrode 435A may be a semi-circular electrode located concentrically around the inner electrode 435A and separated from the inner electrode 435A by a gap. In the seventh configuration of the electrode 400G, said gap is larger than the radius of the inner electrode 435B, for example, two to three times larger. Accordingly, the radius of the outer electrode 435B is larger than that of 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 perimeter of the circular second electrode 435B is approximately 24.131 mm, the radius of the circular second electrode 435B is approximately 17.777 mm, and the ring thickness of the annular first electrode 435A is approximately 5.080 mm. The ends of the first electrode 435A may be rounded, such as having a radius of about 2.555 mm, and the gap between the open ends of the ring of the first electrode 435A may be about 46.846 mm, tip to tip.

[0252] According to 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. According to the embodiments of FIG. 4F and 4G, the inner electrode 430B, 435B is configured for use as a drive electrode (eg, coupled to a current source), and the outer electrode 430A, 435A is configured for use as a signal electrode (eg, provides a signal to a memory or processor). In some test examples, the sixth electrode configuration 400F showed the best performance shown in FIGS. 4A-4G configurations.

[0253] FIG. 5A shows the first electrode, or excitation electrode; and a second electrode, or signal electrode, which may be spaced apart within the test cell of the cartridges of FIG. 1A-3B, or in the test cell or channel of another suitable target detection cartridge as described herein.

[0254] Formation of an aggregate, nucleic acid complex, or polymer, for example, during the amplification process in the test cells of the cartridges of FIG. 1A-3B can affect the wavelength characteristics of one or more electrical signals sent through the channel. As shown in FIG. 5A, the first electrode or excitation electrode 510A is located at a distance from the second electrode or sensor electrode 510B within the test cell 505. The test cell 505 may contain a test solution to be subjected to an amplification process. During some or all of this process, drive voltage 515 may be applied to drive electrode 510A, from which drive voltage 515 is applied to the fluid (preferably all or substantially all liquid) within cell 505.

[0255] After passing through the liquid sample and attenuating due to interaction with it (schematically denoted as resistance R and reactance X), the attenuated drive voltage is sensed or detected at the sensing electrode 510V. The fluid functions as a series resistor R with drive electrode 510A and sensor electrode 510B. The fluid also functions as a series capacitor(s), labeled as reactance X. The raw perceived signal over some or all of the duration of the test as a function of time can be represented by a sinusoidal curve with varying amplitudes similar to those shown in plot 520 .

[0256] The drive voltage 515 may be an alternating current with a predetermined driving frequency. The choice of a particular frequency may depend on, for example, the particular target to be detected, the medium of the sample being tested, the chemistry of the constituents of the amplification process, the temperature of the amplification process, and/or the drive voltage. According to some embodiments of the cartridges of FIG. 1A-3B, the driving frequency may be between 1 kHz and 10 kHz at the lowest possible driving voltage. In one example, in tests performed to identify a target of H. influenzae (10 ⁶ copies per reaction) spiked in 5% whole blood, the sensor drive frequency varied from 100 Hz to 100,000 Hz at 0.15 volts. These tests have shown that the required "signal drop", a portion of the signal artifact indicative of a positive result for the test sample, described in more detail below, becomes easier to detect at values below 100 Hz, and is easiest to detect at values from 1 kHz to 10 kHz. In addition, at frequencies in the range of 1 kHz to 10 kHz, the signal edge can advantageously be identified before the 12 minute test time has elapsed. Advantageously, faster signal edge identification can result in faster test times, which in turn enables faster test results and the ability to perform more tests per day. At frequencies below 1 kHz, the reactive component of the signal (in which the signal edge for the positive sample can be detected) decreases monotonically. The sensor drive frequency can be similarly fine-tuned for other tests to optimize performance, i.e. optimize edge detection. Edge detectability refers to the ability to stably distinguish between a positive sample and a negative sample.

[0257] FIG. 5B is an example graph 525 depicting an impedance signal 530 that can be extracted from the raw signal 520 provided by the sensor electrode 510B. The impedance signal 530 corresponds to the electrical impedance Z of the test cell over a period of time. The impedance Z can be represented by the following equation for complex numbers in Cartesian coordinates:

Z=R+jX

where R corresponds to the resistance of the test cell and is the real part of the above equation, and X corresponds to the reactance of the test cell and is the imaginary part of the above equation (denoted as j).

Accordingly, the test cell impedance can be decomposed into two components, resistance R and reactance X.

[0258] The initial value of the resistance R can be determined by baseline measurement in the test cell before the process or at the beginning of the amplification process. Although the resistance of the test fluid may deviate from the specified base value during the test, the current sensed by the sensor electrode 510B due to the resistance of the fluid under test may be in phase with the signal passing through the drive electrode 510A. Accordingly, changes or deviations in resistance can be identified by the values of the in-phase component of signal 520 over a period of time. Reactivity may result from the effect of inductance in the test fluid, capacitance in the test fluid, or both; this effect may cause a temporary persistence of current in the fluid (eg, the electron current provided by drive electrode 510A). After some time, said stored current flows from the test fluid into the sensing electrode 510V. Because of this delay, the current sensed by the sensing electrode 510V due to the reactivity of the fluid under test may be out of phase with the sensed current resulting from the resistance of the fluid under test. Accordingly, the reactivity values of the fluid under test can be identified from the values of the out-of-phase component of the signal 520 over a period of time. Reactivity may fluctuate throughout the duration of the test due to changes in the chemical constituents of the test fluid as a result of the amplification process. A signal drop (e.g., an increase or decrease in reactivity at or above a threshold or value, and/or within a given time window) indicative of a positive sample can be detected in X reactivity.

[0259] During the test, a sinusoidal excitation of the excitation electrode 510A with a certain amplitude and voltage may occur. The drive electrode 510A is placed in series with the test fluid in the cell, which can be thought of as a resistor R. The resistor (eg, the test fluid) and the electrode form a voltage divider, with the voltage determined by the ratio of the resistors and the chemistry/impedance of the electrode. The resulting voltage waveform sensed by the sensing electrode 510V corresponds to the complex impedance signal 530. In some embodiments, the waveform of, for example, the impedance signal 530 may not be generated, instead, the raw sensed signal 520 may be decomposed into a resistive component and a reactance component according to description in this document. The impedance signal 530 is given as an example of a combined curve representing both the resistance of the test fluid and the reactivity of the test fluid over a period of time. The complex impedance signal 530 can be interpreted as a quadrature modulated waveform (e.g., a combination of an in-phase waveform as a result of the resistance of the fluid under test and an out-of-phase waveform as a result of the reactivity of the fluid under test), where the in-phase and out-of-phase components change over time significantly more than modulating frequency. The in-phase waveform is in phase with the complex waveform of the complex impedance. In some embodiments, a synchronous detector may be used, for example, with a multiplier 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 amplitude and phase.

[0260] To decompose the impedance signal 530 (or the raw sensed signal 520) into its constituent resistance and reactance components, the voltage waveform 520 at the 510V sensor electrode is measured at a frequency greater than its Nyquist frequency (e.g., , twice the maximum frequency of the excitation voltage) and then divided into a common-mode component (resistance) and an out-of-phase component (reactance). The common-mode and out-of-mode voltage components can be calculated using a known series resistance (eg, R value) to calculate the real component of the impedance (resistance) and the imaginary component of the impedance (reactance).

[0261] FIG. 5C is a graph 541 of the resistance component 540A and the reactance component 540B over time (t=3 minutes to t=45 minutes) extracted from the raw signal 520 generated from the positive test example. In the illustration, a signal slope 545 corresponds to a change in Δ _R of reactance 540V over a particular time window T _W . A signal slope of 545 indicates a positive sample. At times before signal edge 545, reactance curve 540V is relatively flat or stable; and similarly, after the signal edge 545, the reactance curve 540V is relatively flat or stable. Accordingly, according to this embodiment, the signal slope 545 for the specific test parameters presented in plot 541 is represented by a decrease in _ΔR in the expected region 535.

[ ₀₂₆₂ ] The amount of reactivity change ΔR that corresponds to a positive sample signal edge 545, as well as the position and/or duration of a particular time window T _W when signal edge 545 is expected to occur, may vary depending on a number of test parameters. These parameters include the specific test target (for example, the rate at which said target is amplified), the frequency of the drive voltage, the configuration of the drive and sensor electrodes (for example, their individual shape and dimensions, the gap separating the electrodes; and the material of the electrodes), the frequency measurements, the amount of amplification agents at the start of the test, the temperature of the amplification process, and the amount of target present in the sample. In some embodiments, the expected characteristics of the signal slope in the positive sample, predetermined, for example, experimentally, can be used to distinguish between positive samples and negative samples. In some embodiments, expected signal slope characteristics can be used to determine the severity or progression of a medical condition, for example, by establishing a correlation between specific signal transition characteristics and specific initial amounts of target in a sample. The predetermined expected characteristics may be fed into the reader, stored therein, and then accessed by a reader configured to receive signals from the sensor electrode(s) of the test cartridge when the test results are determined.

[0263] For a given test, the expected amount of change in reactivity _ΔR and the expected time window T _W of the signal drop 545 for a positive sample can be determined experimentally based on monitoring and analyzing reactivity curves generated by samples for positive control (and optionally samples for negative control). In some embodiments, the test parameters that affect the signal edge may vary and may be fine-tuned to identify parameters that correspond to a distinct signal edge. The reader and cartridge as described herein may be configured to suit the configuration under test and provided with characteristics for the expected signal slope for said test.

[0264] For example, in a series of experimental tests for H. influenza, the test fluid initially included primers for amplification and 1,000,000 copies of the target added, the drive voltage was 200 mV P2P, the test parameters included the beginning of the sweep in the range of 10 kHz and the end of the sweep in the range 10 MHz for drive current frequency, near and far electrode gaps set to 2.55 mm and 5 mm, respectively. The amplification temperature was set to 65.5 degrees Celsius and the arrangement of two electrodes (one for each of the near and far gaps) included platinum electrodes. At low frequencies (10 kHz - 100 kHz), detectable signal spikes were identified starting at about 23 minutes of amplification at about 10 kHz and at about 30 minutes at about 100 kHz, using a 5 mm gap electrode configuration, with a reactivity change of about 3.5-4 ohms at 10 kHz, dropping to about 3.25-3.5 ohms at 100 kHz. At low frequencies (10 kHz - 100 kHz), detectable signal spikes were identified starting from about 25 minutes of amplification at about 10 kHz, and from about 30 minutes at about 100 kHz, using a 2.5 mm gap electrode configuration, with a change in reactivity about 3.5-4 ohms. At higher frequencies, the drop in reactivity of the signal edge was reduced, and the time to identify these smaller signal edges was shifted to a later point in the amplification process. Accordingly, in this example, the test cell in the test cartridge may be configured with a 5 mm electrode gap and the reader may be configured to apply a 10 kHz drive current to the test cartridge during amplification. The reader may be instructed to provide the indicated current and monitor the resulting test cell reactivity throughout the amplification or a window of time around the expected signal break point (23 minutes in this case), for example 20 to 35 minutes. The reader may also be instructed to identify a positive sample based on reactivity showing a change of about 3.5-4 ohms after about 23 minutes of amplification or within a time window around the expected signal edge.

[0265] Once identified, the Δ _R and T _W values can be transferred to readers for use in distinguishing between positive and negative samples in that particular test. According to some examples, such devices can determine if the reactance curve 540V contains the required value and/or has the required slope within the identified time window T _W to match the signal edge. In other embodiments, said reader may analyze the shape of the reactivity curve over a period of time to determine if it contains a signal edge. In some embodiments, the reader may modify the test procedures based on the identified time window T _W when signal edge 545 is expected, such as, for example, by energizing the drive and monitoring the received signal only within said window, which can advantageously save energy and resources for processing compared to continuous monitoring throughout the entire testing period.

[0266] FIG. 5D is a graph 551 of the resistance component and the reactance component extracted from the raw sensory data of the sensing electrode 510B in the positive and negative control example tests. In particular, graph 551 shows a positive sample resistance curve 550A, a positive sample reactivity curve 550B, a positive sample resistance curve 550C, and a positive sample reactivity curve 550D over a 35 minute test. As shown in FIG. 5D, the signal drop in the case of a positive sample occurs approximately 17 minutes after the start of the test, with a relatively flat and stable 550V reactivity curve before the signal drop. Conversely, at the same time, the reactivity curve of the 550D negative sample does not show a signal drop, but maintains a quadratic curvature from about 8 minutes to the end of the test.

[0267] FIG. 5E is a graph 561 of the resistance component 560A and the reactance component 560B over a period of time (t=0 minutes to t=60 minutes from start of amplification) extracted from the raw signal 520 generated from the positive test example. In the illustration, a signal slope 565 corresponds to a change in Δ _R of reactance 560V within a particular time window T _W . A signal slope of 565 indicates a positive sample. At times before signal edge 565, reactivity curve 560V is relatively flat or stable, and similarly, after signal edge 565, reactivity curve 560B is relatively flat or stable, and slightly concave. The signal slope 565 for specific test parameters shown in graph 561 appears as a peak, overshoot or bell curve in the expected region 535 as the reactivity values rise and fall by Δ _R , forming an approximately parabolic curve. As described herein, varying certain test parameters (eg, test cell configuration, chemistry and initial number of amplification constituents, target, and drive current characteristics) can alter the geometry of the signal slope provided by the positive sample. Accordingly, in some embodiments, the geometry of the "trip" on the reactivity vs. time curve may vary from test to test, although for a particular test, the geometry of the curve and/or the time frame of the signal drop remains stable within the parameters of reactivity changes and/or time frames. in all positive samples for the specified test. [0266] FIG. 6 is a schematic block diagram of an example reader 600 that can be used with the cartridges described herein, such as cartridges 100 or 300. Reader 600 includes memory 605, processor 610, communications module 615, user interface 620, heater 625, electrode interface 630, voltage source 635, compressed air energy storage 640, motor 650, and cavity 660 into which a cartridge can be inserted.

[0268] When the test cartridge 100 is inserted into the reader, the cartridge electrode interface 135 is paired with the electrode interface 630 of the reader 600. This can allow reader 600 to detect insertion of the cartridge, for example, by testing for an established communication path. In addition, such communications may allow the reader 600 to identify the embedding of a particular test cartridge 100 and access the appropriate test protocols. Test protocols may include test duration, test temperature, positive sample impedance curve characteristics, and information to display to the user based on various test results obtained. In other embodiments, reader 600 may receive, via user interface 620, an indication that a cartridge is embedded (eg, by user input of a "start test" command and optionally a test cartridge ID).

[0269] The memory 605 includes one or more physical electronic storage devices configured to store computer-executable instructions for controlling the operations of the reader 600 and data generated by using the reader 600. For example, the memory 605 can receive and store data from sensor electrodes paired with the 630 electrode interface device.

[0270] The processor 610 includes one or more hardware processors that execute computer-executable instructions to control the operations of the reader 600 during a test, for example, by providing a user interface 620, controlling a heater 625, controlling a communication module 615, and activating a voltage source 635 compressed air 640 and engine 650. One example of test operations is described in connection with FIG. 7A below. The processor 610 may also be configured with these instructions to determine test results based on data obtained from the drive electrodes of the inserted test cartridge, for example, by performing the process of FIG. 7B described below. Processor 610 may be configured to identify different targets in the same test sample based on signals obtained from different test cells of the same cartridge, or may identify the same target based on individual or cumulative analysis of signals from different test cells.

[0271] The communication module 615 may optionally be contained in the reader 600 and include network hardware components, such as wired or wireless network components, to enable network communication between the reader 600 and remote computing devices. Suitable network components include WiFi, Bluetooth, cellular modems, Ethernet ports, USB ports, and the like. Preferably, network capabilities may allow the reader 600 to send test results and other test data over a network to identified remote computing devices such as hospital information systems and/or laboratory information systems that store electronic medical records, national health agency databases, and computing devices. clinical specialists or other designated personnel. For example, a physician can retrieve test results for a particular patient on a mobile device, laptop, or office desktop as the reader determines the test results, allowing the physician to achieve faster diagnostics and treatment plans. In addition, networking capabilities may allow reader 600 to receive information over the network from remote computing devices, such as updated edge settings for the current test, new edge settings for new tests, and updated or new test protocols.

[0272] The user interface 620 may include a display for presenting test results and other test information to users, as well as user input devices (e.g., buttons, a touchscreen display) allowing the user to enter commands or test data into the reader 600.

[0273] A heater 625 may be positioned adjacent cavity 660 to heat the inserted cartridge to the temperature required for the amplification process. Although the depicted heater 625 is located on one side of the cavity 660, according to some variants of implementation, it may surround the specified cavity.

[0274] As described herein, the voltage source 635 may apply a drive signal at a predetermined voltage and frequency to each drive electrode of the inserted test cartridge. The compressed air energy store 640 can be used to supply pneumatic pressure through the channel 645 to the pneumatic interface 160 of the test cartridge 100, which facilitates the flow of fluid within the test cartridge. Compressed air power storage 640 may store previously compressed air or generate compressed air as needed for reader 600. In other embodiments, other suitable air pumps and pressure-applying mechanisms may be used instead of storing or generating compressed air. The motor 650 may be actuated to move the actuator 655 towards the blister pack 140 of the inserted cartridge and back to break through the blister pack as described above.

[0275] FIG. 7A is a flowchart of an example process 700 for operating a reader during a test as described herein. Process 700 may be performed by reader 600 described above.

[0276] At block 705, reader 600 may detect an inserted assay cartridge 100, 200, 300, for example, in response to user input or in response to a signal path being established when the cartridge is inserted. In some embodiments, said cartridge 100, 200, 300 may include an information element that identifies a particular test or tests to be performed on reader 600 and optionally includes test protocol information.

[0277] At block 710, reader 600 may heat cartridge 100, 200, 300 to a predetermined temperature for amplification. For example, the temperature may be determined from information that is stored on the cartridge 100, 200, 300 or made available from the internal memory of the reader 600 as a result of identifying the cartridge 100, 200, 300.

[0278] At block 715, reader 600 may activate a blister pack piercing mechanism, such as motor 650 and drive 655. Piercing of the blister pack may cause release of liquid contents, including amplification promoting chemicals, from the previously sealed chamber.

[0279] At 720, reader 600 may activate an air pump to move the sample and liquid from the blister pack through the cartridge flow path toward the test cell. As described above, the test cells may include vents to allow liquid to be forced through the flow path of the cartridge, as well as to allow any trapped air to escape. The pneumatic pump may contain compressed air 640 or other suitable pressure source, and may be in fluid communication with pneumatic interface 160.

[0280] At a block 725, the reader 600 may release any trapped air from the test cells, for example, by forcing fluid through the cartridge flow path until a certain resistance is detected (for example, fluid in the flow path is forced through a liquid-tight, gas-permeable vent filter ). Block 725 may optionally include shaking the inserted cartridge to move any entrapped air or gas bubbles up through the liquid and out through the vents. In addition, at block 725, the reader 600 may optionally provide signals to the cartridge that cause the valves located between the test wells to close to avoid mixing of the amplification processes.

[0281] At decision block 730, reader 600 may determine if a predetermined test duration has been exceeded. For example, if a window of time is known when a spike in the positive sample should occur, the test may end at the end of the specified window or at some predetermined time after the end of the specified window. In such a case, process 700 proceeds to an optional decision block 735 or, in embodiments without block 735, to block 740.

[0282] In an optional decision block 735, the reader 600 determines whether to monitor amplification in the test cell by logging data from the sensor electrode of the test cell. For example, reader 600 may be instructed to monitor only the impedance of a test cell within a particular window, or a window during a test. In the event that reader 600 determines that amplification monitoring is not needed in the test well, process 700 loops back to decision block 730.

[0283] In the event that reader 600 detects amplification monitoring in the test cell, process 700 proceeds to block 740. At block 740, reader 600 provides a drive signal to the excitation electrode of the test cell or cells of the inserted cartridge. As described above, said signal may be an alternating current at a specific frequency and voltage.

[0284] At block 745, the reader 600 detects and records data from the sensing electrode of the test cell or cells of the inserted cartridge. In some embodiments, said data may be stored for later analysis, such as after a test is completed. In some embodiments, the reader 600 may analyze said data in real time (eg, while the test is still ongoing) and may stop the test upon identifying a signal edge in a positive sample.

[0285] When the reader 600 at block 730 determines that the test duration has already exceeded the specified duration, the process 700 moves to block 750 to analyze the test data and output the test result. The test result may include an indication that testing the sample for the presence of the target resulted in a positive or negative result, or may more specifically indicate the estimated amount of the target in the test sample.

[0286] FIG. 7B is a flow chart of an exemplary process for analyzing data from a target detection test as described herein, which may be performed by reader 600 as block 750 of FIG. 7A.

[0287] At block 755, reader 600 may access logged signal data obtained from the cell electrode. Even if a cartridge contains multiple cells, data from each cell can be analyzed individually. The test results from the wells can later be analyzed collectively to produce a single test result for a single target based on all tests performed in the cartridge, or to obtain multiple test results for multiple targets.

[0288] At block 760, reader 600 may decompose the signal into a resistance component and a reactance component at some different time points or all test time points. For example, as described above, at each point in time, reader 600 may determine the in-phase and out-of-phase components of the raw sampled voltage waveform, and then may recover those components from the convolution using the known series resistance of the electrode circuit to calculate the common-mode (resistance) and the out-of-phase (reactivity) parts of the test cell impedance.

[0289] At block 765, reader 600 may generate a curve of reactivity values over a period of time. Also at block 765, reader 600 may optionally generate a curve of resistance values over a period of time.

[0290] At block 770, reader 600 may analyze the reactivity curve to identify a signal change indicative of positive test results. As described above with respect to the signal edge of FIG. 5C, reader 600 may expect a change in reactivity greater than a threshold, may expect such a change within a given window of time, may analyze the slope of the reactivity curve at a given time, or may analyze the overall shape of the reactivity curve to determine whether a signal edge is present (e.g., rising or falling signal, before and after which relatively more stable values are observed).

[0291] At decision block 775, based on the analysis performed at block 770, reader 600 may determine whether the desired signal change in the reactivity curve has been identified. If so, process 750 proceeds to block 780 to provide an indication of a positive test result to the user. If not, process 750 proceeds to block 785 to provide an indication of a negative test result to the user. The result can be displayed locally, for example, on the display of the device, or via the network to a designated remote computing device.

Overview of device examples

[0292] Some embodiments of the methods, systems, and compositions of the present invention include devices comprising a drive electrode and a sensor electrode. In some embodiments, said excitation electrode and said sensor electrode measure the electrical properties of the sample. In some embodiments, said electrical properties include complex admittance, impedance, conductivity, resistivity, resistance, and/or dielectric constant.

[0293] In some embodiments, the electrical properties are measured in a sample having electrical properties that do not change during the measurement. In some embodiments, electrical properties are measured in a sample having dynamic electrical properties. In some such embodiments, dynamic electrical properties are measured in real time.

[0294] In some embodiments, a drive signal is applied to the drive electrode. Said drive signal may include direct current or voltage and/or alternating current or voltage. In some embodiments, said drive signal is capacitively coupled to/through the sample. In some embodiments, said excitation electrode and/or said sensor electrode are passivated to prevent direct contact between the sample and the electrode.

[0295] 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 depending on sample volume and/or geometry.

[0296] In some embodiments, said voltage and said frequency of the drive voltage may be fixed or vary during the measurement. For example, the measurement may involve sweeping voltage and frequency during detection, or selection of specific voltage and frequency that can be optimized for each sample. In some embodiments, the drive voltage induces a current at the signal electrode, which may vary depending on device admittance and/or sample characteristics.

[0297] In some embodiments, detection parameters are optimized by modeling admittance, device, and sample in a lumped equivalent circuit consisting of electrode-sample coupling impedances, sample impedance, and inter-electrode parasitic impedance. Lumped equivalent circuit parameters are determined by measuring the admittance of the electrode-sample system at one or more drive frequencies for the device. In some embodiments, the complex (comprising both real and imaginary components) admittance of the electrode-sample system is measured using both magnitude-sensitive and phase-sensitive detection techniques. In some embodiments, detection parameters are optimized by determining frequencies corresponding to transitions between frequency regions by measuring admittance over a wide range of frequencies. In some embodiments, detection parameters are optimized by determining frequencies corresponding to transitions between frequency domains by calculation from predetermined lumped model values.

[0298] According to some embodiments, the admittance of a system in which the electrode is capacitively coupled to the sample contains three frequency regions: a low frequency region, where the impedance due to the coupling of the electrode to the sample predominates, a medium frequency region, where the impedance of the sample predominates, and a high frequency region. frequencies where parasitic interelectrode impedance prevails. The admittance due to the connection of the electrode with the sample has a capacitive nature and is characterized by a value that increases linearly with frequency, with a phase shift of 90 degrees. The admittance in the region of the sample is conductive in nature and does not vary significantly with frequency, with a phase shift of approximately 0 degrees. The admittance of the interelectrode region has a capacitive nature and is characterized by a value that increases linearly with frequency and a phase shift of 90 degrees.

[0299] According to some embodiments, the induced current at the discharge electrode is related to the drive voltage and the complex admittance by the following relationship:

current = (complex admittance) X (voltage)

[0300] According to some embodiments, said device measures both the magnitude of the drive voltage and the magnitude of the induced current to determine the magnitude of the complex admittance. In some embodiments, said device is calibrated to known drive voltages and the magnitude of the induced current is measured. To determine the phase of the complex admittance, said device can measure the relative phase difference between the drive voltage and the induced current.

[0301] According to some implementation options, the indicated magnitude and phase are measured directly.

[0302] In some embodiments, magnitude and phase are measured indirectly, such as using both synchronous and asynchronous detection. The synchronous detector provides the common mode component of the induced current. The asynchronous detector provides the quadrature component of the induced current. Both components can be combined to determine the complex admittance.

[0303] In some embodiments, these electrodes are not passivated.

[0304] In some embodiments, the drive electrode and/or the detection electrode are passivated. Said excitation electrode and/or said detection electrode may be passivated to prevent, for example, unwanted adhesion, contamination, adsorption, or other damaging physical interactions between the electrode and the sample or constituents thereof. In some embodiments, the passivation layer comprises a dielectric material. In some embodiments, passivation provides effective capacitive coupling of the electrodes to the sample. Coupling efficiency is determined by measuring the characteristics of the electrode/sample system, for example, which may include: dielectric properties of the passivating layer, thickness of the passivation layer, area of the interface between the passivation layer/sample, surface roughness of the passivating layer, electrical double layer on the interface between the passivation layer/ sample, temperature, applied voltage and applied frequency, electrical properties of the sample, electrical and/or chemical properties of electrode materials.

[0305] In some embodiments, electrode configuration and fabrication are optimized to reduce unwanted parasitic coupling between electrodes. This can be done by shielding the electric field, using an electrode substrate with a varying dielectric constant, optimizing the topology and/or ground layers.

Overview of examples of devices for the detection of biomolecules

[0306] Some embodiments of the methods, systems, and compositions of the present invention include devices for detecting a target, such as a biomolecule. In some such embodiments, the measurement of the electrical properties of a sample is used as a detection strategy for biomolecular assays.

[0307] In some embodiments, said target is a nucleic acid, protein, small molecule, drug, metabolite, toxin, parasite, intact virus, bacterium, spore, or any other antigen that can be recognized and/or bound by the probe fragment to capture and/or detection.

[0308] In some embodiments, said target is a nucleic acid. In some embodiments, the methods include nucleic acid amplification. In some embodiments, the amplification comprises isothermal amplification. In some embodiments, the nucleic acid amplification reaction is quantified by measuring electrical properties or from a change in the reaction solution. In some embodiments, the electrical properties of the amplification reaction are measured in real time throughout the reaction, or comparative measurements are made by measuring the electrical properties before and after the reaction.

[0309] In some embodiments, the target antigen is detected by specific binding of a detection probe, such as, for example, an antibody, aptamer, or other fragment for molecular recognition and/or binding to the antigen. In an exemplary embodiment, a detection antibody is fused to a nucleic acid sequence to form a chimeric antibody-nucleic acid complex. Said chimeric complex is synthesized prior to analysis for antigen detection. Many different nucleic acids can be conjugated to a single antibody, thus increasing sensitivity to detect binding of the chimeric complex to an antigen. After removing any excess of the chimeric complex not bound to the antigen, the nucleic acid portion of the chimeric complex is amplified and the amplification reaction is quantified by measuring the electrical properties (or changes thereof) of the reaction solution as described herein. Thus, the degree of amplification of the nucleic acids associated with the antigen in the chimeric complex indicates the presence of the target antigen and allows quantitative determination of the antigen. The use of antigen recognition secondary amplification combined with electrical detection provides greater simplicity, sensitivity, and dynamic range than other antigen detection methods.

[0310] In some embodiments, an capture probe, such as an antibody, aptamer, or other moiety for molecular recognition and/or antigen binding, binds to a surface by conjugation or association. Immobilization of the capture probe on the surface allows for the removal of excess unbound reagents and/or antigen by washing. The chimeric complex binds to the surface-captured antigen, allowing the unbound chimeric complex to be removed by washing. Thus, only the captured antigen remains for detection by the chimeric complex. An example implementation is shown in FIG. 8. In some embodiments, said capture probe and detection antibody are the same.

[0311] In some embodiments, said capture probe is immobilized to the surface by covalent conjugation, the use of streptavidin-biotin bonds, or other bioconjugation and molecular immobilization techniques commonly used and familiar to those skilled in the art. In some embodiments, said surface is a flat surface, a scaffold, a filter, a microsphere, a particle of any shape, a nanoparticle, or a granule, or the like. An example implementation is shown in FIG. nine.

Overview of examples of magnetic beads

[0312] Some embodiments of the methods, systems, and compositions of the present invention include magnetic beads or their use. In some embodiments, said microsphere, particle, or granule is magnetic and/or magnetizable. The use of a magnetic support according to such embodiments may facilitate washing of the beads to remove excess antigen and/or non-specifically adsorbed chimeric complex from surfaces. A method that includes the use of a magnetic particle support may include a magnetic amplification immunoassay (MAIA). An example implementation is shown in FIG. ten.

[0313] In some embodiments, magnetic beads are suitable for capturing targets and are used for magnetophoretic manipulation in the context of pure electrical processing (MEMS) of the sample and/or amplification/detection cartridge, and reduce or eliminate dependency on flow/pressure driven mobility in the fluid system . In some embodiments, magnetic beads are used to extract and/or concentrate a target genomic material from a sample. See, for example, source: Tekin, HC, et al., Lab Chip. DOI: 10.1039/c31c50477h, which is incorporated herein by reference in its entirety. An automated microfluidic processing platform suitable for embodiments of the present invention is described in: Sasso, LA., et al., Microfluid Nanofluidics. 13:603-612, which is incorporated herein by reference in its entirety. Examples of beads suitable for use in embodiments of the present invention include Dynabeads® for IVD nucleic acid diagnostics (ThermoFisher Scientific) or Dynabeads® SILANE Viral NA Kit (ThermoFisher Scientific).

Overview of excitation and detection examples ƒC ⁴ D

[0314] In some embodiments, the described devices, systems, and/or methods employ a ƒC ⁴ D approach to monitor nucleic acid amplification in real time. Accordingly, one or more phase-sensitive electrical conductivity measurements may indicate the presence of one or more targets in the sample.

[0315] In some aspects, the method includes sweeping frequencies at specific drive voltages to determine the optimal drive frequency (ƒ _opt ) when amplification-related sample conductivity is at its maximum. At ƒ _opt , the output signal from the sensor corresponds to the minimum of the relative phase difference between the drive voltage and the induced current, which provides a highly sensitive quantification of the biomolecule by conductivity measurements.

[0316] In some embodiments, the ƒC ⁴ D detection system employs at least two electrodes. These two electrodes are placed relatively close to the microchannel where nucleic acid amplification takes place. The PT signal is applied to one of these two electrodes. The electrode to which said signal is applied can be capacitively coupled through a microchannel to the second of the two said electrodes. Accordingly, in some aspects, the first electrode is a signal electrode and the second electrode is a signal electrode.

[0317] In general, the frequency of the detected signal at the signal electrode is identical to the frequency of the AC signal applied to the signal electrode, but is smaller in magnitude and has a negative phase shift. The captured current can then be amplified. In some aspects, the sensed current is converted to voltage. In some aspects, the voltage is rectified. In some aspects, the rectified voltage is converted to a PTT (direct current) signal using a low pass filter. The signal can be offset to zero before being sent to the DAQ system for further processing.

[0318] The above system may be represented by a series of capacitors and resistors. Changes in electrical conductivity that occur during nucleic acid amplification within the channel can cause a decrease in the overall impedance of the system and, accordingly, an increase in the level of the produced pickup signal. Such changes in the level of the resulting signal may occur as one or more peaks in the DAQ system.

[0319] Electronics for generating and demodulating signals are implemented in the form of circuits. For example, a printed circuit board ("PCB"), a ISSN device, or other integrated circuit ("IC") is produced using conventional manufacturing and fabrication techniques. In some aspects, such electronics are designed in whole or in part with single use components and/or disposable components. To achieve the desired results, the varying physical geometry and electrical characteristics (passivation layer thickness, electrode pad area, cross-sectional area and channel length, as well as dielectric strength) of such circuits are used.

[0320] An example nucleic acid detection system includes at least one channel, and detects one or more physical properties, such as pH, optical properties, electrical properties, and/or characteristics, along at least a portion of the length of the channel to determine whether it contains channel a specific nucleic acid of interest and/or a specific nucleotide of interest.

[0321] An exemplary detection system is configured to include one or more channels for placing a sample and one or more sensor connections (e.g., one or more nucleic acid probes), one or more entry ports for introducing a sample and sensor connections into the channel and, in some embodiments, one or more output ports through which the content of the channel may be retrieved.

[0322] One or more sensor compounds (e.g., one or more nucleic acid probes) can be selected such that direct or indirect interaction between the nucleic acid of interest and/or the nucleotide of interest (when present in the sample) and the sensor particles compounds leads to the formation of an aggregate that changes one or more physical properties, such as pH, optical properties or electrical properties and/or characteristics, at least in part of the length of the channel.

[0323] In certain cases, the formation of an aggregate, a nucleic acid complex, or a polymer inhibits or blocks the flow of fluid in the channel and, accordingly, causes a measurable drop in electrical conductivity and electric current when measured along the length of the channel. Similarly, in these cases, the formation of an aggregate, nucleic acid complex, or polymer 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 the formation of the aggregate, nucleic acid complex, or polymer enhances the electrical path along at least a portion of the length of the channel, thereby causing a measurable increase in electrical conductivity and electric current when measured along the length. channel. In these cases, the formation of an aggregate, nucleic acid complex, or polymer causes a measurable decrease in resistivity along the length of the channel.

[0324] In certain cases, the formation of an aggregate, a complex with a nucleic acid, or a polymer affects the characteristics of the waveform of one or more electrical signals sent through the channel. As shown, for example, in FIG. In FIG. 11 shows an alternative or complementary approach to that described above in FIG. 5A-5D. Said first and second electrodes 1116, 1118 are not necessarily in contact with the measurement solution contained within the channel 1104. In this sense, said first and second electrodes 1116, 1118 are capacitively coupled to the solution within the channel 1104. The strength of the capacitive coupling depends on the geometry electrodes, the thickness of the passivating layer and the material of the passivating layer (in particular, its relative dielectric strength).

[0325] According to some aspects, the solution is limited to the channel 1104. The cross-sectional area of the specified channel can be measured in micrometers. Accordingly, said solution behaves like a resistor whose resistance depends on the conductivity of the solution and the geometry of channel 1104.

[0326] In some embodiments, an alternating current/voltage is applied to the drive electrode 1116 and the induced current is measured at the signal electrode 1118. The induced current is proportional to the inter-electrode impedance, which can vary with the conductivity of the solution. As shown, drive voltage 1400 is applied to drive electrode 1116 and induced current 1410 is detected by signal electrode 1118.

[0327] In some embodiments, the sensitivity of the detector depends at least in part on the frequency of the excitation. Accordingly, in some aspects, the maximum sensitivity is observed at a minimum of the absolute value of the phase of the induced current. In this region, the impedance of the chip is determined mainly by the impedance of the fluid. Fluid impedance is a function of fluid conductivity and chip geometry. Information about the complex impedance is important to ensure the maximum sensitivity of the detector and the correct operation of the detector.

[0328] Analysis of the lumped equivalent circuit model showed that detector sensitivity is closely related to the strength of the coupling capacitance, C _WALL , solution resistance, R _LAMP , and parasitic capacitance, C _X . In particular, the change in electrode-to-electrode impedance in relation to the change in conductance is maximum when the drive frequency, ƒ, satisfies the following inequality:

1/(πR _LAMP C _WALL ) << ƒ << 1/(π R _LAMP C _X )

[0329] As shown in FIG. 12, the signal impedance depends on the drive frequency and changes after the LAMP reaction occurs in channel 1104. Also, as seen in FIG. 12, the inequality on the left can determine the frequency range below which the coupling impedance dominates, and changes in the solution impedance become almost imperceptible. The inequality on the right may define a frequency region above which spurious effects predominate and the electrodes 1116, 1118 are effectively shunted.

[0330] As shown in FIG. 13, in both extreme regions, the impedance is condenser and out of phase (approaching 90°) with the drive voltage. Between these two regions, the impedance begins to approach the limit for a simple resistor, and the dependence on impedance with respect to frequency levels out. In fact, the maximum sensitivity of the detector corresponds to the minimum phase of the impedance.

[0331] To clarify the need for synchronous detection, two parallel current paths can be considered in the simplified model: current through the chip through the fluid channel, and parasitic or geometry-driven capacitance. With a drive signal, V, at a given frequency, ƒ, the induced current, I, is:

where Y is the admittance of the chip due to fluid path coupling, C _x is the parasitic capacitance, and j is the imaginary unit. Multiplying by j means that the current through the parasitic path is 90° out of phase with the drive voltage. The measured impedance of the sample chip with respect to the drive frequency is shown in FIG. fourteen.

[0332] In a synchronous detector, the sampled current is multiplied by an in-phase square wave, m, and then subjected to low-pass filtering.

[0333] It is easy to show that the contribution of signals 90° out of phase with the baseband signal will be zero, so parasitic capacitance can be ignored in this analysis. To see the effect of synchronous detection on the current along the fluid path, you can multiply the induced current (minus the parasitic contribution) by the modulating wave

where |Y| - admittance value, ϕ=arg(Y), a H.F.T. means high-frequency terms (for example, higher than ƒ). After low-pass filtering, we can isolate the PTT term of the synchronous output signal:

This expression can be simplified, taking into account the fact that:

what gives:

Alternatively, one can express this in terms of the Z impedance, taking into account the fact that

where the bar denotes complex conjugation. The output signal of the synchronous detector, respectively, becomes equal to:

[0334] Considering the simple circuit models for the chip, the impedance is explicitly calculated and the output signal of the synchronous detector is predicted.

[0335] A simple equivalent circuit model contains two capacitors, C, in series with a resistor R. As discussed above, the resistance R is primarily a function of the microfluidic geometry and the electrical conductivity of the solution. The capacitance is primarily a function of the area of the electrode, the dielectric used for the passivation layer, and the thickness of the passivation layer. The impedance Z of the simplified circuit is given as follows:

The square of the impedance:

|Z| ² \u003d R ² + (πƒC) ^-2

and the expression for the output signal of the synchronous detector:

where the numerator and denominator are multiplied by the square of the electrical conductivity, G=1/R.

[0336] For conductivity meters, the element constant, k, can be given as:

where k is expressed in reciprocal length units. The element constant k is primarily dependent on placement, electrode area, and fluid path, and is not necessarily a simple linear relationship. Then the output signal of the synchronous detector is expressed as follows:

, где:To facilitate the analysis, a dimensionless conductivity parameter can be introduced,

, where:

Thus:

It should be noted that the output data of the detector depends on the dimensionless conductivity,

для

1) Detector response is asymptotically proportional

for

2) The detector response reaches a local maximum s _max =|V|ƒC at

для

3) The response of the detector is asymptotically proportional

for

[0337] Given the dependence of the detector response on dimensionless electrical conductivity, it is important to tightly couple the design of the chip and the detector. Consideration of the previously stated points in terms of actual electrical conductivity leads to the following statements:

1) The detector response is asymptotically proportional to σ for

для

2) The response of the detector is asymptotically proportional

for

3) Detector response becomes non-monotonic at σ=kƒC

[0338] In other words, increasing the driving frequency expands the conduction range in which the output signal of the synchronous detector is linear. The response of the synchronous detector is plotted against the dimensionless conductivity in FIG. fifteen.

[0339] To assess the validity of the lumped model, the detector response was measured for the known conductivity of KCl solutions. The channel of the chip had a size of 2 mm with a cross-sectional area of 0.01 mm ² . The area of each of the two electrodes passivated with a SU8 photoresist layer 10 µm thick was 9 ^mm2 . The calculated cell constant and capacitance were determined, and the excitation frequency was chosen such that the conductivity corresponding to the non-linearity of the detector output signal was approximately 5 mS/cm. The experiment was repeated at excitation frequencies of 10, 15, and 20 kHz.

[0340] The measured conductivity of the chemical components prior to LAMP was approximately 10 mS/cm. Table 1 below shows the calculated values of the minimum frequency of the detector, controlled by the previously set limit, namely:

[0341] The results in the model shown in FIG. 16 show good agreement with detector output over a wide range of conductivities and for given frequency steps. It is important to note that the same two metrics, k and C, are used at each frequency. This model predicts the qualitative behavior of the detector response, namely, the functional form of the response, the dependence of the critical conductivity, at which a nonlinearity is observed at the excitation frequency. The model overestimates the divergence of the frequency-dependent behavior for conductivities beyond the critical conductance.

[0342] As a tool for quickly assessing the electrical conductivity and capacitance of the walls, the effects of surface conductivity and capacitance along with the effects of edge fields can be ignored. A geometric finite element model can be used to further refine this rough estimate.

[0343] The electrode is modeled as a plane-parallel capacitor with area A _E separated by a dielectric with relative dielectric strength ε _r and thickness t. The capacitance is then approximated as:

where ε ₀ is the dielectric constant.

и проводимостью σ. Соответственно, электропроводность пути текучей среды может быть аппроксимирована следующим образом:[0344] The fluid can be modeled as a simple resistor with cross-sectional area A _F , length

and conductivity σ. Accordingly, the electrical conductivity of the fluid path can be approximated as follows:

Based on this, the element constant is also approximated.

[0345] In some aspects, said device is configured to determine an "impedance spectrum" upon insertion of the chip. The specified device may provide for digital control of the excitation frequency. Said device may be capable of providing a fast sweep. This device may include the in-phase and quadrature components of the induced signal, from which the complex impedance can be determined. The suitability of the impedance spectrum is determined, at least in part, based on curve fitting or other heuristic method to determine a properly inserted chip and/or a proper sample insertion. In some aspects, said device is first tested by excitation at a frequency determined by the initial sweep. In some embodiments, said device includes a detector using synchronous detection. Thus, the measured induced currents due to the fluid path (at the minimum phase) can be detected in real time.

Overview of Channel Examples

чем его ширина и глубина. В некоторых случаях, пример соотношений между длиной : шириной может составлять: 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, или 20:1, или находиться в пределах диапазона, определяемого любыми двумя из вышеупомянутых соотношений.[0346] In some embodiments, the channel has the following dimensions: a length measured along the longest dimension (Y-axis) and extending along a plane parallel to the detection system substrate; a width measured along an axis (X-axis) perpendicular to the longest dimension and extending along a plane parallel to said substrate; and a depth measured along an axis (z-axis) perpendicular to a plane parallel to said substrate. An example channel may have a length essentially

than its width and depth. In some cases, an example of length:width ratios might 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 be within the range defined by any two of the above ratios.

[0347] In some embodiments, the channel is configured to have a depth and/or width substantially equal to or less than the diameter of the aggregate, nucleic acid complex, or polymer formed in the channel, preferably while in suspension in the channel. , through interaction between the nucleic acid of interest and the sensor compound particles (eg, one or more nucleic acid probes) used to detect said nucleic acid of interest.

[0348] In some embodiments, the channel is configured to have a width along the X-axis in the range of from about 1 nm to about 50,000 nm, or a width within the range defined by any two numbers in the above range, but not limited to the specified range examples. An example channel has a length along the Y-axis in the range of about 10 nm to about 2 cm, or a length within the range defined by any two numbers in the above range, but not limited to these example ranges. The exemplary channel has a depth along the z-axis in the range of about 1 nm to about 1 µm, or a depth within the range defined by any two numbers in the above range, but not limited to these example ranges.

[0349] In some embodiments, the channel has any suitable cross-sectional shape (for example, a cross-sectional shape along the x-z plane), including, but not limited to, annular, elliptical, rectangular, square, D-shaped (as a result of isotropic etching ) etc.

[0350] In some embodiments, the channel has a length in the range of 10 nm to 10 cm, such as at least or equal to 10 nm, 50 nm, 100 nm, 200 nm, 400 nm, 600 nm, 800 nm, 1 µm , 10 µm, 50 µm, 100 µm, 300 µm, 600 µm, 900 µm, 1 cm, 3 cm, 5 cm, 7 cm, or 10 cm, or a length within the range defined by any two of the above lengths. In some embodiments, the channel has a depth in the range of 1 nm to 1 µm, such as at least or equal to 1 nm, 5 nm, 7 nm, 10 nm, 50 nm, 100 nm, 200 nm, 400 nm, 600 nm , 800 nm, 1 µm, 10 µm, 20 µm, 30 µm, 40 µm, 50 µm, 100 µm, 500 µm, or 1 mm, or a depth within the range defined by any two of the above depths. In some embodiments, the channel has a width in the range of 1 nm to 50 µm, such as, for example, 1 nm, 5 nm, 7 nm, 10 nm, 50 nm, 100 nm, 200 nm, 400 nm, 600 nm, 800 nm , 1 µm, 10 µm, 20 µm, 30 µm, 40 µm, 50 µm, 100 µm, 500 µm, or 1 mm, or a width within the range defined by any two of the widths mentioned above.

[0351] In some embodiments, the channels are formed in a cartridge later inserted into the device. In some aspects, said cartridge may be a disposable cartridge. In some aspects, said cartridge is made from economical plastic materials. In some aspects, at least a portion of the cartridge is made from paper and fluidic laminates.

[0352] An embodiment of a detection system 2100 that is used to detect the presence or absence of a particular nucleic acid and/or a particular nucleotide in a sample is depicted in FIG. 17A-17B. In FIG. 17A is a plan view of the system, and FIG. 17B is an elevation view of the system in cross section. The detection system 2100 includes a substrate 2102 that is positioned along a substantially horizontal xy plane. In some embodiments, substrate 2102 may be formed from a dielectric material such as silicon dioxide. Other examples of substrate materials 2102 include, but are not limited to, glass, sapphire, or diamond.

[0353] the Substrate 2102 supports or includes a channel 2104 containing at least the inner surface 2106 and the inner space 2108 to accommodate the fluid. In some cases, channel 2104 is etched into the top surface of substrate 2102. Examples of materials for interior surfaces 2106 of channel 2104 include, but are not limited to, glass or silica.

[0354] In certain embodiments, channel 2104 and substrate 2102 are formed from glass. Under biological conditions, the use of glass-derived implants is difficult due to the slow dissolution of glass in biological fluids and the adhesion of proteins and small molecules on the glass surface. In certain non-limiting embodiments, a method for modifying glass surfaces for nucleic acid detection and analysis is surface modification using a self-assembling monolayer. In some embodiments, at least a portion of the inner surface 2106 of the channel 2104 is pre-treated or covalently modified by incorporating a material or coating with a material that provides specific covalent bonding of the compound to the inner surface. In some embodiments, the sliding cover 2114 closing the channel may also be covalently modified with a material.

[0355] Examples of materials that are used to modify the inner surface 2106 of channel 2104 include, but are not limited to, a silane compound (e.g., trichlorosilane, alkylsilane, triethoxysilane, perfluorosilane), zwitterionic sultone, poly(6-9)ethylene glycol (PEG), perfluorooctyl , fluorescein, aldehyde or graphene compound. Covalent modification of the inner surface of the channel reduces the non-specific absorption of certain molecules. According to one example, covalent modification of the inner surface can provide covalent binding of sensor compound molecules to the inner surface while preventing non-specific absorption by the inner surface of other molecules. For example, poly(ethylene glycol) (PEG) is used to modify the inner surface 2106 of the channel 2104 to reduce non-specific adsorption of materials to said inner surface.

[0356] In some embodiments, said channel 2104 is nanofabricated or microfabricated with a well-defined, smooth inner surface 2106. Surface Modification Methods for Fused Silica Micro- and Nanochannels," MRS Proceedings, 1659, pp 15-26. doi:10.1557/opl.2014.32, the contents of which are hereby incorporated by reference in their entirety and expressly.

[0357] The first end section of the channel 2104 includes an input port 2110 or is in fluid communication with an input port 2110, and the second end section of the channel 2104 includes an output port 2112 or is in fluid communication with an output port 2112. According to certain non-limiting embodiments, ports 2110 and 2112 are offered at the ends of the 2104 channel.

[0358] In some embodiments, the top surface of the substrate 2102 with channel 2104 and ports 2110, 2112 is covered and sealed with a sliding cover 2114. In some embodiments, rigid plastic is used to form the channels, including the top; a semi-permeable membrane may also be used.

[0359] The first electrode 2116 is electrically connected at the first end section of the channel 2104, such as at or near the input port 2110. The second electrode 2118 is electrically connected at the second end section of the channel 2104, such as at or near the port of the output 2112. First and second electrodes 2116, 2118 are electrically connected to a power source or voltage source 2120 to provide a potential difference between the first and second electrodes. Thus, the potential difference is applied to at least part of the length of the channel. With fluid present in channel 2104 and subjected to an applied potential difference, electrodes 2116, 2118 and said fluid create a complete electrical path.

[0360] The power supply or voltage source 2120 is configured to apply the 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) as well as in a second, opposite direction (along the Y-axis). In one example, in the case of a first direction of the electric field or potential difference, the positive electrode is connected at the first end section of the channel 2104, such as at or near the port of the input 2110, and the negative electrode is connected at the second end section of the channel 2104, such as at or near the port. output port 2112. In another example, in the case of a second, opposite direction of the electric field or potential difference, the negative electrode is attached at the first end section of the channel 2104, for example, at or near the input port 2110, and the positive electrode is attached at the second end section of the channel 2104 , for example, at or near output port 2112.

[0361] In some embodiments, the power supply or voltage source 2120 is configured to provide a FET signal. The frequency of the PT signal can change dynamically. In some aspects, the power supply or voltage source 2120 is configured to provide an electrical signal at a frequency of 10-10 ⁹ Hz. In some aspects, the power supply or voltage source 2120 is configured to provide an electrical signal at a frequency of 105-107 Hz.

[0362] The first and second end sections of channel 2104 (e.g., at or near input port 2110 and output port 2112) are electrically coupled to nucleic acid detection circuits 2122 programmed or configured to detect values of one or more electrical properties of channel 2104 to determine if a particular nucleic acid and/or nucleotide is present or absent in channel 2104. Electrical property values are detected over a single period of time (e.g., within a specified period of time after the introduction of the sample and one or more sensor compounds into the channel) or within several different periods of time (eg, before and after the introduction of both the sample and one or more sensory connections into the canal). In some aspects, electrical property values are detected continuously over a set period of time from sample injection by LAMP amplification. Examples of detectable electrical properties include, but are not limited to, electric current, conduction voltage, resistance, frequency, or waveform. Certain examples of nucleic acid detection circuits 2122 include a processor or computing device, or are configured as a processor or computing device, such as device 1700 shown in FIG. 18. Certain other 2122 nucleic acid detection circuits include, but are not limited to, an ammeter, voltmeter, ohmmeter, or oscilloscope.

[0363] In one embodiment, the nucleic acid detection circuit 2122 includes a measurement circuit 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 equalization 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 select just one of these values after they reach equilibrium (for example, after the cessation of changes exceeding a certain threshold of dispersion or tolerance).

[0364] The nucleic acid detection circuitry 2122 may also include a comparison circuitry 2126 programmed or configured to compare two or more channel electrical property values, such as an electrical property reference value (e.g., measured to the point where both the sample and all sensor compounds have already been introduced into the channel) and the value of the electrical property (for example, measured after the sample and all sensor compounds have been introduced into the channel). Comparison circuit 2126 may use said comparison to determine if a nucleic acid is present or absent in a channel. In one embodiment, a comparison circuit 2126 calculates the difference between a measured electrical property value and a reference electrical property value, and compares the difference to a predetermined value indicative of the presence or absence of a nucleic acid in a channel, and this information is used to diagnose or predict a disease state, or the presence or the absence of infection in the subject.

[0365] In some embodiments, when both a sample and a sensing compound are introduced into a channel, the comparison circuit 2126 is programmed or configured to compare a first value of an electrical property (e.g., an amount of electric current) when an electric field or potential difference is applied through the channel in the first direction along the length of the channel, with a second value of the electrical property (for example, the magnitude of the electric current) when applying an electric field or potential difference through the channel in the second, opposite direction along the length of the channel. In one embodiment, the comparison circuit 2126 calculates the difference between the values of said first and second values, and compares said difference with a predetermined value (e.g., determines if said difference is essentially zero) indicative of the presence or absence of a nucleic acid in the channel. For example, if the indicated difference is essentially zero, this indicates the absence of nucleic acid, which may be in the channel in dispersed, polymeric form or aggregate form. Where the difference is substantially non-zero, this indicates the presence of nucleic acid, which may be present in the channel in dispersed form, polymeric form, or aggregate form.

[0366] In some embodiments, the nucleic acid detection circuitry 2122 is programmed or configured to determine the absolute concentration of the nucleic acid in the sample, and/or the relative concentration of the nucleic acid relative to one or more additional substances in the sample.

[0367] In some embodiments, comparison circuit 2124 and equalization circuit 2126 are configured as separate circuits or modules, while in other embodiments they are configured as a single integrated circuit or module.

[0368] The nucleic acid detection circuit 2122 includes an output 2128 that may, in some embodiments, communicate with one or more external devices or modules. For example, the nucleic acid detection circuitry 2122 may communicate the electrical property reference value and/or one or more measured electrical property values to one or more of: a processor 2130 for further calculations, processing, and analysis, a persistent information storage device, or a memory 2132. to store values; and/or a display device 2134 for displaying said values to the user. In some embodiments, nucleic acid detection circuitry 2122 generates an indication of whether the sample includes a nucleic acid and passes this indication to processor 2130, persistent storage or memory 2132, and/or display device 2134.

[0369] In an example of the method of using the system of FIG. 17A and FIG. 17B, one or more sensor compounds (eg, one or more nucleic acid probes) and a sample are sequentially or simultaneously introduced into the channel. When the flow of the fluid and/or the flow of charged particles in the fluid is not inhibited (for example, due to the absence of aggregates), conductive particles or ions in the fluid move along at least a portion of the length of the channel 2104 along the Y axis from the input port 2110 to the output port 2112 The movement of conductive particles or ions produces or generates a first or "reference" electrical property value or range of values (e.g., electrical current, conductivity, resistivity, or frequency) detected by nucleic acid detection circuitry 2122 along at least a portion of the length of channel 2104. According to In some embodiments, the equalizing circuit 2124 periodically or continuously monitors the electrical property value over a period of time until the values reach equilibrium. The equalization circuit 2124 then selects one of the values as the reference value of the electrical property to avoid being affected by temporal changes in said electrical property.

[0370] As used herein, a "reference" electrical property value refers to the value or range of values of the electrical property of a channel prior to insertion of the sample and all sensor compounds (eg, one or more nucleic acid probes) into the channel; that is, the reference value is a value characterizing said channel prior to any interaction between the nucleic acid in the sample and all sensor compounds. In some cases, the reference value is found during the period of time after the introduction of the sensor compound into the channel, but before the introduction of the sample and additional sensor compounds into the channel. In some cases, the reference value is found during the period of time after the introduction of the sensor compound and sample into the channel, but before the introduction of additional sensor compounds into the channel. In some cases, the reference value is found during the period of time before the introduction of the sample or sensor compounds into the canal. In some cases, the reference value is predetermined and stored on a durable storage medium from which it can be accessed.

[0371] In some cases, the formation of an electrically conductive aggregate, polymer, or complex with a nucleic acid in a channel (for example, as a result of interactions between the nucleic acid of interest in the sample and one or more nucleic acid probes) enhances the electrical path along at least a portion of the length channel 2104. In this case, the nucleic acid detection circuit 2122 detects a second value or range of values of an electrical property (e.g., electric 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 provides a waiting or adjustment period after the introduction of the sample and all sensor connections into the channel, until the second value of the electrical property is detected. Said waiting or adjustment period allows the aggregate, polymer, or nucleic acid complex to form in the channel, preferably while in suspension in the channel, and allows the resulting aggregate, polymer, or nucleic acid complex to change the electrical properties of the channel, preferably while in suspension in the channel. channel.

[0372] In some embodiments, the equalization circuit 2124 periodically or continuously monitors the electrical property value for a period of time following the introduction of the sample and all sensor connections until the values reach equilibrium. The equalization circuit 2124 may then select one of the values as the second electrical property value to avoid being affected by temporal changes in said electrical property.

[0373] The comparison circuit 2126 compares the second electrical property value with the electrical property reference value. If said circuit determines that the difference between the second value and the reference value corresponds to the specified range of current increase or conductivity (or decrease in resistivity), nucleic acid detection circuit 2122 determines that an aggregate, polymer, or complex with a nucleic acid is present in the channel and, accordingly, the target the nucleic acid is present or detected in the sample. Based on this, the presence or absence of a target and a disease or infectious condition in a subject can be diagnosed or identified.

[0374] According to certain other embodiments, when the fluid flow in the channel and/or the flow of charged particles in the fluid is partially or completely blocked (for example, due to the formation of an aggregate, polymer, or complex with a nucleic acid), the conductive particles or ions in the fluid are unable to move freely along at least a portion of the length of the channel 2104 along the Y axis from the input port 2110 to the output port 2112. Obstructed or stopped movement of conductive particles or ions produces or generates a third electrical property value or range of values (e.g., electric current or signal, conductivity , resistivity, or frequency) detected by the nucleic acid detection circuitry 2122 along at least a portion of the length of the channel 2104. The third electrical property value is detected in addition to or instead of the second electrical property value. In some embodiments, the nucleic acid detection circuitry 2122 may wait for a wait or adjustment period after the introduction of both the sample and all sensor compounds into the channel until a third electrical property value is detected. Said waiting or adjustment period allows the aggregate, polymer or nucleic acid complex to form in the channel, allows the resulting aggregate, polymer or nucleic acid complex to change the electrical properties of the channel.

[0375] In some embodiments, the equalization circuit 2124 periodically or continuously monitors electrical property values over a period of time after sample and all sensor connections are introduced until said values reach equilibrium. Then, the equalization circuit 2124 selects one of the values as the third value of the electrical property in order to avoid the effect of temporal changes in the electrical property.

[0376] The comparison circuit 2126 compares the third electrical property value with the electrical property reference value. If said circuit determines that the difference between the third value and the reference value falls within a predetermined range of current reduction or conductance (or increase in resistivity), the nucleic acid detection circuit 2122 determines that an aggregate, polymer, or nucleic acid complex is present in the channel and accordingly the target nucleic acid is identified as being present in the sample.

[0377] Fluid flow along the length of the channel depends on the size of the aggregate, polymer or nucleic acid complex relative to the dimensions of the channel, and the formation of an electrical double layer (EDL) on the inner surface of the channel.

[0378] In general terms, a DEL is a region of total charge between a charged solid (e.g., the inner 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 content of the channel). DES is observed both around the inner surface of the channel and around any nucleic acid particles and nucleic acid aggregates, polymers, or complexes inside the channel. Counterions from the electrolyte are attracted by the charge of the inner surface of the channel and induce a region of full charge. DES affects the flow of ions within the channel and around the analyte particles and aggregates, polymers or nucleic acid complexes of interest, preventing any counterions from passing along the length of the channel, which causes diode-like behavior.

[0379] To mathematically determine the characteristic length of the DEL, the Poisson-Boltzmann (“PB”) equation and/or the Poisson-Nernst-Planck (“PNP”) equations are solved. These solutions are coupled to the Navier-Stokes (NS) fluid flow equations to create a non-linear system of coupled equations that are analyzed to understand the operation of the example system.

[0380] Considering the dimensional interactions between the channel surface, the DEL, and nucleic acid aggregates, polymers, or complexes, exemplary channels are configured and designed with carefully chosen dimensional parameters to substantially inhibit the flow of conducted ions along the length of the channel when an aggregate, polymer, or complex is formed in the channel. with a nucleic acid of a certain predetermined size. In certain instances, an exemplary channel is configured to have a depth and/or width substantially equal to or less than the diameter of the particle aggregates formed in the channel during nucleic acid detection. According to some implementation options, the dimensions of the DEL are also taken into account when choosing the dimensional parameters of the channel. In certain instances, an exemplary channel is configured to have a depth and/or width substantially equal to or less than the dimensions of the EDL generated around the inner surface of the channel and the aggregate, polymer, or nucleic acid complex in the channel.

[0381] In some embodiments, prior to application of the detection system, the channel contains no sensor connections (eg, one or more nucleic acid probes). Thus, the manufacturer of the discovery system may not pre-process or modify the specified channel to include the sensing connection. In this case, in use, the user introduces one or more sensor compounds, for example, in an electrolyte buffer, into the channel and detects the reference value of the electrical property of the channel with the sensor connection, but in the absence of a sample.

[0382] In certain other embodiments, the channel is pretreated or modified such that at least a portion of the inner surface of the channel includes or is coated with a sensor compound (e.g., one or more nucleic acid capture probes) prior to application of the detection system. In one example, a manufacturer detects an electrical property reference value of a channel modified by a sensor connection and, when used by a user, can use the stored electrical property reference value. Thus, the manufacturer of the detection system may pre-process or modify the channel to include a sensor connection. In this case, the user needs to introduce the sample and one or more additional sensor compounds into the channel.

[0383] Some examples of detection systems include one channel. Some other examples of detection systems include multiple channels on the same substrate. Such detection systems may include any suitable number of channels, including but not limited to at least or exactly 2, 3, 4, 5, 6, 7, 8, 9, or 10 channels, or multiple channels within a range, defined by any two of the above numbers.

[0384] According to one embodiment, the detection system includes a plurality of channels, where at least two channels operate independently of each other. The channel example 2104 and associated components of FIG. 17A-17B are reproduced on a single substrate to obtain such a multi-channel detection system. These multiple channels are used to detect one nucleic acid in one sample, different nucleic acids in one sample, the same nucleic acid in different samples; and/or different nucleic acids in different samples. According to another implementation variant, the detection system includes a plurality of channels, where at least two channels operate in cooperation with each other. In some aspects, these channels have different shapes depending on the target to be detected.

Overview of device examples for point-of-care applications

[0385] In some embodiments, said device is portable and configured to detect one or more targets in a sample. As shown in FIG. 19, said device includes a processor 900 configured to drive an ƒC ⁴ D circuit 905. Said ƒC ⁴ D circuit 905 includes a signal generator 907. The signal generator 907 is configured to provide one or more signals through channel 2104 or a test cell. as described above. Signal generator 907 couples to preamplifier 915 to amplify one or more signals from signal generator 907. Said one or more signals pass through multiplexer 909 and through channel 2104. The signal from channel 2104 is amplified at post amplifier 911 and demodulated by demultiplexer 913. Analog to Digital converter 917 receives the signal and sends a digital signal to processor 900. Processor 900 includes circuitry configured to measure, balance, compare, and the like to determine if a desired target has been detected in a sample. In some embodiments, said analog-to-digital conversion may occur first. In some such implementations, the induced waves may be fully sampled and digitally demodulated in software.

[0386] The processor 900 is also paired with one or more heating elements 920, in some embodiments. Said one or more heating elements 920 may be resistance heating elements. One or more heating elements 920 are configured to heat the sample and/or solution in the channel 2104. In some embodiments, said sample is heated to a temperature greater than or equal to 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80° С, 85°С, 90°С, 95°С, 100°С, 105°С, 110°С, 115°С, 120°С, 130°С, 140°С, 150°С, 160°С, 170°C, 180°C, 190°C, 200°C, 210°C, 220°C, 230°C, 240°C, 250°C, 260°C, either to any temperature or to any temperature range between the above two values. In some embodiments, said sample is cooled to a temperature below or equal to 40°C, 35°C, 30°C, 25°C, 20°C, 15°C, 10°C, 5°C, 0°C, -5 °C, -10°C, -15°C, -20°C, or up to any temperature or up to any temperature range between the above two values. In view of the foregoing, processor 900 and/or other circuitry is configured to sense the temperature 925 of the sample and/or channel 2104, and control one or more heating elements 920 until the desired heating set point 930 is reached. In some aspects, the entire conduit 2104 is configured to be heated by one or more heating elements 920. In other aspects, only portions of the conduit 2104 are configured to be heated by one or more heating elements 920.

[0387] The processor 900 is configured to accept user input 940 from one or more user inputs such as keyboards, touch screens, buttons, switches, or microphones. Data is output (950) and logged (951), communicated to the user (953), sent to the cloud storage system 952, and the like. The data is sent to another device for processing and/or further processed according to some embodiments. For example, ƒC ⁴ D data can be sent to the cloud and later processed to determine the presence or absence of the target(s) in the sample.

[0388] In some aspects, said device is configured to draw relatively low power. For example, said device may require as little as 1-10 watts of power. In some aspects, said device requires 7 watts of power or less. Said device is configured to process data, communicate wirelessly with one or more other devices, send and detect signals through a channel, heat a sample/channel, and/or detect and display input/output via a touchscreen display.

[0389] In some embodiments, the sample collection device, the sample preparation device, and the fluid cartridge are formed as separate physical devices. Accordingly, the first sample collection device is used to collect the sample. Said sample may contain saliva, mucosal secretion, blood, plasma, feces or cerebrospinal fluid. The sample is then transferred to a second sample preparation device. The sample preparation device includes the components and reagents required for nucleic acid amplification. After sample preparation, it is transferred to a third device containing a fluid cartridge where amplification, excitation and ƒC ⁴ D measurements take place. In some embodiments, sample collection and sample preparation are performed by a single device. In some embodiments, sample preparation and fluid cartridge reside in the same device. In some embodiments, one device is configured to allow sampling, sample preparation, amplification of at least a portion of the sample, and analysis of the sample using ƒC ⁴ D.

Overview of Compact Fluid Cartridge Examples

[0390] In some aspects, said device includes a removable fluid cartridge that can be paired with another associated device. Said removable fluid cartridge is configured for use as a disposable cartridge. In some embodiments, said cartridge includes a plurality of channels. Channels can have different shapes. In some aspects, four channel shapes are used and repeated to ensure accuracy. In some aspects, more than four channel shapes are used, repeated to ensure accuracy. In some aspects, each channel is configured to detect one unique target. In other aspects, each channel is configured to detect the same targets. In some embodiments, said cartridge includes one or more heating elements. In general, a fluid cartridge may include at least one channel configured to provide ƒC ⁴ D analysis.

[0391] According to some aspects, the specified cartridge includes a multilayer lamellar structure. One or more channels are stamped and/or laser cut into the substrate. The substrate, in some embodiments, includes a polypropylene film. One or both sides of the film are covered with glue. Said layer with channels is fixed over a polyamide heating coil to heat the entire channel or part of the channel. Said channel at least partially covers the hydrophilic PET layer. Printed electrodes can be located under the specified layer of PET. In some aspects, at least one thermistor per channel is provided to provide temperature feedback.

[0392] In other aspects, said cartridge comprises an injection molded plastic. One or more channels are located inside the injection molded plastic. A layer of PET or PET film covers the channels completely or partially, applying PET by laser welding on PET on injection molded plastic. Injection molding can provide advantages such as rigidity and three-dimensional structure, as well as fabrication of features such as valves and a frame for ease of handling. The cartridge may optionally include printed electronics and/or heating elements and/or thermistors depending on the particular design.

[0393] An example of an embodiment of a fluid cartridge 500 is shown in FIG. 20. As shown, the specified cartridge 2500 includes four layers. PCB (printed wiring board)/PWB (printed wiring diagram) layer 2501 with applied electrodes 2505. The electrodes can be passivated with a 30 nm thick layer of titanium dioxide using methods such as atomic deposition. The PCB/PWB layer may include access points 2506 for screws or other holding means to hold the four layers together. The power supply and detection circuits can be interfaced with the PCB/PWB layer. Seal layer 2510 with notches 2513 and 2514, and access points 2506. The seal layer may be made from a material such as fluorosilicone. The bottom rigid layer of the substrate 2520, which includes the access points 2506 and the inlet ports 2522. The upper rigid layer 2530, which includes the access points 2506 and the inlet ports 2522. Both the lower and upper rigid layers can be made from a material such as acrylic. Four channels are formed when the four layers are assembled by fixing with screws or other holding means through several access points 2506 of several layers. Cutouts 2513 and 2514 form the sides of the channels. Notch 513 forms a channel with two trapezoidal ends, and notch 2514 forms a channel with substantially straight sides. Parts of the PCB/PWB layer 2501, including the electrodes 2505, form the bottom of the channels. The bottom rigid layer 2520 forms the top of the channels, and the inlet ports 2522 form the inlet and outlet ports for the channels. The upper layer inlet ports 2522 and the upper rigid layer inlet ports provide reagent access to all channels. In some embodiments, a channel with two trapezoidal ends may have a volume of from about 30 µl to about 50 µl. In some embodiments, a substantially straight-sided channel may have a volume of from about 20 µl to about 30 µl. Such volumes can be adjusted by varying the compression of at least the sealing layer. In FIG. 21 is a plan view of the fluid cartridge 2500 of FIG. 20 and shows access points 506 for screws or other restraints, inlet ports 2522 communicating with channels 2550, and electrodes 2505. FIG. 22 shows an example of dimensions for two electrodes 2505. FIG. Figure 23 shows an example of dimensions for a 2550 conduit with two trapezoidal ends. According to some implementation options, the specified channel is heated to a temperature of 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70 ° C, 71 ° C, 72 ° C, 73 ° C, 74 ° C or 75 ° C, or up to a temperature within the range determined by any two of the above numbers, and pressurize it. In some aspects, said channel may be pressurized to 1, 2, 3, 4, 5, or 6 atmospheres, or pressure within a range defined by any two of the above pressures.

[0394] In some embodiments, the fluidic device channel can be adapted or configured to hold a sample volume greater than or equal to 1 µl, 2 µl, 3 µl, 4 µl, 5 µl, 6 µl, 7 µl, 8 µl , 9 µl, 10 µl, 20 µl, 30 µl, 40 µl, 50 µl, 60 µl, 70 µl, 80 µl, 90 µl, 100 µl, 200 µl, 300 µl, 400 µl, 500 µl, 600 µl, 700 µl, 800 µl, 900 µl or 1000 µl, or with a volume or volume range between any two of the above volumes. In some embodiments, the channel of the fluid device may be adapted to be pressurized. In some embodiments, the sample in the channel may be pressurized to 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 a pressure corresponding to any range between any two of the above pressures. In some embodiments, the channel of the fluid device can be adapted for temperatures greater than or equal to -20°C, -15°C, -10°C, -5°C, 0°C, 5°C, 10°C, 15° С, 20°С, 25°С, 30°С, 35°С, 40°С, 45°С, 50°С, 55°С, 60°С, 65°С, 70°С, 85°С, 80°C, 85°C, 90°C, 95°C, 100°C, 105°C, 110°C, 115°C, 120°C, 130°C, 140°C, 150°C, 160° C, 170°C, 180°C, 190°C, 200°C, 210°C, 220°C, 230°C, 240°C, 250°C, 260°C, or any temperature or any temperature range between any two of the above temperatures.

Overview of sample collection examples

[0395] In some embodiments, the methods, systems, and apparatus as described herein employ a simplified direct sample collection process. This reduces the number of steps between sample collection and analysis. In other words, in some embodiments, it is desirable to minimize the number of sample transfers and/or user manipulation of the sample in order to avoid sample contamination. In some aspects, the devices as described herein are configured to be compatible with various sample collection methods to suit all types of testing environments. Accordingly, homogeneous vessel-to-chip interfaces are used in some aspects. When sampling systems are adjusted, the detection hardware remains the same regardless of the type of sample taken and analyzed.

Overview of Analysis Examples

[0396] Some embodiments of the methods, systems, and compositions of the present invention include simple lysis/amplification/detection of targets from crude samples in a single vessel. Some implementation options include amplification based on immune responses to detect non-nucleic acid targets. Some implementation options include reagents added to the reaction, which provide an increase in the change in conductivity. Some implementation options include isothermal amplification strategies such as LAMP, SDA and/or RCA. In some embodiments, the detection targets are biomarkers such as proteins, small molecules such as pharmaceuticals or drugs, or biological weapons such as toxins. Detection of such targets can be achieved by conjugating immune binding reagents such as antibodies or aptamers to nucleic acids that participate in the isothermal amplification reaction. In some embodiments, additives for the amplification reaction may increase the change in the conductivity of the solution, correlating with the results of the quantitative determination of the target. The use of additives can provide higher sensitivity and dynamic range of detection.

[0397] Some embodiments of the methods proposed according to the present invention provide one or more of the following desirable features in the collection and processing of samples: no centrifugation; portability; low cost; single use; wall outlets may not be required; the use may be simple and/or intuitive; only relatively minor technical skills may be required for use; it may be possible to extract RNA and/or DNA from a small volume sample (eg 70 µl); it may be possible to stabilize the RNA and/or DNA prior to amplification; thermostable reagents that do not require a cold chain for storage can be used; the possibility of compatibility with the analysis of samples with a low level of detail (for example, samples containing 1000 copies/ml or less) and/or a dynamic range that allows the detection of viral load, for example, differing by at least 4 orders of magnitude.

[0398] Some embodiments of the proposed methods, systems and compositions include the collection and processing of samples for use in a diagnostic device as described herein. Examples of a collected sample, also referred to as a biological sample, may include, for example, plant, blood, serum, plasma, urine, saliva, ascitic fluid, cerebrospinal fluid, semen, lung lavage fluid, sputum, mucus, mucus secretions, feces, body fluid containing cells or nucleic acids, a solid medium containing cells or nucleic acids, tissue, and the like. Methods for obtaining specimens may include finger prick, heel prick, venipuncture, nasal aspirate in adults, nasal aspirate in children, nasopharyngeal lavage, nasopharyngeal aspirate, swab, collection of colonic contents in a container, tissue biopsy, or sampling by lavage. Additional examples include environmental samples such as a soil sample and a water sample.

Overview of Amplification Examples

[0399] Some embodiments of the methods, systems, and compositions of the present invention include amplification of target nucleic acids. Methods for amplifying nucleic acids are well known and include methods characterized by changes in temperature during the reaction, such as PCR.

[0400] Additional examples include isothermal amplification, characterized in that the reaction can occur at a substantially constant temperature. In some embodiments, isothermal amplification of target nucleic acids results in changes in the conductivity of the solution. There are several types of nucleic acid isothermal amplification methods, such as nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), loop amplification (LAMP), Invader assay, rolling ring amplification (RCA), signal mediated amplification of RNA technology (SMART, "signal mediated amplification of RNA technology))), helicase amplification (HDA), recombinase polymerase amplification (RPA), signal amplification with nicking endonuclease (NESA) and activation of nanoparticles using nicking endonuclease (NENNA), target recycling using exonucleases, splice-terminal amplification strategies or using Y-probes, cleaved DNAzyme, and deoxyribozyme amplification, template-driven chemistries that result in signal amplification, non-covalent catalytic reactions with DNA, hybridization chain reactions (HCR), and detection via self-assembly DNA probe ov with obtaining supramolecular structures. See, for example, Yan L., et al., Mol. BioSyst, (2014) 10: 970-1003, which is expressly incorporated herein and in its entirety by reference.

[0401] In the LAMP example, the two primers in the forward primer set are referred to as the inner (F1c-F2, where "c" means "complementary") and the outer (F3) primers. At 60°C, the F2 region of the internal primer first hybridizes to the target and is extended by DNA polymerase. The outer F3 primer then binds to the same target strand at F3c and the polymerase extends F3c by expelling the newly synthesized strand. The displaced strand forms a loop-on-stem structure at the 5' end by hybridization of the F1c and F1 regions. At the 3' end, the reverse primer set can hybridize to the indicated strand and the polymerase generates a new strand with loop-on-stem structures at both ends. "Dumbbell" structured DNA enters the cycle of exponential amplification, and by repeated lengthening and displacement of strands, strands with several inverted repeats of the target DNA can be obtained. In some embodiments of the methods of the present invention, the components for LAMP include 4 primers, DNA polymerase, and dNTPs. Examples of the use of LAMP include viral pathogens, including Dengue fever virus (M. Parida, et al., J. Clin. Microbiol, 2005, 43, 2895-2903) Japanese encephalitis (M. M. Parida, et al., J. Clin. Microbiol., 2006, 44, 4172-4178), chikungunya fever (M. M. Parida, et al., J. Clin. Microbiol., 2007, 45, 351-357), West Nile fever (M. Parida, et al., J. Clin. Microbiol., 2004, 42, 257-263), severe acute respiratory syndrome (SARS) (T. C. T. Hong, Q. L. Mai, D. V. Cuong, M. Parida, H. Minekawa, T. Notomi, F. Hasebe and K. Morita, J. Clin. Microbiol., 2004, 42, 1956-1961) and highly pathogenic avian influenza (HPAI) H5N1 (M. Imai, et al., J. Virol. Methods, 2007, 141, 173-180); each of the sources listed is expressly and in its entirety incorporated herein by reference.

[0402] In the SDA example, the probe includes two parts: a Hinc II recognition site at the 5' end and another segment that includes sequences complementary to the target. DNA polymerase can extend the primer and include deoxyadenosine-5'-[α-thio]triphosphate (dATP[αS]). The Hinc II restriction endonuclease then "nicks" the probe strand at the Hinc II recognition site, since said endonuclease cannot cleave another strand that includes a thiophosphate modification. Digestion with an endonuclease exposes the 3'-OH, which is then extended by DNA polymerase. The newly generated strand still contains the Hinc II nicking site. Subsequent nicking of the newly synthesized duplex followed by DNA polymerase-mediated elongation is repeated several times and this results in an isothermal amplification cascade. In some embodiments of the methods of the present invention, the SDA components include 4 primers, DNA polymerase, REase HincII enzyme, dGTP, dCTP, dTTP, and dATPαS. Examples of the use of SDA include the genomic DNA of Mycobacterium tuberculosis (source: M. Vincent, et al., EMBO Rep., 2004, 5, 795-800, which is expressly and fully incorporated herein by reference).

[0403] In the NASBA example, forward primer 1 (P1) consists of two parts, one of which is complementary to the 3' end of the target RNA, and the other to the sequence of the T7 promoter. When P1 binds to the target RNA (RNA (+)), reverse transcriptase (RT) extends the primer into the complementary DNA (DNA (+)) of the target RNA. 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) which contains the T7 promoter sequence. After said initial phase, the system enters the amplification phase. T7 RNA polymerase generates multiple strands of RNA (RNA (-)) based on dsDNA, and the reverse primer (P2) binds to the newly formed RNA (-). RT extends the reverse primer and RNase H degrades the RNA in the RNA-cDNA duplex to produce ssDNA. The newly produced cDNA (DNA(+)) then becomes the template for P1 and the cycle repeats. In some embodiments of the methods of the present invention, the NASBA components 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, 4034-4041), hepatitis C virus RNA (M. Damen, et al., J. Virol. Methods, 1999, 82, 45-54), human cytomegalovirus mRNA (F. Zhang, et al., J. Clin. Microbiol., 2000, 38, 1920-1925), bacterial species 16S RNA (S. A. Morre, et al. , J. Clin. Pathol.: Clin. Mol. Pathol., 1998, 51, 149-154) and enterovirus genomic RNA (J. D. Fox, et al., J. Clin. Virol., 2002, 24, 117-130) . Each of the above references is expressly incorporated herein by reference in its entirety.

[0404] Additional examples of isothermal amplification methods include: self-sustaining sequence replication reaction (3SR); 90-I; BAD Amp; cross-priming amplification (CPA); exponential isothermal amplification reaction (EXPAR); isothermal chimeric primer initiated nucleic acid amplification (ICAN); isothermal amplification with multiple displacement (IMDA); mediated by SDA ligation; amplification with multiple displacement; polymerase helical reaction (PSR); exponential restriction cascade amplification (RCEA); Smart Amplification Process (SMAP2); single primer amplification reaction (SPIA); transcription-based amplification system (TAS); transcription-mediated amplification (TMA); ligase chain reaction (LCR); and/or multiple displacement cross-amplification (MCDA).

Overview of Immune Isothermal Amplification Examples

[0405] Some embodiments of the methods, systems, and compositions of the present invention include the use of immune isothermal amplification to detect non-nucleic acid targets. In some such embodiments, primers suitable for use in an isothermal amplification method are linked to an antibody, or fragment thereof, or an aptamer. As used herein, an "aptamer" may include a peptide or oligonucleotide that specifically binds to a target molecule. In some embodiments, said antibody or said aptamer may be linked to primers suitable for use in an isothermal amplification method via covalent or non-covalent bonds. In some embodiments, primers suitable for use in an isothermal amplification method may be linked to an antibody or aptamer via biotin and streptavidin linkers. In some embodiments, primers suitable for use in the isothermal amplification method can be linked to the antibody or aptamer using THUNDER-LINK (Innova Biosciences, UK).

[0406] In some embodiments, the target antigen binds to an antibody or aptamer, and primers associated with said antibody or aptamer are a substrate for isothermal amplification and/or initiate isothermal amplification. See, for example, Pourhassan-Moghaddam et al., Nanoscale Research letters, 8:485-496, which is hereby expressly incorporated by reference in its entirety. In some embodiments, the target antigen is captured in a "sandwich" form between two antibodies or aptamers (ATs), a capture antibody and a detection antibody that specifically bind to the target antigen. The capture AT, previously immobilized on the surface of the solid support, captures the target Ag, and the detection AT, which is associated with primers suitable for use in the isothermal amplification method, is attached to the captured Ag. After washing, isothermal amplification is carried out, and the presence of amplified products indirectly indicates the presence of target Ag in the sample.

Overview of Examples of Enhancement of Conductivity Changes

[0407] Some embodiments of the methods, systems, and compositions of the present invention include amplifying changes in solution conductivity as a result of nucleic acid amplification. In some embodiments, pyrophosphate ("PPi") chelation that results from nucleic acid amplification can be used to amplify changes in solution conductivity as the amplification reaction continues. Without wishing to be bound by a particular theory, the conductivity changes that may occur during nucleic acid amplification may be based on precipitation of magnesium cations and PPi ions from solution. Some embodiments of the methods proposed according to the present invention may include increasing the change in conductivity by changing the equilibrium states, which otherwise leads to the precipitation of magnesium cations and PPi ions. In some embodiments, this is done by adding molecules that compete with magnesium cations for PPi. In some such embodiments, high ionic mobility compounds are provided that contribute significantly to the net conductivity of a solution. Accordingly, removing said compound from solution by precipitating said PPi compounds provides a dramatic change in the conductivity of the solution. In some embodiments of the methods of the present invention, compounds/complexes that can bind PPi and result in changes and/or amplification of changes in solution conductivity as amplification continues, include Cd ²⁺ -cyclen-coumarin; complexes of Zn ²⁺ with bis(2-pyridylmethyl)amine (DPA) unit; DPA-2Zn ²⁺ -phenoxide; acridine-DPA-Zn ²⁺ ; DPA-Zn ²⁺ -pyrene; and azacrown-Cu ²⁺ complexes. See, for example, references: Kim SK et al., (2008) Accounts of Chemical Research 42: 23-31; and Lee DH, et al., (2007) Bull. Korean Chem. soc. 29:497-498; Credo GM et al., (2011) Analyst 137:1351-1362; and Haldar BC (1950) "Pyrophosphato-Complexes of Nickel and Cobalt in Solution" Nature 4226:744-745, each expressly incorporated herein by reference in its entirety.

[0408] Some embodiments include compounds such as 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG). MESG is used in pyrophosphate detection kits such as the EnzChek® Pyrophosphate Assay Kit (ThermoFischer Scientific), where the enzyme purine nucleoside phosphorylase (PNP) converts MESG to ribose-1-phosphate and 2-amino-6-mercapto-7-methylpurine in the presence of an inorganic phosphate. Enzymatic conversion of MESG leads to a shift in the absorption maximum from 330 nm to 360 nm. PNP catalyzes the conversion of pyrophosphate to two phosphate equivalents. The phosphate then goes into the MESG/PNP reaction and is detected by an increase in absorbance at 360 nm. Additional sensitivity is provided by the amplification of one molecule of pyrophosphate to obtain two molecules of phosphate. Another kit includes the PIPER Pyrophosphate Assay Kit (ThermoFischer Scientific).

[0409] In some embodiments, amplification of solution conductivity changes resulting from nucleic acid amplification include compounds that bind amplified DNA. In some such embodiments, as amplification continues, charge carrier substances bind to increasing amounts of amplified DNA, resulting in an absolute decrease in the conductivity of the solution. In some embodiments, charged carrier substances may include positively charged molecules commonly used for staining/as dyes for DNA/RNA, such as ethidium bromide, crystal violet, SYBR, which bind to nucleic acids by electrostatic attraction. Coupling these small charged molecular species to larger and less mobile amplification products can decrease the conductivity of the solution by effectively reducing the mobility of the charged dye molecules. It should be noted that although this electrostatic attraction is a mechanism often used in DNA staining for gel electrophoresis, the molecules that bind to the amplicons are not necessarily the compounds traditionally used for DNA staining. Since these molecules are used because of their function as charge carriers (contributing to the conductivity of the solution) as well as their ability to bind to the amplicon, they do not necessarily have the ability to stain DNA in any way.

[0410] Some embodiments include the use of antibodies or aptamers associated with the nanoparticle. In some such embodiments, the presence of the target antigen results in antibody aggregation and a change in the conductivity of the solution. Without being bound by a particular theory, the effective electrical conductivity of colloidal nanosuspensions in a liquid may exhibit a complex dependence on electrical double layer (EDL) characteristics, volume content, ionic concentrations, and other physicochemical properties. See, for example, Angayarkanni SA., et al., Journal of Nanofluids, 3: 17-25, which is expressly and fully incorporated herein by reference. Antibody-conjugated nanoparticles are well known in the art. See, for example, sources: 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 hereby expressly incorporated by reference in its entirety. Examples of nanoparticles that are suitable for use in the methods proposed according to the present invention include γ-Al ₂ O ₃ , SiO ₂ , TiO ₂ and α-Al ₂ O ₃ and gold nanoparticles, see for example, source: Abdelhalim, MAK. , et al., International Journal of the Physical Sciences, 6:5487-5491, expressly incorporated herein by reference in its entirety. The use of antibodies bound to the nanoparticles can also enhance the signal generated at the surface when measured using electrochemical impedance spectroscopy (EIS). See, for example, Lu J., et al., Anal Chem. 84:327-333 which is hereby incorporated by reference in its entirety.

[0411] Some embodiments of the methods, systems, and compositions of the present invention include the use of enzyme-linked antibodies or aptamers. In some embodiments, the activity of the enzyme provides a change in the conductivity of the solution. In some such embodiments, a change in conductivity is detected by charge transfer to the substrate in contact with the assay components.

Overview of examples of viral targets

[0412] Some embodiments of the methods, systems and compositions proposed according to the present invention include the detection of certain viruses and viral targets. The viral target may include a viral nucleic acid, a viral protein, and/or a product of viral activity such as an enzyme or activity thereof. Examples of viral proteins that are detected using the methods and devices of the present invention 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 gene encoding the aforementioned viral proteins are also detected using the methods and devices described herein. The nucleotide sequences of such targets are easily obtained from public databases. Primers suitable for isothermal amplification are easily designed based on the nucleic acid sequences of the desired viral targets. Antibodies and aptamers to the proteins of such viruses are also readily available commercially and/or using techniques well known in the art. Examples of viruses that are detected using the methods, systems and compositions proposed according to the present invention include DNA viruses, such as double-stranded DNA viruses and single-stranded viruses; RNA viruses such as double-stranded RNA viruses, single-stranded (+) RNA viruses, and single-stranded (-) RNA viruses; as well as reverse transcribed viruses such as single strand reverse transcribed RNA viruses and double strand reverse transcribed DNA viruses. Viruses that are detected using this technology include animal viruses such as human viruses, domestic animal viruses, livestock viruses or plant viruses. Examples of human viruses that are detected using the methods, systems and compositions of the present invention include those listed in Table 2 below, which also provides examples of nucleotide sequences from which primers suitable for amplification can be easily designed.

Overview of examples of bacterial targets

[0413] Some embodiments of the methods, systems and compositions of the present invention include the detection of certain bacteria and bacterial targets. A bacterial target includes a bacterial nucleic acid, a bacterial protein, and/or a product of bacterial activity such as toxins and enzyme activities. Nucleotide sequences pointing to certain bacteria are easily obtained from public databases. Primers suitable for isothermal amplification are readily designed based on the nucleic acid sequences of such bacterial targets. Antibodies and aptamers to certain bacterial proteins are readily obtained commercially and/or using techniques well known in the art. Examples of bacteria that are detected using the methods, systems and compositions proposed according to the present invention include gram-negative or gram-positive bacteria. Examples of bacteria that are detected using the methods, systems and compositions of the present invention include: Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium Salmonella typhi Salmonella paratyphi Salmonella enteritidis Shigella dysenteriae Shigella Jlexneri Shigella sonnei Enterobacter cloacae Enterobacter aerogenes Klebsiella pneumoniae Klebsiella oxytoca Serratia marcescens Francisella tularensis Morganella morganii , Providencia rettgeri, Providencia stuartii, Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertus sis Bordetella bronchiseptica Haemophilus influenzae Haemophilus parainfluenzae Haemophilus haemolyticus Haemophilus parahaemolyticus Haemophilus ducreyi Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Kingella, Moraxella, Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis, группу гомологии Bacteroides 3452A, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, Bacteroides splanchnicus, Clostridium difficile , Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium leprae, Corynebacterium diphtheriae, Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes , Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus hyicus, subspecies hyicus, Staphylococcus haemolyticus, Staphylococcus hominis and/or Staphylococcus saccharolyticus. Additional examples include B. anthracis, B. globigii, Brucella, E. herbicola, or F. tularensis.

Overview of examples of antigenic targets

[0414] Some embodiments of the methods, systems and compositions of the present invention include the detection of certain antigenic targets. Antigens are detected using antibodies, their binding fragments, or aptamers coupled to primers configured to allow amplification, such as isothermal amplification. Antibodies and aptamers for certain antigens are readily available commercially and/or using techniques well known in the art. As used herein, "antigen" includes a compound or composition that specifically binds an antibody, its binding fragment, or an aptamer. Examples of antigens that are detected using the methods, systems and compositions of the present invention include proteins, polypeptides, nucleic acids and small molecules such as pharmaceutical compounds. Additional examples of analytes include toxins such as ricin, abrin, botulinum toxin, or staphylococcal enterotoxin B.

Review of examples of parasitic targets

[0415] Some embodiments of the methods, systems, and compositions of the present invention include the detection of certain parasitic targets. The parasitic target includes a parasite nucleic acid, a parasite protein, and/or a parasite activity product, such as a toxin and/or an enzyme, or an enzyme activity. Nucleotide sequences indicating certain parasites are readily available from public databases. Primers suitable for isothermal amplification are readily designed based on the nucleic acid sequences of such parasitic targets. Antibodies and aptamers to proteins of certain parasites are readily obtained commercially and/or using techniques well known in the art. Examples of parasites that are detected using the methods, systems and compositions of the present invention include certain endoparasites such as protozoan organisms such as Acanthamoeba spp. Babesia spp., B. divergens, B. bigemina, B. equi , B. microfti, B. duncani, Balamuthia mandrillaris, Balantidium coli, Blastocystis spp., Cryptosporidium spp., Cyclospora cayetanensis, Dientamoeba fragilis, Entamoeba histolytica, Giardia lamblia, Isospora belli, Leishmania spp., Naegleria fowleri, Plasmodium falciparum, Plamodium vivax , Plasmodium ovale curtisi, Plasmodium ovale wallikeri, Plasmodium malariae, Plasmodium knowlesi, Rhinosporidium seeberi, Sarcocystis bovihominis, Sarcocystis suihominis, Toxoplasma gondii, Trichomonas vaginalis, Trypanosoma brucei, or Trypanosoma cruzi. Certain helminth organisms such as Bertiella mucronata, Bertiella studeri, Cestoda, Taenia multiceps, Diphyllobothrium latum, Echinococcus granulosus, Echinococcus multilocularis, E. vogeli, E. oligarthrus, Hymenolepis nana, Hymenolepis diminuta, Spirometra erinaceieuropaei, Taenia saginata, or Taenia saginata solium. Certain trematode organisms such as Clonorchis sinensis; Clonorchis viverrini, Dicrocoelium dendriticum, Echinostoma echinatum, Fasciola hepatica, Fasciola gigantica, Fasciolopsis buski, Gnathostoma spinigerum, Gnathostoma hispidum, Metagonimus yokogawai, Metorchis conjunctus, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Paragonimus westermani; Paragonimus africanus; Paragonimus caliensis; Paragonimus kellicotti; Paragonimus skrjabini; Paragonimus uterobilateralis, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni and Schistosoma intercalatum, Schistosoma mekongi, Schistosoma sp, Trichobilharzia regenti or Schistosomatidae. Certain ascaris organisms such as Ancylostoma duodenale, Necator americanus, Angiostrongylus costaricensis, Anisakis, Ascaris sp. Ascaris lumbricoides, Baylisascaris procyonis, Brugia malayi, Brugia timori, Dioctophyme renale, Dracunculus medinensis, Enterobius vermicularis, Enterobius gregorii, Halicephalobus gingivalis, pathogen of loiasis, Mansonella streptocerca, Onchocerca volvulus, Strongyloides stercoraliaeis, Thelazia, Toxara californiensis, The catarociporniensis , Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa, Trichuris trichiura, Trichuris vulpis or Wuchereria bancrofti. Other parasites such as Archiacanthocephala, Moniliformis moniliformis, Linguatula serrata, Oestroidea, Calliphoridae, Sarcophagidae, Cochliomyia hominivorax (family Calliphoridae), Tungapenetrans, Cimicidae: Cimex lectularius or Dermatobia hominis. Additional examples of parasites include ectoparasites such as Pediculus humanus, Pediculus humanus corporis, Pthirus pubis, Demodex folliculorum/brevis/canis, Sarcoptes scabiei or arachnids such as Trombiculidae or Pulex irritans or arachnids such as Ixodidae and/or Argasidae.

Overview of examples of target miRNAs

[0416] Some embodiments of the methods, systems, and compositions of the present invention include the detection of certain microRNA (miRNA) targets. miRNAs include small non-coding RNA molecules that function in RNA silencing or post-transcriptional regulation of gene expression. Some miRNAs are associated with dysregulation in various human diseases that are driven by abnormal epigenetic patterns, including abnormal DNA methylation and histone modification patterns. For example, the presence or absence of certain siRNAs in a sample from a subject is indicative of a disease or disease state. Primers suitable for siRNA detection and suitable for isothermal amplification are easily designed based on siRNA nucleotide sequences. MiRNA nucleotide sequences are easily obtained from public databases. Examples of miRNA targets that are detected using the methods, systems and compositions proposed according to the present invention 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, hsa-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, hsa-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-17-5p, hsa-miR-17-92, hsa-miR-18, hsa-miR-18a, hsa- miR-18b, hsa-miR-181a, hsa-miR-181a-1, hsa-miR-181a-2, hsa-miR-181b, 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-miR-199a-1, hsa-miR-19 9a-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-1, 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, 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, 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-337-3р, 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-409-3р, 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, hsa-miR-452, hsa-miR-451, hsa-miR-452, hsa-miR-452, hsa-miR-483, hsa-miR-483-3р, 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-493-3р, 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- miR-512-5p, 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-542-3р, hsa-miR-542-5p, hsa-miR-550, hsa-miR-551a, hsa-miR-561, hsa-miR-563, 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-615-3р, 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-654, 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-769-3р, hsa-miR-802, hsa-miR-885-3р, hsa-miR-9, hsa-miR-9-1, hsa-miR-9-3, hsa-miR-9-3р, 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-99b.

Overview of examples of agricultural analytes

[0417] Some embodiments of the methods, systems and compositions proposed according to the present invention include the detection of certain analytes of agricultural importance. Agricultural analytes include nucleic acids, proteins or small molecules. Nucleotide sequences indicating certain analytes of agricultural importance are readily available from public databases. Primers suitable for isothermal amplification are easily designed based on the nucleic acid sequences of such agricultural analytes. Antibodies and aptamers to proteins of certain agricultural analytes are readily available commercially and/or using techniques well known in the art.

[0418] Some embodiments of the methods and devices of the present invention are used to identify the presence of an organism or product of said organism in a meat product, fish product, or yeast product 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 a particular organism in a food product.

[0419] Some embodiments of the methods, systems, and compositions of the present invention include pesticide detection. In some embodiments, the pesticides are found in samples such as soil samples or food samples. Examples of pesticides that are detected using the devices and methods described herein include herbicides, insecticides, or fungicides. Examples of herbicides include 2,4-dichlorophenoxyacetic acid (2,4-D), atrazine, glyphosate, mecoprop, dicamba, paraquat, glufosinate, metham sodium, dazomet, ditopyr, pendimethalin, EPTC, trifluralin, flazasulfuron, metsulfuron-methyl, diuron , nitrofen, nitrofluorfen, acifluorfen, mesotrione, sulcotrione, or nitisinone. Examples of insecticides that are detected using the devices and methods described herein include organochlorines, organophosphates, carbamates, pyrethroids, neonicotinoids, or ryanoids. Examples of fungicides that are detected using the devices and methods described herein include carbendazim, diethophencarb, azoxystrobin, metalaxyl, metalaxyl-M, streptomycin, oxytetracycline, chlorothalonil, tebuconazole, zineb, mancozeb, tebuconazole, myclobutanil, triadimefon, fenbuconazole, deoxynivalenol or mancozeb.

Overview of examples of biomarkers

[0420] Some embodiments of the methods, systems and compositions of the present invention include the detection of certain biomarkers of certain disorders. Biomarkers may include nucleic acids, proteins, protein fragments, and antigens. Some biomarkers may include the target proposed according to the present invention. Examples of disorders include cancer such as breast cancer, colon cancer, gastric cancer, gastrointestinal stromal tumors, leukemias and lymphomas, lung cancer, melanomas, brain cancer, and pancreatic cancer. Some implementation options may include detecting the presence or absence of a biomarker, or the level of a biomarker in a sample. Said biomarker may indicate the presence, absence, or stage of a particular disorder. Examples of biomarkers include estrogen receptor, progesterone receptor, HER-2/neu, rEGFR, KRAS, UGT1A1, C-KIT, CD20, CD30, FIP1L1-rTRF-alpha, rTRF, Philadelphia chromosome (BCR/ABL), PML/RAR-alpha , TPMT, UGT1A1, EML4/ALK, BRAF, and elevated levels of certain amino acids such as leucine, isoleucine, and valine.

EXAMPLES

Example 1 Pre/Post ƒC4D LAMP detection in PDMS

[0421] The reaction mixture for LAMP was prepared according to the standard NEB protocol, using the untranslated 5' region of the H. influenza genome as a target. Aliquots of the mixture were placed in the pre-amplification vessel (- control) and post-amplification vessel (+ control).

The preamplification vessel was inactivated by heating at 85°C for 20 minutes to prevent amplification. Postamplification vessel was amplified at 63°C for 60 minutes. Aliquots from each vessel were applied sequentially, alternating two vessels, at room temperature, onto a polydimethylsiloxane (PDMS)/v.1.1 glass chip, with real-time data collection. In FIG. 24 is a graph showing sensor voltage over a period of time.

Example 2 Pre/Postamp ƒC4D Whole Blood PDMS Detection

[0422] The reaction mixture was prepared using the untranslated 5' region of the H. influenza genome as a target, with 0%, 1% and 5% whole blood (by volume). Aliquots of the mixture were placed in the pre-amplification vessel (- control) and post-amplification vessel (+ control). The preamplification vessel was inactivated by heating at 85°C for 20 minutes to prevent amplification. Postamplification vessel was amplified at 63°C for 60 minutes. Aliquots from each vessel were applied sequentially, alternating two vessels, at room temperature, onto a PDMS/glass chip v.1.1, with real-time data collection. In FIG. 25, fig. 26 and FIG. 27 are plots of sensor voltage over time before amplification (- control) and after amplification (+ control) for 0%, 1%, and 5% whole blood, respectively.

Example 3 Filtration before/after LAMP amplification

[0423] Samples were obtained according to example 1. Before measurement, all samples (with the exception of one control) were filtered by centrifugation with a filter with a cut-off mass of 50 kD. Aliquots from each vessel were applied sequentially, alternating two vessels, at room temperature, onto a PDMS/glass chip v.1.1, with real-time data collection. The filtering improved the signal-to-noise ratio and the change in conductance. In FIG. 28 and FIG. 29 are plots of sensor voltage over time before amplification (- control) and after amplification (+ control) with 0% whole blood, for an unfiltered sample and a filtered sample, respectively.

Example 4 Detection of Conductivity for 10 ³ -10 ⁶ Target Copies

[0424] The reaction mixture was prepared using the untranslated 5' region of the H. influenza genome as a target. Detection was performed using the ƒC ⁴ D instrument. Data were averaged over 3 replicates. In FIG. 30 is a plot of time versus target load; error bars show the standard deviation. None of the matrix negative controls showed no signal when heated for 60 minutes.

Example 5 Pre/postamplification detection of ƒC4D with whole blood in PDMS

[0425] The reaction mixture was prepared using the untranslated 5' region of the H. influenza genome as a target, with 0% or 1% whole blood (by volume). Aliquots of the mixture were placed in the pre-amplification vessel (- control) and post-amplification vessel (+ control). The preamplification vessel was inactivated by heating at 85°C for 20 minutes to prevent amplification. Postamplification vessel was amplified at 63°C for 60 minutes. Aliquots from each vessel were applied sequentially, alternating two vessels, at room temperature, onto a PDMS/glass chip v.1.1, with real-time data collection. In FIG. 31 is a graph of conductivity for various samples from the pre-amplification vessel (- control) and the post-amplification vessel (+ control).

Example 6 Hepatitis B Surface Antigen Detection Using MAIA

[0426] A biotinylated polyclonal capture probe antibody (anti-HBsAg) was conjugated to streptavidin-functionalized 1 μm magnetic microspheres (Dynal T1). Chimeric detection complexes were synthesized by conjugation of a biotinylated polyclonal capture probe (anti-HBsAg) with streptavidin, and conjugation of the streptavidin-antibody complex with a biotinylated target DNA. The antibody-functionalized beads captured HBsAg from solution. HBsAg was detected by binding of the chimeric complex of antibody and DNA, followed by amplification of the DNA-template part of the chimeric complex. In FIG. 32 depicts the binding of an antigen to a nucleic acid conjugated antibody. In FIG. 33 is a graph showing the detection of hepatitis B surface antigen.

Example 7 Detection with Low Ionic Strength Buffer

[0427] A commercial amplification solution and a T10 amplification solution were prepared from the reagents listed in Table 3 and Table 4, respectively. Commercial amplification solution has generally been used for general amplification reactions. The T10 amplification solution is characterized by a reduced content of Tris-HCl and the absence of ammonium sulfate. 400 μl of each solution was prepared, and approximately 15 μl of each solution was added to different channels of the experimental cartridge. The solutions were heated to 63.0°C. Data was collected using a data collection panel.

[0428] The results are shown in FIG. 34. The T10 amplification buffer provided at least 30% stronger signal compared to the signal provided by the commercial amplification solution.

Example 8 Fluid Cartridge Impedance Characteristics

[0429] The fluid cartridge channels shown in FIG. 17A was filled with a reference buffer with a conductivity of 1288 mS/cm and the drive frequency was adjusted from less than about 100 Hz to more than about 1 MHz and the impedance ("|Z|") or arg Z was measured as a function of frequency. The results are shown in FIG. 35 as either |Z| or arg Z depending on the frequency.

Example 9 Amplification of Nucleic Acids Containing HCV Sequences

[0430] Samples containing nucleic acids containing hepatitis C virus (HCV) sequences were amplified in a series of experiments using LAMP under various conditions, and cycle threshold (C _t ) values were determined along with standard deviations (SD), and relative standard deviation in % (RSD). Nucleic acids included synthetic nucleic acids containing the HCV sequence; synthetic RNA containing the HCV sequence. All reactions contained 5% Tween-20. For experiments with reactions containing approximately one million copies of synthetic nucleic acids containing the HCV sequence, the mean Ct was 856, with an SD of 15 and an RSD of 1.72%.

[0431] Plasma samples containing synthetic RNA containing the HCV sequence were amplified using LAMP under various conditions, including: no treatment, after heat treatment before the addition of said synthetic RNA, with heat after the addition of said synthetic RNA, and with the addition of 100 mM DTT. Each reaction contained approximately 25,000 copies of the nucleic acid. Table 5 summarizes the results.

[0432] Addition of 100 mM DTT or heat treatment of plasma prior to addition of said synthetic RNA improved amplification as seen by RSD compared to untreated samples. The addition of DTT or heat treatment of the plasma prior to addition of said synthetic RNA also resulted in faster amplification (approximately 50 times faster) than untreated samples (P=0.03 and 0.002, respectively).

[0433] Plasma samples containing HCV (SeraCare, Milford, Massachusetts) were amplified using LAMP under various conditions, including: heat treatment of plasma, addition of 100 mM DTT, addition of SDS and/or DTT. Table 6 summarizes the results.

[0434] Treatment of plasma with heat or addition of DTT improved amplification results compared to untreated plasma as seen from RSD values. The addition of either 0.05% or 0.1% SDS reduced the reproducibility and rate of amplification compared to plasma that was untreated, heat treated, or to which DTT was added.

Example 10 Amplification of Clinical Specimens Containing HCV

[0435] Clinical plasma samples containing HCV were amplified using LAMP using various concentrations of DTT. WarmStart Master Mix for LAMP (New England Biolabs) was used to obtain samples in quadruplicate. Samples included: 5% plasma (SeraCare, Milford, Massachusetts) containing approximately ~20k copies of HCV per reaction, 50 U per mouse RNase inhibitor reaction, at various concentrations of Tween and DTT. Samples containing synthetic nucleic acids containing the HCV sequence (1 million copies per reaction) were tested with 1% and 5% Tween. No-target controls (NTC) were also tested. LAMP was run at 67°C and the results were measured using the Zeus QS3 system for 60 cycles of 1 min/cycle, data was recorded during each cycle, and after the completion of LAMP, an ascending/descending melting curve was used. The results are summarized in Table 7.

[0436] Samples containing 5% Tween had improved amplification compared to samples containing 1% Tween, as seen from the RSD values. A similar study was carried out by additionally varying the concentration of Tween in the reaction tubes. The results are summarized in Table 8.

[0437] Reaction volumes with higher concentrations of Tween and DTT had better reproducibility of amplification results of HCV samples, in particular, there were fewer extreme outliers in replicates, fewer failed amplification reactions, and lower RSD values for amplified replicates. At 5 mM DTT and 10 mM DTT, non-amplified repeats were absent at any concentration of Tween. Similarly, there were no failed replicates or extreme outliers at 4% and 5% Tween, except when using low (1 mM and below) DTT concentrations.

Example 11 Cartridge Target Amplification

[0438] A series of three experiments was carried out using a cartridge substantially similar to the cartridge shown in FIG. 2 with six cells each containing a ring electrode. Each cell was associated with a measured channel. Samples included target nucleic acids containing Haemophilus influenza (Hinf) or hepatitis B virus (HBV) sequences. Samples were amplified using LAMP, and impedance changes were measured.

[0439] The wells were prepared by preheating the cartridge to 72°C for 20 minutes, filling each well with 25 µl of "template and primer free control buffer" (NTPC), blocking said buffer with mineral oil, heating the cartridge to 72°C for 20 minutes, removing bubbles from the cells, cooling the cartridge at room temperature for 10 minutes. Samples were introduced to the bottom of the pre-filled wells and the cartridge was placed under conditions of 67° C. or 76.5° C. to conduct LAMP in a particular experiment. For studies with Hinf, a frequency of 60 kHz was used. Samples and corresponding wells/channels for each cartridge are listed in Table 9. Target sequences and primers are listed in Table 10. Reaction components are listed in Table 11.

[0440] Data for LAMP carried out on a cartridge at 65°C is shown in FIG. 36A and 36B. In FIG. 36A is a graph of the out-of-phase portion of the attenuated drive signal detected in the test cell of the cartridge of FIG. 2 where the x-axis is time and lines are marked which correspond to LAMP on NTPC samples, Hinf examples and synthetic HBV. In FIG. 36B is a plot of the in-phase portion of the attenuated drive signal detected in the test cell of the cartridge of FIG. 2 where the lines correspond to synthetic HBV (channels (Ch) 1-3), NTPC (channel 4) and Hinf (channels 5-6). Samples containing synthetic HBV were not amplified on the cartridge at 65°C. The labeled sample with Hinf shows an example of a spike indicating a positive sample.

[0441] Data for LAMP carried out on a cartridge at 67°C is shown in FIG. 36C and 36D. In FIG. 36C is a graph of the out-of-phase portion of the attenuated drive signal detected in the test cell of the cartridge of FIG. 2 where the x-axis is time and lines are marked which correspond to LAMP on NTPC samples, Hinf examples and synthetic HBV. In FIG. 36D is a graph of the in-phase portion of the attenuated drive signal detected in the test cell of the cartridge of FIG. 2, where the lines correspond to synthetic HBV (channels 1-3), NTPC (channel 4) and Hinf (channels 5-6). Samples containing synthetic HBV were amplified on the cartridge at 67° C. in approximately 49 minutes. The labeled sample with Hinf shows an example of a spike indicating a positive sample.

[0442] LAMP data carried out on the cartridge at 67°C are shown in FIG. 36E and 36F. In FIG. 36E is a graph of the out-of-phase portion of the attenuated drive signal detected in the test cell of the cartridge of FIG. 2, where the x-axis is time, and lines are marked that correspond to LAMP on samples for NTPC, and examples of Hinf and synthetic HBV. In FIG. 36F is a graph of the in-phase portion of the attenuated drive signal detected in the test cell of the cartridge of FIG. 2, where the lines correspond to synthetic HBV (channels 1-3), NTPC (channel 4) and Hinf (channels 5-6). Samples containing synthetic HBV were amplified on the cartridge at 67° C. in approximately 46 minutes.

[0443] Samples were also tested using quantitative PCR using the Applied Biosystems QuantStudio™ 3 real-time PCR system at 67°C. Threshold cycles (C _t ) were calculated using QS3 software from Thermo Fisher with a set threshold of 10 ⁴ and the same set base value for all sets of the same reactions. Table 12 shows the median Ct values for samples containing Hinf or synthetic HBV.

Systems Implementation and Terminology

[0444] In embodiments as described herein, systems, methods, and apparatus are provided for detecting the presence and/or amount of a target analyte. One skilled in the art will appreciate that these embodiments may be implemented in hardware or a combination of hardware and software and/or firmware.

[0445] The signal processing and reader control functions described herein may be stored as one or more instructions on a processor-readable or computer-readable medium. The term "computer-readable medium" refers to any available medium that a computer or processor can connect to. For example, without limitation, such media may include RAM, ROM, EEPROM, flash memory, CD-ROM or other optical disk storage media, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store the required program code in the form of instructions or a data structure, and to which a computer can connect. It should be noted that the computer-readable medium may be tangible and durable. The term "computer program product" refers to a computing device or processor in combination with code or instructions (eg, a "program") that can be executed, processed, or computed by said computing device or processor. As used herein, the term "code" may refer to software, instructions, code, or data executable or executable by a computing device or processor.

[0446] The various illustrative logical blocks and modules described in connection with the embodiments herein may be implemented or implemented by a machine, such as a general purpose processor, a digital signal processor (DSP), a user programmable application specific integrated circuit (ASIC). gate array (FPGA) or other programmable logic device, discrete logic element or transistorized logic circuit, discrete hardware components, or any combination thereof designed to perform the functions described in this document. A general purpose processor may be a microprocessor, however, in the alternative, said processor may be a controller, microcontroller, combinations thereof, or the like. A processor may also be implemented as a combination of computing devices, such as a combination of a DSP and a microprocessor, a collection of microprocessors, one or more microprocessors in combination with a DSP core, or any other such configuration. While the description herein is primarily applicable to digital technology, a processor may also include primarily analog components. For example, any of the signal processing algorithms described herein may be implemented in analog circuitry. The computing environment may include any type of computer system, including, but not limited to, a microprocessor-based computer system, a large computer, a digital signal processor, a portable computing device, a personal organizer, a device controller, and an in-device computing mechanism, among others. .

[0447] The methods described herein include one or more steps or steps to implement the described method. Said method steps and/or actions may be interchanged without departing from the scope of the claims. In other words, if a specific order of steps or actions is not required for the correct performance of the described method, the order and/or application of specific steps and/or actions can be modified without departing from the scope of the claims.

[0448] The term "comprising" in this document is synonymous with the terms "including", "comprising" or "characterized" by something, is inclusive or non-limiting, and does not exclude additional elements or steps not mentioned.

[0449] In the above description, some of the methods and materials proposed according to the present invention are disclosed. The present invention contemplates modifications to methods and materials, as well as changes to process methods and equipment. Such modifications will become apparent to those skilled in the art upon examination of this document or practice of the present invention as described in the document. Accordingly, the present invention is not intended to be limited to the specific embodiments described herein, but to cover all modifications and alternatives falling within the scope and spirit of the present invention.

[0450] All sources cited herein, including, but not limited to, published and unpublished applications, patents, and references, are incorporated herein in their entirety by reference and thus form part of the present invention. To the extent that publications and patents or patent applications incorporated by reference conflict with the invention disclosed herein, the present description is intended to supersede any such conflicting material and/or take precedence.

Claims (112)

1. A system for detecting a target agent, comprising: an analytical cartridge comprising a test cell containing an excitation electrode and a sensor electrode, wherein the test cell is configured to contain a sample containing the target agent undergoing the amplification process, wherein said target agent contains a nucleic acid; and reader, including: an area configured to receive an analytical cartridge, a heater positioned so as to heat the assay cartridge in use inside the cavity, a memory storing at least machine-readable storage instructions, and a processor configured by the specified instructions so that at least: causing the assay cartridge to be heated by the heater to a predetermined temperature to perform the amplification process within the test cell; to apply the excitation current to the excitation electrode for at least part of the time of the amplification process, receive a signal from the sensor electrode corresponding to the excitation current after its decay due to interaction with at least the sample inside the test cell, decompose the specified signal into a component of active resistance and a component of reactance, analyze the reactance component to determine the presence of a signal drop with respect to time and at a certain frequency of the specified signal, indicating a positive sample containing the target agent, for at least part of the time of the amplification process, and output a positive test result in response to determining the occurrence of a signal drop; or output a negative test result in response to determining the absence of a signal edge. 2. The system according to claim. 1, characterized in that the specified analytical cartridge additionally contains: a sample input area configured to receive a sample; and a flow channel that fluidly connects the sample injection area to the test cell. 3. The system of claim 2, wherein the assay cartridge further comprises: a sealed chamber containing the liquid components of the amplification process, located in the area of the analytical cartridge having an opening leading to the flow channel, and the sample injection area is located between the specified hole and the test cell along the flow channel; and a fluid pneumatic conduit fluidly connecting the pneumatic interface device to the area of the analytical cartridge, at the same time, the dried components of the amplification process are placed in the specified test cell. 4. The system of claim. 3, in which the reader includes a pneumatic system configured to supply pressure through a pneumatic interface device, a processor further configured by these instructions so that at least: actuating a motor coupled to an actuator positioned to break through the sealed chamber and cause liquid constituents to enter the area of the analytical cartridge; and activate the pneumatic system, causing the liquid components to enter the flow channel and transfer the sample from the sample injection area to the test cell. 5. The system according to claim 4, characterized in that said analytical cartridge further comprises a mixing chamber located between the sample input area and the test cell along the flow channel, configured to mix the liquid components and the sample into a substantially uniformly mixed test fluid. 6. The system of claim 1 wherein the assay cartridge comprises a first electrode interface device including a first pad leading to a drive electrode and a second pad leading to a sensor electrode. 7. The system of claim. 6, characterized in that the specified reader contains a second interface with the electrode, made with the possibility of connection with the first interface with the electrode, while the analysis cartridge enters the area of the reader. 8. The system according to claim 7, characterized in that said reader further comprises a voltage source configured to generate a drive current, and the second electrode interface includes: the third pad, the location of which provides interfacing with the first pad, and the specified third pad is associated with a voltage source; and the fourth pad, the location of which provides interfacing with the second pad, and the specified fourth pad is associated with the memory. 9. The system according to claim 1, characterized in that for decomposing said signal into a resistance component and a reactance component, the processor is additionally configured with instructions so that at least: measure a signal with a frequency greater than its Nyquist frequency, where said signal corresponds to the impedance of said sample; decompose said signal into an in-phase component and an out-of-phase component; and calculate a resistance component based on the common mode component; and calculate a reactance component based on the out of phase component. 10. The system of claim. 1, characterized in that, for analyzing the reactance component, the processor is further configured with said instructions to at least evaluate the predetermined expected edge characteristics from the memory. 11. The system of claim 10, wherein said predetermined expected signal edge characteristics stored in the memory include a window of time over the duration of the amplification process when said signal edge should occur as predicted. 12. The system of claim 10, wherein said predetermined expected edge characteristics stored in the memory include a threshold change in the value of the reactance component. 13. The system of claim 10, wherein said predetermined expected ramp characteristics stored in the memory include a threshold slope of the reactance component curve corresponding to reactance component values measured during at least a portion of the time the amplification process is running. 14. The system of claim. 1, characterized in that said amplification process includes bringing the processed sample into contact with the capture probe. 15. The system of claim 14, wherein said capture probe is selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, and a nucleic acid. 16. The system of claim 14, wherein said capture probe contains a detectable nucleic acid. 17. The system of claim 1, wherein said amplification process comprises isothermal amplification. 18. The system of claim. 1, characterized in that said amplification process includes loop isothermal amplification (LAMP). 19. The system according to claim. 1, characterized in that the specified temperature for performing the amplification process inside the test cell is above 30°C. 20. The system according to claim. 1, characterized in that the specified temperature for performing the amplification process inside the test cell is in the range from 60°C to 70°C. 21. The system of claim 3, wherein the liquid constituents of the amplification process comprise a component selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, a nucleic acid such as a primer, a buffer, and an enzyme such as a polymerase. 22. The system of claim 3, wherein the dried components of the amplification process contain a component selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, a nucleic acid such as a primer, a buffer, and an enzyme such as a polymerase. 23. A device for examining a sample for a target agent, comprising: a sample input area configured to receive a sample containing the target agent; a test cell comprising an excitation electrode and a sensor electrode, said test cell being configured to: contain the sample during the amplification process, apply current to the sample during the amplification process using the excitation electrode, and to receive, using the sensor electrode, a signal corresponding to the current after its attenuation due to interaction with at least the sample inside the test cell; a flow channel that fluidly connects the sample input area with the test cell; and a processor configured to: receive the specified signal, decompose the specified signal into a component of active resistance and a component of reactance, analyze the reactance component to determine the presence of a signal drop with respect to time and at a certain frequency of the specified signal, indicating a positive sample containing the target agent, for at least part of the time of the amplification process. 24. The device according to claim 23, further comprising: a sealed chamber containing the liquid components of the amplification process, located in the area of the device having an opening leading to the flow channel, while the sample input area is located between the specified hole and the test cell along the flow channel; and dried components of the amplification process placed in the test cell. 25. The device according to claim 24, further comprising: a tip configured to pierce the sealed chamber and cause liquid constituents to enter said area; and a pneumatic fluid channel connecting the pneumatic fluid interface device with the area of the specified device, while the specified pneumatic channel for the fluid medium is configured to apply pressure to the specified area, which leads to the ingress of liquid components into the flow channel and the transfer of the sample received from sample entry area, into the test cell. 26. The device according to claim 25, further comprising a mixing chamber located between the sample injection area and the test cell along the flow channel, while said mixing chamber is configured to mix the liquid components and the sample into a substantially evenly mixed test fluid. 27. The device according to claim 23, characterized in that said analytical cartridge comprises a first electrode interface device, including a first contact pad leading to the excitation electrode and a second contact pad leading to the sensor electrode. 28. The device according to claim. 23, further comprising a circuit board including an excitation electrode and a sensor electrode, characterized in that the sample introduction area and at least part of the flow channel form a single part of a liquid-impervious material, and in that said circuit board attached to a part of a liquid-impervious material. 29. The apparatus of claim 28, further comprising a lid located over the liquid impervious material and a circuit board containing an opening located above the sample entry area, and a cap configured to seal said opening with the possibility of subsequent opening. 30. Apparatus according to claim 29, characterized in that the sides of the test cell form a round hole in the liquid-impermeable material and in that the bottom of the test cell is formed by a circuit board. 31. The device according to claim 30, characterized in that said excitation electrode and said sensor electrode are located at the bottom of the test cell, away from the sides of the test cell. 32. The apparatus of claim 28, wherein said drive electrode and said sensor electrode are configured to be substantially flush with the bottom layer of the circuit board. 33. The apparatus of claim. 23, further comprising a vent configured to release gas from the test cell, characterized in that said vent is covered with a liquid-tight, gas-permeable filter. 34. The device according to claim 23, characterized in that said excitation electrode comprises a circular electrode located in the center of the cell, and in that said sensor electrode comprises an annular electrode located concentrically around the excitation electrode. 35. The device according to claim 34, characterized in that said annular electrode is separated from said circular electrode by a gap approximately equal to the radius of the annular electrode. 36. The device according to claim 34, characterized in that said annular electrode is separated from said circular electrode by a gap the size of which is at least twice the radius of the annular electrode. 37. The device according to claim 23, characterized in that said excitation electrode comprises a first semicircular electrode, and said sensor electrode comprises a second semicircular electrode separated by a gap from the first semicircular electrode, characterized in that the rectilinear parts of said first and second semicircular electrodes face each other to a friend through the gap. 38. The device according to claim 23, characterized in that said excitation electrode comprises a first linear electrode, and said sensor electrode comprises a second linear electrode separated by a gap from the first linear electrode. 39. The device according to claim 23, characterized in that said excitation electrode comprises a first square electrode, and said sensor electrode comprises a second square electrode separated by a gap from the first linear electrode. 40. The device according to claim 23, characterized in that said amplification process includes bringing the processed sample into contact with a capture probe. 41. The device of claim 40, wherein said capture probe is selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, and a nucleic acid. 42. The device according to claim 40, characterized in that said capture probe contains a detectable nucleic acid. 43. The device according to claim 23, characterized in that said amplification process includes isothermal amplification. 44. The device according to claim 23, characterized in that said amplification process includes loop isothermal amplification (LAMP). 45. The device according to claim 23, characterized in that said test cell is configured to heat the sample to a temperature above 30°C. 46. The device according to claim. 23, characterized in that the specified test cell is configured to heat the sample to a temperature in the range from 60°C to 70°C. 47. The device according to claim 24, characterized in that said liquid constituents of the amplification process contain a component selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, a nucleic acid such as a primer, a buffer, and an enzyme such as a polymerase. 48. The device according to claim 24, characterized in that said dried components of the amplification process contain a component selected from the group consisting of an antibody or antigen-binding fragment, a protein receptor, a nucleic acid such as a primer, a buffer and an enzyme such as a polymerase. 49. A method for detecting a target agent, including: providing a cartridge containing a test cell including an excitation electrode and a sensor electrode; introducing a sample containing the target agent into said cartridge; inserting the cartridge into the reader; conducting amplification of the target agent contained in the sample inside the test cell; applying an excitation signal from the reader to the excitation electrode; sensory detection, using an excitation electrode, of a signal from the test cell corresponding to the impedance of the sample being amplified; signal transmission to the reader; and detection of the target agent based on the reader's analysis of the reactive part of the impedance to identify a signal drop with respect to time and at a certain frequency of said signal indicating a positive test result. 50. The method of claim 49, further comprising: supplying a sample to a sample input area of the cartridge; breaking through the sealed chamber within the cartridge to release the liquid constituents of the amplification process into the flow channel of the cartridge; and allowing the liquid constituents and the sample to flow along the flow channel into the test cell, thereby mixing the liquid constituents and the sample to form a test fluid. 51. The method of claim 50, further comprising moistening the dried components of the amplification process contained within the test cell with the test fluid. 52. The method of claim 49, further comprising expelling gas entrained in the test fluid through a vent in fluid communication with the test cell. 53. The method of claim 49, further comprising identifying a signal edge based on a portion of the curve generated based on the reactive portion that differs in one or more of the first and second of more than a threshold value change and temporal location within a given time window in the amplification process. 54. The method of claim 49, wherein said amplification process includes bringing the treated sample into contact with a capture probe. 55. The method of claim 54, wherein said capture probe is selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, and a nucleic acid. 56. The method of claim 54, wherein said capture probe contains a detectable nucleic acid. 57. The method of claim 49, wherein said amplification comprises isothermal amplification. 58. The method of claim 49, wherein said amplification comprises loop isothermal amplification (LAMP). 59. The method according to p. 49, characterized in that the amplification of the target agent includes heating the sample to a temperature exceeding 30°C. 60. The method according to p. 49, characterized in that the amplification of the target agent includes heating the sample to a temperature in the range from 60°C to 70°C. 61. The method of claim 50, wherein said liquid components of the amplification process comprise a component selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, a nucleic acid such as a primer, a buffer, and an enzyme such as a polymerase. 62. The method of claim 51, wherein said dried amplification process components comprise a component selected from the group consisting of an antibody or antigen-binding fragment thereof, a protein receptor, a nucleic acid such as a primer, a buffer, and an enzyme such as a polymerase.

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