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WO2024256522A1 - Dispositif de dosage électronique - Google Patents

Dispositif de dosage électronique Download PDF

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Publication number
WO2024256522A1
WO2024256522A1 PCT/EP2024/066331 EP2024066331W WO2024256522A1 WO 2024256522 A1 WO2024256522 A1 WO 2024256522A1 EP 2024066331 W EP2024066331 W EP 2024066331W WO 2024256522 A1 WO2024256522 A1 WO 2024256522A1
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WO
WIPO (PCT)
Prior art keywords
electrode
region
solution
analyte
potential
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/066331
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English (en)
Inventor
Paul Davis
YueHua DOU
Stephen Edwards
Stephanie Mcintyre
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mologic Ltd
Original Assignee
Mologic Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mologic Ltd filed Critical Mologic Ltd
Publication of WO2024256522A1 publication Critical patent/WO2024256522A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Definitions

  • the disclosure relates to the field of assay devices.
  • results of the assay procedure typically require interpretation by a user.
  • a lateral flow test may show a coloured line in the event that an analyte is present in a sample.
  • the user may interpret the intensity of the line to determine whether an analyte is present and/or to give some indication of the concentration of analyte in the sample.
  • Interpretation of the results of the assay procedure may introduce further variation into the measurements.
  • a method of detecting the presence or absence of an analyte in a sample using an electronic assay device wherein the electronic assay device is configured to output an electronic signal indicative of the presence of an analyte in a sample.
  • the electronic assay device comprises a sample receiving region.
  • the electronic assay device further comprises a detection reagent comprising a catalytic agent.
  • the electronic assay device further comprises capture molecules.
  • the electronic assay device further comprises a test region comprising an electrode assembly and a test line configured to bind to the capture molecules.
  • the electronic assay device further comprises a battery configured to be activated by contact with a liquid.
  • the method comprises depositing a sample and a solution on the sample receiving region, such that the analyte, where present, binds to the detection reagent via a first binding site of the analyte and binds to the capture molecule via a second binding site of the analyte; and the solution is incident on the battery, activating the battery and triggering a sequence of potentials applied to the electrode assembly; and the bound analyte, where present, and the solution are incident on the test region, wherein the solution carries indicator molecules to the test region.
  • the method further comprises applying a first potential to the electrode assembly to produce hydrogen peroxide in the test region.
  • the method further comprises generating oxidised indicator molecules at the test region, wherein the oxidised indicator molecules are produced via hydrogen peroxide oxidation of the indicator molecules in the presence of the catalytic agent.
  • the method further comprises applying a second potential to the electrode assembly such that the oxidised indicator molecules are reduced, generating the indicator molecules and a charge transfer.
  • the method further comprises measuring the charge transfer, wherein the charge transfer is indicative of the presence or absence of the analyte.
  • the method further comprises outputting data indicative of the charge transfer.
  • the presence or absence of an analyte in a sample may be accurately detected.
  • the presence or absence of an analyte in a sample may be accurately detected without further intervention from a user, meaning that the accuracy of the detection is not vulnerable to variation in timing by a user or other variations between assay procedures.
  • the result of the assay may be measured electronically without the need for user interpretation.
  • the solution may comprise the indicator molecules.
  • the indicator molecules may be added in a solution such that the indicator molecules are carried to the test region.
  • the indicator molecules may be in the same solution as the sample, reducing the number of steps performed by the user.
  • the first potential may be applied to the electrode assembly in the presence of an electron donor.
  • An electron donor or redox mediator may can lower the size of the potential required for the reaction to produce hydrogen peroxide to take place.
  • Either the solution may comprise the electron donor; or the electrode assembly may comprise the electron donor.
  • the electron donor may be present at the test region.
  • the electron donor may be carried to the test region by the solution.
  • the electrode assembly comprises the electron donor, the electron donor is present at the test region.
  • the detection reagent may be in a dried state on the electronic assay device and depositing the solution may release the detection reagent.
  • the detection reagent may be present without the user needing to add it.
  • this reduces the number of steps carried out by the user and so reduced variation between assays.
  • the capture molecule may be in a dried state on the electronic assay device and depositing the solution may release the capture molecule.
  • the capture molecules may be present without the user needing to add them.
  • this reduces the number of steps carried out by the user and so reduced variation between assays.
  • the capture molecule may bind to the test line via an intermediate molecule.
  • the capture molecules may be carried to the test line by a solution and may be fixed at the test line by the intermediate molecule.
  • the sample receiving region may comprises a first region configured to receive the sample and a buffer solution; and a second region, configured to receive a reagent solution comprising the electron donor and the indicator molecule.
  • the sample and a buffer solution may be added to the device separately to the reagent solution.
  • any reaction between the solutions before it is required by the method may be avoided.
  • the method may comprise depositing the sample and a buffer solution on the first region and depositing the reagent solution on the second region.
  • the sample and a buffer solution may be added to the device separately to the reagent solution.
  • any reaction between the solutions before it is required by the method may be avoided.
  • the first region may comprise the capture molecule and depositing the buffer onto the first region may release the capture molecule.
  • the capture molecule may be added to the solution to bind with the analyte, where present.
  • the capture molecule and the analyte may be carried by the buffer solution to the test region, providing enough time for all the analyte present to bind to capture molecules.
  • the first region may comprise the detection reagent and depositing the buffer onto the first region may release the detection reagent.
  • the detection reagent may be added to the solution to bind with the analyte, where present.
  • the detection reagent and the analyte may be carried by the buffer solution to the test region, providing enough time for all the analyte present to bind to the detection reagent.
  • the first region may comprise a first pad comprising the capture molecule and a second pad comprising the detection reagent, wherein the method may comprise depositing the sample and solution on the first pad and wherein the sample and solution may subsequently flow through the second pad.
  • the electrode assembly may comprise a first working electrode and a counter electrode, wherein the second potential may be applied between the first working electrode and the counter electrode.
  • the first potential may be applied between the first working electrode and the counter electrode.
  • the electrode assembly may further comprise a second working electrode, wherein the first potential may be applied between the second working electrode and the counter electrode.
  • the electrode assembly may further comprise a second working electrode and a third working electrode, wherein the first potential may be applied between the second working electrode and the third working electrode.
  • the first working electrode may be adjacent to the counter electrode, and the second working electrode may be adjacent to the third working electrode.
  • the electrode assembly may further comprise a first reference electrode.
  • a reference value for the measurement may be obtained using the first reference electrode.
  • the first potential may be applied between the first working electrode and the counter electrode, and the second potential may be referenced to the reference electrode.
  • the first working potential may be applied between the first working electrode and the second working electrode and the second potential may be applied: between the first working electrode and the counter electrode; and subsequently between the reference electrode and the counter electrode.
  • a control value may be obtained by subsequently applying the second potential between the reference electrode and the counter electrode.
  • the electrode assembly may further comprise a second reference electrode, wherein the first potential may be applied between the first working electrode and the counter electrode and wherein the second potential may be applied: between the first working electrode and the counter electrode; subsequently between the first reference electrode and the counter electrode; and subsequently between the second reference electrode and the counter electrode.
  • a first and second control value may be obtained by subsequently applying the second potential between the first reference electrode and the counter electrode and subsequently applying the second potential between the second reference electrode and the counter electrode.
  • the first working electrode may be between the first reference electrode and the second reference electrode.
  • the test region may be aligned with the first working electrode.
  • the hydrogen peroxide may be produced at the test region.
  • the test region may intersect with the counter electrode.
  • the analyte is bound at the region where the second potential is applied.
  • the counter electrode may be shaped such that the counter electrode is adjacent to each other electrode of the electrode assembly.
  • the data indicative of charge transfer may be further indicative of a concentration of the analyte such that the method further comprises determining the concentration of the analyte.
  • the amount of analyte present in a sample may be quantified without need for user interpretation.
  • the detection reagent may comprise a molecular binding reagent labelled with the catalytic agent.
  • the catalytic agent may comprise catalytic platinum nanoparticles.
  • the catalytic agent may comprise horse-radish peroxidase.
  • the capture molecule may comprise Biotin-Ab1.
  • the electron donor may comprise a quinone.
  • the electron donor may comprise flavin mononucleotide.
  • the indicator molecule may comprise 3,3',5,5'-Tetramethylbenzidine, TMB.
  • the intermediate molecule may comprise Polystreptavidin, PSA.
  • an electronic assay device configured to output an electronic signal indicative of the presence of an analyte in a sample.
  • the electronic assay device comprises a detection reagent comprising a catalytic agent configured to bind to a first binding site of the analyte, where present.
  • the electronic assay device further comprises capture molecules configured to bind to a second binding site of the analyte, where present.
  • the electronic assay device further comprises a test region comprising an electrode assembly and a test line configured to bind to the capture molecule.
  • the electronic assay device further comprises a battery configured to be activated by contact with a liquid.
  • the electronic assay device further comprises a sample receiving region configured to receive a sample and a solution such that the solution is incident on the test region and on the battery.
  • the electronic assay device further comprises a microprocessor configured to initiate a sequence of potentials applied to the electrode assembly upon activation of the battery. After a first period of time from activation of the battery, the microprocessor is further configured to apply a first potential to the electrode assembly to produce hydrogen peroxide in the test region, wherein the hydrogen peroxide oxidises indicator molecules in the presence of the catalytic agent to produce oxidised indicator molecules, wherein the indicator molecules are carried to the test region by the solution.
  • microprocessor is further configured to apply a second potential to the electrode assembly such that the oxidised indicator molecules are reduced, generating the indicator molecules and a charge transfer.
  • the microprocessor is further configured to measure the charge transfer, wherein the charge transfer is indicative of the presence or absence of the analyte.
  • the microprocessor is further configured to output data indicative of the charge transfer.
  • Figure 1 shows a schematic diagram of an electronic assay device according to an embodiment of the present disclosure.
  • Figure 10 shows a schematic diagram of an electronic assay device according to an embodiment of the present disclosure.
  • An electronic assay device is provided according to an aspect of the disclosure, wherein the electronic assay device is configured to output an electronic signal indicative of the presence of an analyte in a sample.
  • the electronic assay device allows detection of the presence or absence of analyte in a sample.
  • a sample and a solution are deposited on the sample receiving region 110.
  • the solution is incident on the battery, activating the battery and triggering a sequence of potentials applied to the electrode assembly.
  • the solution may be deposited as a single solution, or as more than one solution. In an event that the solution is deposited as more than one solution, the more than one solution may be deposited in different regions or in the same region. In use, the solution is incident on the test region, and therefore on the electrode assembly.
  • the analyte 210 binds to the detection reagent 220 via a first binding site of the analyte and binds to the capture molecule 130 via a second binding site of the analyte.
  • the first potential may be applied to the electrode assembly to produce hydrogen peroxide, without the presence of an electron donor. In other embodiments, the first potential may be applied to the electrode assembly in the presence of an electron donor to produce hydrogen peroxide.
  • the solution may comprise the electron donor. In other embodiments, the electrode assembly may comprise the electron donor.
  • the electron donor may be mixed with the electrode material when the electrodes of the electrode assembly are prepared.
  • the sample receiving region may be in a different lateral position on the assay device than the test region.
  • the sample and solution may flow from the sample receiving region to the test region via substrate 140.
  • the substrate 140 may comprise or support a lateral flow strip, membrane or other means for allowing fluid flow.
  • the electrode assembly is configured to be in fluidic contact with the substrate when the solution reaches the test region. The distance of the test line 120 from the sample receiving region 110 may affect the time taken to carry out an assay and the sensitivity of the assay.
  • a longer distance between the test line 120 and the sample receiving region 110 increases the time taken for the sample and the solution to travel from the sample receiving region 110 to the test line 120, and so increases the time taken to carry out an assay.
  • the analyte, where present, and the capture molecules and detection reagent may be present in the solution as it travels from the sample receiving region 110 to the test line 120.
  • a longer distance between the test line 120 and the sample receiving region 110 provides more time for the sample molecules to bind to the capture molecules, increasing the sensitivity of the assay.
  • the detection reagent may be in a dried state on the electronic assay device. Depositing the solution onto the sample receiving region releases the detection reagent. The detection reagent may flow with the solution to the test region. The analyte may bind to the detection reagent at the location of release of the detection reagent, or as the solution flows to the test region, or at the test region.
  • the capture molecules may be in a dried state on the electronic assay device. Depositing the solution may release the capture molecules, such that the capture molecules flow to the test region with the solution. The capture molecules may bind to the test line via intermediate molecules, wherein the intermediate molecules are located on the test line. In certain embodiments, the deposited solution may comprise the indicator molecules, such that the indicator molecules flow to the test region with the solution. In other embodiments, the indicator molecules may be in a dried state on the electronic assay device. Depositing the solution may release the capture molecules, such that the capture molecules flow to the test region with the solution.
  • a sample and a solution are deposited on the sample receiving region 110.
  • the solution comprises the indicator molecules.
  • the solution is incident on the battery, activating the battery and triggering a sequence of potentials applied to the electrode assembly.
  • the solution may be deposited as a single solution, or as more than one solution. In an event that the solution is deposited as more than one solution, the more than one solution may be deposited in different regions or in the same region.
  • the capture molecules and the detection reagent may be in a dried state on the sample receiving region 110. The capture molecules and the detection reagent may be released by the solution.
  • the analyte 210 binds to the detection reagent 220 via a first binding site of the analyte and binds to the capture molecule 130 via a second binding site of the analyte.
  • the solution is further incident on the test region, and therefore on the electrode assembly.
  • the bound analyte, where present, binds to the test line.
  • a first potential is applied to the electrode assembly to produce hydrogen peroxide in the test region.
  • Indicator molecules are present at the test region, having been carried to the test region by the solution.
  • An oxidised indicator molecule is generated at the test region, wherein the oxidised indicator molecule is produced via hydrogen peroxide oxidation of the indicator molecule in the presence of the catalytic agent.
  • a second potential is applied to the electrode assembly such that the oxidised indicator molecule is reduced, generating the indicator molecule and a charge transfer. The charge transfer is measured, wherein the charge transfer is indicative of the presence or absence of the analyte. Data indicative of the charge transfer is output.
  • a sample and a solution are deposited on the sample receiving region 110.
  • the solution does not comprise the indicator molecules.
  • the solution is incident on the battery, activating the battery and triggering a sequence of potentials applied to the electrode assembly.
  • the solution may be deposited as a single solution, or as more than one solution. In an event that the solution is deposited as more than one solution, the more than one solution may be deposited in different regions or in the same region.
  • the capture molecules, the detection reagent and the indicator molecules may be in a dried state on the sample receiving region 110.
  • the capture molecules, the detection reagent and the indicator molecules may be released by the solution.
  • the solution travelling from the sample receiving region to the test region therefore comprises the capture molecules, detection reagent and indicator molecules.
  • the analyte 210 binds to the detection reagent 220 via a first binding site of the analyte and binds to the capture molecule 130 via a second binding site of the analyte.
  • the solution is further incident on the test region, and therefore on the electrode assembly.
  • the bound analyte, where present, binds to the rest region.
  • a first potential is applied to the electrode assembly to produce hydrogen peroxide in the test region.
  • Indicator molecules are present at the test region, having been carried to the test region by the solution.
  • An oxidised indicator molecule is generated at the test region, wherein the oxidised indicator molecule is produced via hydrogen peroxide oxidation of the indicator molecule in the presence of the catalytic agent.
  • a second potential is applied to the electrode assembly such that the oxidised indicator molecule is reduced, generating the indicator molecule and a charge transfer. The charge transfer is measured, wherein the charge transfer is indicative of the presence or absence of the analyte. Data indicative of the charge transfer is output.
  • the sample receiving region 110 may comprise one region configured to receive both the sample and solution.
  • the sample and solution may be received by the same region, at the same time or at different times.
  • the sample receiving region 110 may comprise a sample pad.
  • the solution may comprise a buffer solution.
  • the solution may comprise a buffer solution and a reagent solution.
  • the sample receiving region 110 may comprise more than one sub-region. The more than one subregion may each be configured to receive one or more of the sample and the solution.
  • the solution may comprise a buffer solution and a reagent solution.
  • the buffer solution may be deposited with the sample.
  • the reagent solution may be deposited separately to the sample and buffer solution.
  • the reagent solution may comprise the indicator molecules.
  • a first sub-region of the sample receiving region 110 may be configured to receive the sample and buffer solution and a second sub-region of the sample receiving region 110 may be configured to receive the reagent solution.
  • the sample receiving region 110 may comprise more than one pad, each pad comprising a sub-region of the sample receiving region 110.
  • the battery may be configured to be activated by the buffer solution.
  • the battery may be configured to be activated by the reagent solution.
  • the solution may comprise an electrolyte solution configured to activate the battery.
  • the sample receiving region 310 of an assay device 300 may comprise a first sub-region 320 and a second sub-region 330, wherein the first sub-region 320 is configured to receive the sample and a buffer solution (indicated by arrow 321) and wherein the second sub-region 330 is configured to receive a reagent solution (indicated by arrow 331).
  • the reagent solution may comprise the indicator molecules.
  • the indicator molecules may be in a dried state on the second sub-region 330, such that the indicator molecules are released when the reagent solution is deposited on the second sub-region 330.
  • the reagent solution may further comprise the electron donor.
  • the method may comprise depositing the sample and a buffer solution on the first subregion 320 and depositing the reagent solution on the second sub-region 320.
  • the first sub-region 320 and the second sub-region 330 may be separated such that the reagent solution added to the second sub-region 330 is not incident on the first sub-region 320.
  • a barrier 340 may be located between the first sub-region 320 and the second sub-region 330 such that fluid cannot be transferred between the first sub-region 320 and the second sub-region 330 or vice versa, but such that fluid can still flow from the first subregion 320 to the test line 120 and from the second sub-region 330 to the test line 120.
  • the first sub-region 320 may comprise the capture molecule in a dried state, wherein depositing the buffer onto the first region releases the capture molecule.
  • the first subregion 320 may comprise the detection reagent in a dried state, wherein depositing the buffer onto the first sub-region 320 releases the detection reagent.
  • the sample receiving region 410 of an assay device 400 may comprise a first sub-region 420 configured to receive the sample and the buffer solution (indicated by arrow 421).
  • the first sub-region 420 may comprise the capture molecules in a dried state.
  • the sample receiving region 410 may further comprise a second sub-region 430 comprising the detection reagent in a dried state, wherein the sample and solution are deposited on the first sub-region 420 and wherein the sample and solution subsequently flow through the second sub-region 430.
  • the first sub-region 420 and the second sub-region 430 may each comprise a pad.
  • the sample receiving region 410 may further comprise a third sub-region 440, wherein the third sub-region 440 is configured to receive a reagent solution (indicated by arrow 441).
  • the reagent solution may comprise the indicator molecules.
  • the indicator molecules may be in a dried state on the third sub-region 440, such that the indicator molecules are released when the reagent solution is deposited on the third subregion 440.
  • the reagent solution may further comprise the electron donor.
  • the method may comprise depositing the sample and a buffer solution on the first sub-region 420 and depositing the reagent solution on the third sub-region 440.
  • the third sub-region 440 may be separated from the first subregion 420 and the second sub-region 430 such that the reagent solution added to the third sub-region 440 is not incident on the first sub-region 420 or the second sub-region 430.
  • a barrier 450 may be located between the third sub-region 440 and the first and second sub-regions 420, 430 such that fluid cannot be transferred between the third subregion 440 and the first and second sub-regions 420, 430 or vice versa.
  • the barrier 450 may be located such that fluid can still flow from the first sub-region 420 to the second subregion 430 and then to the test line 120 and from the third sub-region 440 to the test line 120.
  • the first and second sub-regions 420, 430 may comprise separate pads.
  • the pads may be located as shown in Figure 4, with the pad of the first sub-region 420 above and partially overlapping the pad of the second sub-region 430.
  • the pads may be arranged in different configurations.
  • the pad of the first sub-region 420 may be directly above and substantially overlapping the pad of the second sub-region 430.
  • the pad of the first sub-region 420 may be adjacent to the pad of the second sub-region 430.
  • the first and second sub-regions 420, 430 may be on the same pad.
  • the sample and buffer solution flow from the first sub-region 320 or
  • the reagent solution may be incident on the battery and may flow from the second sub-region 330 or third sub-region
  • an absorbent pad or sink pad may be placed at the opposite end of the substrate to the sample receiving region. This may be used to absorb excess solution or wash solutions.
  • a wash solution may be added to the sink pad after the indicator molecule reaches the test region, such that the oxidised indicator molecule is concentrated around the test region and does not flow away.
  • the method comprises depositing a sample and a solution on the sample receiving region at step 510.
  • the analyte where present, binds to the detection reagent via a first binding site of the analyte and binds to the capture molecule via a second binding site of the analyte.
  • indicator molecules are incident on the test region.
  • the solution is incident on the battery, activating the battery at step 540 and triggering a sequence of potentials applied to the electrode assembly.
  • step 550 of the method comprises applying a first potential to the electrode assembly to produce hydrogen peroxide in the test region.
  • the method further comprises generating an oxidised indicator molecule at the test region at step 560, wherein the oxidised indicator molecule is produced via hydrogen peroxide oxidation of the indicator molecule in the presence of the catalytic agent.
  • step 570 of the method comprises applying a second potential to the electrode assembly such that the oxidised indicator molecule is reduced, generating the indicator molecule and a charge transfer.
  • the method further comprises measuring the charge transfer at step 580, wherein the charge transfer is indicative of the presence or absence of the analyte.
  • the method comprises outputting data indicative of the charge transfer.
  • the solution deposited at step 510 may comprise the indicator molecules.
  • the solution deposited at step 510 may comprise an electron donor.
  • the electron donor may be incident on the test region at step 530.
  • the first potential may be applied to the electrode assembly in the presence of the electron donor.
  • the first potential may be applied to the electrode assembly in the presence of the electron donor, wherein the electrode assembly comprises the electron donor.
  • first potential may be applied to the electrode assembly without an electron donor being present.
  • steps 530 and 540 may occur in the order shown in Figure 5A, or in the opposite order, or at the same time.
  • step 510 may comprise a single step of depositing a sample and a solution on the sample receiving region, such that the analyte, where present, binds to the detection reagent via a first binding site of the analyte and binds to the capture molecule via a second binding site of the analyte at step 520, and the solution is incident on the battery at step 540.
  • the solution may follow one path, which allows the analyte to bind to the detection reagent and capture molecule and be incident on the test region and also allows the solution to be incident on the battery.
  • Each analyte of the sample 621 binds to a capture molecule 631 and to a detection reagent 641.
  • the capture molecule 631 binds to the intermediate molecule 691, such that the analyte and detection reagent 641 are captured at the test region.
  • the test region may change colour upon incidence of a liquid, so test region 690 may change colour when the sample 621, buffer solution 622, capture molecules 631 and detection reagent 641 reach the test region 690 after step 510.
  • Electrode assembly 692 is shown below the substrate 680, but may be located above the substrate 680.
  • the detection reagent 641 may be in a dried form on the first subregion 630 of the sample receiving region 610, such that the buffer solution 622 releases the detection reagent 641 from the first sub-region 630, and the sample 621, buffer solution 622 and detection reagent 641 subsequently flow to the second sub-region 640.
  • the capture molecules 631 may be in a dried form on the second sub-region 640.
  • the buffer solution 622 releases the capture molecules 631 from the second sub-region 640.
  • the capture molecules and the detection reagent may be in dried form on the same sub-region of the sample receiving region.
  • the capture molecules and the detection reagent may be dried onto the same pad.
  • the capture molecules and the detection reagent may be interspersed with one another, or may be dried onto adjacent sections of the pad.
  • the reagent solution may be deposited onto the same sub-region of the sample receiving region as the sample.
  • the reagent solution may be deposited with the sample.
  • the reagent solution may be mixed with the sample.
  • the reagent solution may be deposited separately to the sample but onto the same sub-region of the sample receiving region as the sample.
  • a solution may be deposited such that the solution is incident on the battery, wherein the solution comprises an electrolyte configured to activate the battery.
  • the solution may be deposited on the battery.
  • a solution received by the sample receiving region may be diverted or split such that the solution is incident on the battery.
  • the buffer solution or the reagent solution may be used to activate the battery.
  • Steps 510 to 540 may occur in a different manner, depending on the architecture of the assay device. The result of steps 510 to 540 is that a complex including the analyte (if present) and the detection reagent is captured at the test line, the battery has been activated and the reagent solution 651 has reached the test region.
  • step 550 comprises applying a first potential to the electrode assembly.
  • the first potential is applied in the presence of the electrode donor to produce hydrogen peroxide in the test region.
  • hydrogen peroxide may be produced according to the following equations:
  • Kc is the rate constant for chemical step 2.
  • the production of hydrogen peroxide may be precisely timed by applying the first potential to the electrode assembly after a first period from activation of the battery and for a particular duration.
  • Step 560 comprises generating an oxidised indicator molecule at the test region at step 560.
  • the oxidised indicator molecule is produced via hydrogen peroxide oxidation of the indicator molecule in the presence of the catalytic agent.
  • the oxidised generator molecule Box may be generated according to the following equation: c Equation 3: BRD + B 2 O2 > Box
  • a second potential is applied to the electrode assembly such that the oxidised indicator molecule is reduced, generating the indicator molecule and a charge transfer.
  • the reduction may occur according to the following equation: e -1
  • Equation 4 B ox - > B RD
  • the charge transfer is measured at step 580, wherein the charge transfer is indicative of the presence or absence of the analyte.
  • the charge transfer is proportional to the of BRD produced according to Equation 4, which is proportional to the quantity of Box produced according to Equation 3 and is therefore proportional to the quantity of analyte bound at the test line.
  • the measurement of charge transfer may therefore be used to detect the presence or absence of an analyte (for example by determining whether the charge transfer is above or below a threshold) and/or to quantify the analyte. As will be described, this may be carried out using the electrode assembly. In certain embodiment, steps 570 and 580 may occur simultaneously. Electrodes of the electrode assembly to which the second potential is applied may also be used to measure the charge transfer, such that the second potential is used for generating and measuring the charge transfer.
  • the method comprises outputting data indicative of the charge transfer.
  • the measurement of the charge transfer may be carried out using chronoamperometry.
  • a potential may be applied to a working electrode and to a counter electrode such that the working electrode potential at the working electrode is controlled to be a constant potential value as measured against a reference electrode.
  • the electrode assembly may be in fluidic contact with the substrate so the electrode assembly is in a solution.
  • the reference electrode may have a constant half-cell potential in the solution.
  • the working electrode potential is held at that constant potential for a fixed time period, and the current flowing through the working electrode and counter electrode is measured.
  • the measured current value (in Amps) may be multiplied by the time period (in seconds) to provide the total charge value in Coulombs used in the reduction reaction.
  • the charge value is proportional to the amount of oxidised indicator molecule present during the reduction.
  • the charge value is therefore proportional to the quantity of analyte bound at the test line.
  • the reference electrode and the counter electrode may be the same electrode or different electrodes. In an event that the reference electrode is a separate electrode to the counter electrode, no current flows through the reference electrode as the circuit only measures its potential.
  • the assay device comprises a battery that is activated by liquid.
  • the battery once activated the battery is configured to provide power such that voltages can be applied to the electrode assembly, such as in steps 550 and 570.
  • the battery is further configured to provide power such that the data indicative of charge transfer (output in step 590) is written to memory.
  • the electrode assembly may be positioned adjacent to the substrate.
  • the electrode assembly may be positioned such that when the substrate is wet, the electrode assembly is in fluidic contact with the substrate.
  • the electrode assembly may be above or below the substrate.
  • the electrode assembly may be planar, such that the electrode assembly is parallel to the substrate. Examples of electrode assemblies will be described with reference to Figures 7 and 8, although other electrode assemblies are also possible.
  • the position of the test line is indicated by a white rectangle overlaid on the electrode assembly. This is merely indicative of the position of the test line relative to the electrodes, and is not intended to be precise or to scale.
  • the shapes of the electrodes in Figures 7 and 8 are examples only.
  • Figure 7A shows an electrode assembly 710 with a position of the test line indicated by rectangle 711.
  • the electrode assembly 710 comprises a counter electrode 712 and a first working electrode 713.
  • the test line 711 is aligned with the first working electrode 713.
  • the first potential Vi is applied between the first working electrode 713 and the counter electrode 712.
  • the second potential V2 is applied between the first working electrode 713 and the counter electrode 712.
  • Figure 7B shows an electrode assembly 720 with a position of the test line indicated by rectangle 721.
  • the electrode assembly 720 comprises a counter electrode 722, a first working electrode 723 and a second working electrode 724.
  • the test line 721 is aligned with the first working electrode 723.
  • the first potential Vi is applied between the second working electrode 724 and the counter electrode 722.
  • the second potential V2 is applied between the first working electrode 723 and the counter electrode 722.
  • the first working electrode 723 may be positioned adjacent to the counter electrode 722.
  • the counter electrode 722 may be L-shaped such that the counter electrode 722 is adjacent to both the first working electrode 723 and to the second working electrode 724.
  • the first working electrode 723, and therefore the position of the test line may be between the counter electrode 722 and the second working electrode 724.
  • Figure 7C shows an electrode assembly 730 with a position of the test line indicated by rectangle 731.
  • the electrode assembly 730 comprises a counter electrode 732, a first working electrode 733, a second working electrode 734 and a third working electrode 735.
  • the test line 731 is aligned with the first working electrode 733.
  • the first potential Vi is applied between the second working electrode 734 and the third working electrode 735.
  • the second potential V2 is applied between the first working electrode 733 and the counter electrode 732.
  • the first working electrode 733 may be positioned adjacent to the counter electrode 732.
  • the second working electrode 734 may be positioned adjacent to the third working electrode 735.
  • the first working electrode 733, and therefore the position of the test line may be between the counter electrode 732 and the second and third working electrodes 734, 735.
  • Figure 7D shows an electrode assembly 740 with a position of the test line indicated by rectangle 741.
  • the electrode assembly 740 comprises a counter electrode 742, a first working electrode 743 and a reference electrode 744.
  • the test line 741 is aligned with the first working electrode 743.
  • the first potential Vi is applied between the first working electrode 743 and the counter electrode 742.
  • the second potential V2 is applied between the first working electrode 743 and the counter electrode 742, referenced to the second working electrode 744.
  • the first working electrode 743 may be positioned adjacent to the counter electrode 742.
  • the first working electrode 743, and the test line may be between the counter electrode 742 and the reference electrode 744.
  • Figure 8A shows an electrode assembly 810 with a position of the test line indicated by rectangle 811.
  • the electrode assembly 810 comprises a counter electrode 812, a first working electrode 813 and a reference electrode 814.
  • the test line 811 is aligned with the first working electrode 813.
  • the first potential Vi is applied between the first working electrode 813 and the counter electrode 812.
  • the second potential V2A is applied between the first working electrode 813 and the counter electrode 812.
  • the second potential V2B is subsequently also applied between the reference electrode 814 and the counter electrode 812 to provide a control value.
  • the first working electrode 813 may be positioned adjacent to the counter electrode 812.
  • the counter electrode 812 may be L- shaped such that the reference electrode 814 is adjacent to the counter electrode 812 and the first working electrode 813 is between the counter electrode 812 and the reference electrode 814.
  • Figure 8B shows an electrode assembly 820 with a position of the test line indicated by rectangle 821.
  • the electrode assembly 820 comprises a counter electrode 822, a first working electrode 823, a first reference electrode 824 and a second reference electrode 825.
  • the test line 821 is aligned with the first working electrode 823.
  • the first potential Vi is applied between the first working electrode 823 and the counter electrode 822.
  • the second potential V2A is applied between the first working electrode 823 and the counter electrode 822.
  • the second potential V2B is subsequently also applied between the first reference electrode 824 and the counter electrode 822 to provide a first control value.
  • the second potential V2C is subsequently also applied between the second reference electrode 825 and the counter electrode 822 to provide a second control value.
  • the first working electrode 823 may be adjacent to the counter electrode 822. As shown in Figure 8B, the first working electrode may also be between the first reference electrode 824 and the second reference electrode 825. The first reference electrode 824 and the second reference electrode 825 may be adjacent to the counter electrode 822.
  • the test line at which the analyte is captured may be aligned with the first working electrode.
  • the test line may also overlap the counter electrode.
  • the hydrogen peroxide generation may occur upstream of the test line at which the analyte, where present, is captured, such that the generated hydrogen peroxide flows to the bound analyte.
  • Fluidic contact between the electrode assembly and the substrate may be achieved in any appropriate way.
  • an electrode sheet comprising the electrode assembly may be brought into contact with the substrate such that when the substrate is wet, the electrode assembly is in fluidic contact with the substrate.
  • the electrode assembly may be held in place to maintain fluidic contact.
  • pressure may be applied to maintain fluidic contact.
  • spring fingers may be used to hold the electrode sheet in place.
  • the electrode assembly may be connected to the potentiostat circuit in a similar way.
  • the electrode sheet may comprise electrical contacts. When the electrode sheet is brought into contact with the substrate, the electrical contacts may align with conductive tracks on the substrate or a base board. The pressure applied to keep the electrode assembly in fluidic contact with the substrate may also be used to maintain an electrical connection between the electrical contacts and the conductive tracks.
  • the electrode assembly may be printed onto a base board.
  • the base board may be coated with the substrate material, such that good fluidic contact is achieved.
  • the electrode assembly may be connected permanently to conductive tracks on the base board.
  • the electrode assembly may be screen printed onto a base, such as a polymer base. In certain embodiments, the electrode assembly may be fabricated on a PCB.
  • the electrodes of the electrode assembly may be composed of one or more of carbon, silver, platinum and gold.
  • the counter electrode may be silver.
  • the working electrode(s) may be carbon.
  • a gap between adjacent electrodes may be large enough to ensure that the adjacent electrodes do not short together during the manufacturing process.
  • the gap between the adjacent electrodes may be small enough such that a potential applied at the counter electrode to maintain a potential at the working electrode is below a threshold potential.
  • the gap between the adjacent electrodes may be small enough such that noise is below a threshold.
  • a gap between two electrodes between which a potential is applied may be between 0.1 mm and 1 mm.
  • the solution deposited at the sample receiving region may be split, with part of the solution being diverted to activate the battery.
  • the solution may be deposited via sample port 910 of an electronica assay device 900.
  • the solution and the sample may travel down the assay strip 920, comprising the test region.
  • Part of the solution may be diverted down an activation fluid channel 930 such that that part of the solution is incident on the battery 940.
  • the activation fluid channel 930 may be filled with a porous material.
  • the detection reagent may comprise a molecular binding agent labelled with redox active molecules or structures.
  • the redox active molecules or structures are the catalytic agent for the reaction in which the hydrogen peroxide (H2O2) oxidises the indicator molecule.
  • the redox active molecules or structures may comprise the enzyme horse-radish peroxidase, or catalytic platinum nanoparticles.
  • the molecular binding reagent is configured to bind to the analyte, and may comprise an antibody.
  • the molecular binding reagent may comprise Biotin.
  • the test region may comprise polystreptavidin configured to bind to Biotin.
  • the electron donor may comprise flavin mononucleotide, a quinone or a derivative of a quinone. Flavin mononucleotide is photosensitive. Quinones (Q) have the further advantage of being cheaper and produce hydrogen peroxide efficiently. The production of hydrogen peroxide follows a 2 electron electrochemical reduction, as follows:
  • Suitable quinones are 2-hydroxy-1 ,4-naphthoquinone, 2- meth oxy- 1,4- naphthoquinone, 9,10-phenanthrenequinone, 9,10-anthraquinone, 1,4-naphthoquinone, 1 ,2-Naphthoquinone, 2-methoxy-1 ,4-naphthoquinone, 5,8-Dihydroxy-1 ,4-naphthoquinone and 1 ,4-dihydroxyanthraquinone.

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Abstract

L'invention concerne un dispositif de dosage électronique et un procédé de détection de la présence ou de l'absence d'un analyte dans un échantillon à l'aide d'un dispositif de dosage électronique. Le dispositif de dosage électronique est conçu pour émettre un signal électronique indiquant la présence d'un analyte dans un échantillon. Le dispositif de dosage électronique comprend une région de réception d'échantillon, un réactif de détection comprenant un agent catalytique, des molécules de capture, une zone de test et une batterie conçue pour être activée par contact avec un liquide. La zone de test comprend un ensemble électrode et une ligne de test conçue pour se lier aux molécules de capture. Une séquence de potentiels est appliquée à l'ensemble électrode lors de l'activation de la batterie. Un premier potentiel est appliqué à l'ensemble électrode pour produire du peroxyde d'hydrogène dans la zone de test. Un deuxième potentiel est appliqué à l'ensemble électrode pour réduire les molécules indicatrices oxydées, générant un transfert de charge.
PCT/EP2024/066331 2023-06-13 2024-06-13 Dispositif de dosage électronique Pending WO2024256522A1 (fr)

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GB2308804.0 2023-06-13
GBGB2308804.0A GB202308804D0 (en) 2023-06-13 2023-06-13 Electronic assay device

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WO2024256522A1 true WO2024256522A1 (fr) 2024-12-19

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3387926B2 (ja) * 1993-07-23 2003-03-17 ロシュ ダイアグノスティックス コーポレーション 電位差式バイオセンサー及びその使用方法
US20130334042A1 (en) * 2010-12-06 2013-12-19 Syngenta Limited Pathogen sensor
WO2018106129A1 (fr) * 2016-12-09 2018-06-14 Digital Sensing Limited Capteurs électrochimiques et leurs procédés d'utilisation
WO2022011001A1 (fr) * 2020-07-07 2022-01-13 The Regents Of The University Of California Procédé de détection d'antigènes à partir d'échantillons cliniques directs

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3387926B2 (ja) * 1993-07-23 2003-03-17 ロシュ ダイアグノスティックス コーポレーション 電位差式バイオセンサー及びその使用方法
US20130334042A1 (en) * 2010-12-06 2013-12-19 Syngenta Limited Pathogen sensor
WO2018106129A1 (fr) * 2016-12-09 2018-06-14 Digital Sensing Limited Capteurs électrochimiques et leurs procédés d'utilisation
WO2022011001A1 (fr) * 2020-07-07 2022-01-13 The Regents Of The University Of California Procédé de détection d'antigènes à partir d'échantillons cliniques directs

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