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WO2024256533A1 - Procédé et dispositif de détection d'un analyte dans un échantillon - Google Patents

Procédé et dispositif de détection d'un analyte dans un échantillon Download PDF

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Publication number
WO2024256533A1
WO2024256533A1 PCT/EP2024/066352 EP2024066352W WO2024256533A1 WO 2024256533 A1 WO2024256533 A1 WO 2024256533A1 EP 2024066352 W EP2024066352 W EP 2024066352W WO 2024256533 A1 WO2024256533 A1 WO 2024256533A1
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WO
WIPO (PCT)
Prior art keywords
analyte
molecule
sample
solid support
minutes
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Pending
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PCT/EP2024/066352
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English (en)
Inventor
Stephen John Edwards
Stephanie Jane MCINTYRE
YueHua DOU
Paul Davis
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Mologic Ltd
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Mologic Ltd
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Publication date
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Publication of WO2024256533A1 publication Critical patent/WO2024256533A1/fr
Anticipated expiration legal-status Critical
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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 invention relates to methods for detecting an analyte in a sample.
  • the invention also relates to devices for detecting an analyte in a sample.
  • the invention relates to analyte detection kits.
  • Analyte detection methods and devices are useful in many settings and may, for example, find applications in diagnostic fields. Certain methods for detection of an analyte in a sample, wherein a user manually adds required reagents at various time points, are known. Similarly, it is also known to provide certain assay devices for detecting the presence of an analyte in a sample, wherein a user deposits a sample on the assay device and then after a predetermined time in the assay procedure, adds a reagent or other substance necessary for the assay test.
  • the manual nature of such methods and devices requires accurate timing by the user, and is vulnerable to variations between assay procedures. The results of the assay procedure may lack accuracy as a result of these variations.
  • the manual nature of detection methods is time-consuming for a user, who must be available to add required reagents at various time points into the reaction mixture.
  • the results of the assay procedure are typically in the form of a visual output that requires 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.
  • Such user interpretation of the results of the assay procedure may introduce further variation into the measurements.
  • the present invention provides a method for detecting an analyte in a test sample.
  • the method may comprise the steps: a) bringing the test sample into contact with a detection reagent to form an analytedetection reagent complex; b) capturing the analyte-detection reagent complex at a capture zone; c) generating or mobilising hydrogen peroxide; d) generating an oxidized indicator molecule through oxidation of an indicator molecule by the hydrogen peroxide; e) reducing the oxidised indicator molecule to produce a charge transfer; and f) measuring the charge transfer to detect the presence or absence of the analyte in the sample.
  • the analyte-detection reagent complex may be captured via a capture molecule immobilized at a capture zone.
  • the capture zone may be a capture zone of a solid support.
  • the invention also provides a method for detecting an analyte in a test sample, the method may comprise the steps: a) bringing the test sample into contact with a detection reagent to form an analytedetection reagent complex; b) capturing the analyte-detection reagent complex via a capture molecule immobilized at a capture zone of a solid support; c) generating or mobilising hydrogen peroxide; d) generating an oxidized indicator molecule through oxidation of an indicator molecule by the hydrogen peroxide; e) reducing the oxidised indicator molecule to produce a charge transfer; and f) measuring the charge transfer to detect the presence or absence of the analyte in the sample.
  • the invention provides a method for detecting an analyte in a test sample, the method may comprise the steps: a) bringing the test sample into contact with a detection reagent to form an analytedetection reagent complex, the detection reagent comprising: (i) a binding molecule; and (ii) a catalytic agent; b) capturing the analyte-detection reagent complex via a capture molecule immobilized at a capture zone of a solid support; c) generating or mobilising hydrogen peroxide; d) generating an oxidized indicator molecule through oxidation of an indicator molecule by the hydrogen peroxide in the presence of the catalytic agent; e) reducing the oxidised indicator molecule to produce a charge transfer; and f) measuring the charge transfer to detect the presence or absence of the analyte in the sample.
  • the capture molecule may be immobilizable at the capture zone. That is to say that the capture molecule may initially be in solution before it reaches the capture zone, where it is immobilized (either by itself or bound to the analyte-detection complex). However, preferably, the capture molecule is immobilized at the capture zone.
  • the invention provides a device configured to perform the method of the invention.
  • the device may comprise:
  • the invention may provide a device comprising:
  • a solid support comprising: i. a sample application zone for receiving a test sample; ii. a capture zone comprising an immobilized capture molecule, wherein the capture molecule is capable of capturing an analyte which is bound to a detection reagent;
  • the device may comprise:
  • a solid support comprising: i. a sample application zone for receiving a test sample; ii. a capture zone downstream of the sample application zone and comprising an immobilized capture molecule, wherein the capture molecule is capable of capturing an analyte which is bound to a detection reagent;
  • the “means for detecting a charge transfer” is capable to detecting a charge transfer which is indicative of the presence of an analyte in a test sample. It will be understood that reference to “a test sample” means the test sample that the device or kit is used to analyse.
  • the invention provides an analyte detection kit.
  • the analyte detection kit may comprise:
  • the analyte detection kit may comprise:
  • a solid support comprising: i. a sample application zone for receiving a test sample; ii. a capture zone downstream of the sample application zone and comprising an immobilized capture molecule, wherein the capture molecule is capable of capturing an analyte which is bound to a detection reagent;
  • the invention provides use of the device of the invention or the analyte detection kit of the invention for detecting an analyte in a test sample.
  • the analyte may, for example, be a biomarker indicative of a microorganism, such as a virus.
  • the virus may, for example, be a respiratory virus, preferably a coronavirus. Accordingly, the methods, devices or kits provided herein may, for example, be used in the diagnosis of an infection, such as a coronavirus infection.
  • the present inventors have surprisingly discovered that in situ mobilisation or generation of hydrogen peroxide reduces manual input required by a user when detecting an analyte in a sample.
  • a method in which hydrogen peroxide is mobilised or generated in situ improves reproducibility and accuracy of analyte detection as compared to methods in which hydrogen peroxide is added manually by a user. This is because the user does not need to add hydrogen peroxide at a specified time point during the detection method.
  • the inventors have also discovered that the method of mobilising or generating hydrogen peroxide can be utilized in a context of a device, such us a lateral flow strip. Unexpectedly, the present inventors have discovered that with the help of electric potential generated on a working electrode in a device, it is possible accurately to control production of hydrogen peroxide at a specific location in the device. This discovery makes the detection method and the detection device particularly suitable for use in automatic systems (i.e. systems which require little or no user input) for detection of an analyte in the sample.
  • an electric potential that is “about” -600mV can include values between (and including) - 540mV and -660mV.
  • analyte refers to any biological material.
  • the analyte may be any material associated with a healthy state, disease or injury or otherwise altered physiological condition. It may for example be a biomarker that is indicative of a disease state or of the presence of a particular pathogen.
  • the analyte may, for example, be a protein, a peptide, a modified protein, a peptide nucleic acid molecule (PNA), an antigen, an antibody, a metabolite, an enzyme, a nucleic acid molecule (e.g. DNA or RNA), a receptor moiety, a natural or synthetic compound, or a microorganism (e.g. a virus or bacterium) or fragment thereof.
  • PNA peptide nucleic acid molecule
  • the analyte may, for example, originate from a virus, a bacterium, a fungus, a plant, or an animal.
  • An analyte of viral origin may be a capsid protein, such as a nucleocapsid protein, or a nucleocapsid protein antigen.
  • the analyte may be a SARS- CoV-2 nucleocapsid protein antigen.
  • the analyte may, for example, be a viral nucleic acid molecule.
  • An analyte of bacterial origin may be a ligand or receptor from bacterial cell wall or cell membrane.
  • An analyte of bacterial origin may be a bacterial nucleic acid molecule.
  • An analyte of animal origin may be an analyte from a human, e.g. a molecule associated with a particular cancer or other disease of interest (e.g. an infection by a respiratory virus, such as coronavirus).
  • the “detecting” of an analyte may be qualitative, semi-quantitative or quantitative.
  • the method may be used to confirm the presence of an analyte, or to confirm that the analyte is present above a pre-determined threshold level; or to confirm the absence of an analyte, or confirm that any analyte, if present, is below a pre-set threshold.
  • the method may detect/determine/measure the concentration of the analyte in a quantitative manner.
  • test sample refers to any sample, including environmental and clinical samples.
  • the sample may be a biological sample and may, in particular, be a sample collected from a subject.
  • the subject may be an animal, for example, a human.
  • the subject may suffer from a disease or disorder.
  • the subject may have symptoms of a disease or disorder.
  • the sample may, for example, be collected by drawing blood; colleting a swab sample, for example from the mouth, nose, vagina or anus; collecting a urine sample; collecting a faecal sample; collecting a lung lavage sample’ or collecting a sample from a foetus.
  • test sample may, for example, comprise or be selected from more or more of the following: a saliva sample, a nasal swab sample, a sputum sample, a blood sample, a wound sample, a urine sample, a faecal sample, a lung lavage sample, an amniotic fluid sample, a peritoneal fluid sample, an ascites sample, or a breast milk sample.
  • the sample may be, or comprise, a fluid and/or a solid.
  • it may be a solution, emulsion, or suspension.
  • the test sample used in the methods provided herein or used with the devices or kits provided herein is or comprises a fluid.
  • the methods provided herein may include a step of sample preparation, which may, for example, comprise contacting the sample with a solvent, such as water or a buffer.
  • the test sample may further comprise a solvent, such as a reagent buffer.
  • a solvent such as a reagent buffer.
  • the solvent or reagent buffer may comprise an indicator molecule as described herein, a chloride, water, and/or an electron carrier as described herein.
  • the test sample may comprise an analyte of interest.
  • the method, devices and kits described herein intend to determine or detect the presence or absence of said analyte in a test sample.
  • the methods provided herein may include a step of obtaining a suitable sample from a subject.
  • the methods do not include such a step and instead employ a “provided” sample that was previously obtained from a subject.
  • the sample may be a provided sample.
  • charge transfer refers to the amount of charge produced in the process of transforming an indicator molecule from its oxidised form to a reduced form. As mentioned elsewhere herein, this charge transfer is indicative of the presence of an analyte in a test sample.
  • the indicator molecule is reduced by application of a specific electric potential.
  • the electric potential may be applied, controlled and/or measured by at least two electrodes, one being a working electrode (i.e. the electrode may switch the potential to a “detection mode”).
  • the charge transfer may be measured by various means known to the skilled person.
  • the charge transfer may be measured by chronoamperometry.
  • the charge transfer may be measured on at least one working electrode.
  • the charge transfer is measured using a counter electrode, a reference electrode and a working electrode.
  • the charge transfer may be measured on three working electrodes.
  • electron carrier refers to any chemical entity that carries electrons between two different compounds or molecules.
  • the electron carrier may be used in the proximity of an electrode.
  • the electron carrier is used to generate hydrogen peroxide by 2- electron electrochemical reduction.
  • the electron carrier may act as an electron donor in its reduced form.
  • the electron carrier may, for example, be a quinone, flavin mononucleotide (FMN), or nicotinamide adenine dinucleotide.
  • the electron carrier may be 2-hydroxy-1 ,4-naphthoquinone (abbreviated as “Q”), 2-methoxy-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, or 1 ,4-dihydroxyanthraquinone.
  • Q 2-hydroxy-1 ,4-naphthoquinone
  • Q 2-methoxy-1 ,4-naphthoquinone
  • the electron carrier is 2- hydroxy-1 ,4-naphthoquinone.
  • 2-hydroxy-1 ,4-naphthoquinone is unexpensive and does not show sensitivity to light unlike FMN, which makes it particularly suitable for use in the method, devices and kits described herein.
  • electron donor refers to any chemical entity that donates electrons to another compound or molecule.
  • the electron donor must be capable of being used to generate hydrogen peroxide, for example capable of generating hydrogen peroxide by 2-eletron electrochemical reduction.
  • the electron donor may, for example, be generated from an electron carrier in the proximity of an electrode, which applies, control or adjusts the electric potential to a specific value (e.g. -0.6 V); the resulting electron donor may then be used to generate hydrogen peroxide.
  • the electron donor is used to generate hydrogen peroxide by 2-electron electrochemical reduction.
  • the electron donor may, for example, be a reduced form of a quinone, of a flavin mononucleotide (FMN), or of a nicotinamide adenine dinucleotide.
  • the electron donor may be a reduced form of a molecule selected from 2-hydroxy-1 ,4-naphthoquinone (abbreviated as “H2Q”), 2-methoxy- 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, or 1 ,4- dihydroxyanthraquinone.
  • the electron donor is a reduced form of 2-hydroxy-1 ,4- naphthoquinon
  • Step 1 wherein Kc is the rate constant for the chemical step (step 2).
  • Step 1 is a fast electron transfer reaction
  • the chemical step (step 2) is a rate-determining reaction.
  • the electron carrier e.g.
  • a quinone initially accepts electrons to generate the electron donor (H2Q) (i.e. the reduced form of the electron carrier, e.g. the reduced form of quinone).
  • the electron donor is further reduced or disproportionated to generate peroxide.
  • the electrode may adjust the electric potential to a specific value (e.g. - 0.6V) (called herein a “sensing mode”) which allows the electron donor to donate electrons to oxygen found in a sample (e.g. in a sample buffer) by 2-electron electrochemical reduction to generate hydrogen peroxide.
  • a specific value e.g. - 0.6V
  • At least one means one or more. Thus, it encompasses one, two, three, four, five, six, seven, eight, nine, ten or more electrodes, and so on. It will now be readily apparent to the skilled person that the method, devices and kits may rely on the use of two, three, four, five, six, seven, eight, nine, ten or more electrodes.
  • the electrodes may be carbon electrodes and/or silver electrodes. The number of carbon and silver electrodes does not need to be the same. For example, the method, device and kit may rely on the presence of five carbon electrodes and two silver electrodes.
  • the present invention provides a method for detecting the presence or absence of an analyte in a test sample.
  • the method may comprise the steps: a) bringing the test sample into contact with a detection reagent to form an analytedetection reagent complex; b) capturing the analyte-detection reagent complex at a capture zone; c) generating or mobilising hydrogen peroxide; d) generating an oxidized indicator molecule through oxidation of an indicator molecule by the hydrogen peroxide e) reducing the oxidised indicator molecule to produce a charge transfer; and f) measuring the charge transfer to detect the presence or absence of the analyte in the sample.
  • binding should be understood to mean that the binding is specific unless otherwise specified.
  • the binding molecule of the detection reagent should be capable of specifically binding to the analyte.
  • step a) preceding step b), which in turn precedes step c) and so on.
  • step b) preceding step b
  • step c) preceding step c
  • one or more steps may be initiated simultaneously.
  • Logical orders will be understood by the skilled person based on the teaching herein; for example, it is logical that step d) can only occur once step c) has mobilised or generated sufficient hydrogen peroxide; or that step f) can only be performed once steps a) through to e) have been initiated.
  • the detection reagent may comprise a binding molecule and a catalytic agent.
  • the binding molecule may be conjugated to the catalytic agent directly or indirectly (e.g. via a linker molecule).
  • the binding molecule may be any molecule capable of binding to an analyte in the test sample.
  • the binding molecule of the detection reagent may thus be referred to as being capable of binding to a first binding site of the analyte.
  • the binding molecule may, for example, be a protein, a peptide, an antibody or a fragment thereof, an aptamer, an enzyme, a receptor, a biotin molecule, or a streptavidin molecule.
  • the binding molecule is an antibody or a binding-fragment thereof.
  • the antibody may be a monoclonal antibody or a polyclonal antibody and may be monospecific, bispecific or multi-specific.
  • the antibody or a fragment thereof may be specific and/or selective for the analyte.
  • the antibody or fragment thereof may have high specificity and/or selectivity for SARS-CoV-2 nucleocapsid protein antigen.
  • the catalytic agent may be any redox active molecule or structure or a suitable enzyme.
  • the catalytic agent may improve generation of an oxidized indicator molecule as compared to the generation of an oxidized indicator molecule in the absence of the catalytic agent, as discussed elsewhere herein.
  • the catalytic agent may be selected from peroxidases, catalytic nanoparticles, N-oxyl radicals, oxoammonium cations, amine cation radicals, thiyl radicals, quinones, dioxiranes and oxaziridines, and hypervalent iodine compounds.
  • the catalytic agent may be a horseradish peroxidase or a platinum nanoparticle (such as a catalytic platinum nanoparticle).
  • the catalytic agent is a platinum nanoparticle.
  • the binding molecule is an antibody and the catalytic agent is a platinum nanoparticle.
  • the detection reagent comprises a platinum nanoparticle and an antibody which binds to the analyte in the test sample.
  • the catalytic agent may catalyse the reaction of generating an oxidized indicator molecule from i) an indicator molecule (e.g. an indicator molecule in the reduced state); and ii) hydrogen peroxide.
  • the step of bringing the test sample into contact with a detection reagent should typically be performed before step b).
  • the step of bringing the test sample into contact with a detection reagent may be performed before the sample is added to the solid support. That is to say that the step of bringing the test sample into contact with a detection reagent may, for example, be performed by incubating the test sample and the detection reagent for a predetermined period to allow the formation of the analyte-detection reagent complex.
  • the predetermined period may be at least 1 second, at least 5 seconds, at least 10 seconds, at least 15 seconds, at least 20 seconds, at least 30 seconds, at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes.
  • the predetermined period is preferably less than 24, 15 or 12 hours and typically less than 10, 9, 8, 7, 6, 5 or 4 hours.
  • the predetermined period may, for example be from 1 second to 4 hours, from 5 second to 2 hours, or from 5 second to about 90 minutes, for example about 10 seconds to about 15 minutes.
  • the step of bringing the test sample into contact with a detection reagent may be performed on a solid support.
  • the analyte-detection reagent complex may be captured at the capture zone.
  • the capture zone may comprise a capture molecule.
  • the capture molecule may be immobilized at the capture zone.
  • the capture molecule must be capable of capturing the analyte-detection reagent complex, either directly or indirectly. It may, for example, comprise (or essentially consist of) a protein, a peptide, an antibody or a fragment thereof, an aptamer a receptor, a biotin molecule, or a streptavidin molecule.
  • the capture molecule (of the capture zone) is capable of binding to the analyte part of the analyte-detection complex.
  • the analyte-detection reagent complex is captured at the capture zone directly.
  • the capture molecule is capable of binding to a binding site of the analyte.
  • the detection reagent is also capable of binding to a binding site of the analyte.
  • the binding molecule of the detection reagent is capable of binding to a first binding site of the analyte and the capture molecule is capable of binding to a second binding site of the analyte.
  • the capture molecule (of the capture zone) is capable of binding to a further binding molecule.
  • said further binding molecule may bind to the analyte part of the analyte-detection reagent complex and the analyte-detection reagent complex is captured at the capture zone indirectly.
  • the capture molecule is capable of binding to a further binding molecule, which in turn is capable of binding to a binding site of the analyte.
  • the detection reagent is also capable of binding to a binding site of the analyte.
  • the binding molecule of the detection reagent is capable of binding to a first binding site of the analyte and the further binding molecule is capable of binding to a second binding site of the analyte.
  • the capture molecule is preferably a streptavidin molecule (or other avidin molecule).
  • the further binding molecule may comprise a capture site which allows for binding to the capture zone (e.g. the capture molecule of the capture zone).
  • the capture site which allows for binding to the capture zone is preferably different to the site of the further binding molecule which binds with the analyte or analyte-detection reagent complex.
  • the further binding molecule may comprise (or essentially consist of) a protein, a peptide, an antibody or a fragment thereof, an aptamer, a receptor, a biotin molecule, or a streptavidin molecule.
  • the further binding molecule comprises a biotin molecule.
  • the further binding molecule comprises a biotin molecule and an antibody or a fragment therefore.
  • the biotin molecule may be conjugated to the antibody or a fragment thereof.
  • the antibody may be a monoclonal antibody or a polyclonal antibody.
  • the capture zone may comprise avidin (e.g. streptavidin) molecules arranged to interact with biotin molecules that form (part of) the further binding molecules.
  • avidin e.g. streptavidin
  • the further binding molecule may be specific and/or selective for the analyte part of the analyte-detection reagent complex.
  • At least one step of the method described herein may be performed on a solid support.
  • at least steps b)-d) of the method are performed on a solid support.
  • Steps e) and/or f) may also be performed on a solid support.
  • the step of bringing the test sample into contact with a detection reagent i.e. step a)
  • step a) may, in some embodiments, be performed before the sample is applied to a solid support as described above.
  • step (a) may be performed on the solid support, in which case the solid support may comprise the detection reagent in pre-deposited form.
  • the zone comprising the detection reagent may be at or downstream of the sample application zone, provided it is upstream of the capture zone.
  • the solid support may define a liquid flow path of the test sample.
  • the solid support may form part of a lateral flow strip or device.
  • the solid support may comprise a sample application zone and a capture zone, wherein the sample application zone must be upstream of the capture zone.
  • the capture zone may also be a detection zone.
  • the solid support may comprise a detection zone downstream of the capture zone.
  • the solid support may further comprise a control zone downstream of the capture zone and/or the detection zone.
  • the function of the control zone may be to show that the method works or has worked.
  • the control zone may comprise an immobilized molecule which interacts with and/or binds to a detection reagent in the absence of the binding to the analyte (or in the absence of the analyte in the test sample).
  • the immobilized molecule at the control zone may comprise (or essentially consist of) a protein, a peptide, an antibody or a fragment thereof, an aptamer, a receptor, a biotin molecule, or a streptavidin molecule.
  • the solid support may comprise any of the reagents, i.e. one or more of the reagents may have been predeposited on and/or into the solid support.
  • a pre-deposit may, for example, be achieved by spraying a reagent onto or embedding a reagent on or in the solid support.
  • the detection reagent may be pre-deposited on or in the solid support.
  • the indicator molecule may be pre-deposited on or in the solid support.
  • the electron carrier may be pre-deposited on or in the solid support.
  • Each of these reagents may pre-deposited at a different or the same zone or location along the solid support (e.g. a lateral flow strip).
  • the indicator molecule may be pre-deposited on or in the solid support at (or near) the capture zone.
  • the detection reagent may be pre-deposited on or in the solid support at (or near) the sample application zone or downstream of the sample application zone and upstream of the capture zone.
  • the electron carrier may be predeposited on or in the solid support at (or near) the capture zone.
  • any of the reagents used in the method may be added directly to or onto the solid support.
  • the electron carrier may be added to the solid support (e.g. the capture zone of the solid support) before the method is performed or while the method is being performed.
  • the reagents used in the method have been added to the test sample in step a) and/or are pre-deposited on the solid support.
  • the solid support may comprise an “indicator pad” that is physically separated from the pad(s) that comprise(s), or to which is added, the detection reagent and/or the further binding molecule.
  • indicator pad is meant a pad on or within which the indicator molecule has been pre-deposited, or to which the indicator molecule is added. The physical separation may be achieved through the use of a suitable physical barrier, which may for example be configured to dissolve as explained elsewhere herein. An exemplary set up is illustrated in Figure 2.
  • the method described herein is performed on a lateral flow strip or in a lateral flow device.
  • the further binding molecule may be pre-deposited in or on the solid support.
  • the further binding molecule may be pre-deposited in or on the solid support downstream of the sample application zone.
  • the further binding molecule may be pre-deposited in or on the solid support downstream of the sample application zone and upstream of the capture zone. This localisation of further binding molecules allows for solubilisation or otherwise mobilisation of the further binding molecules once the test sample is added to the solid support (e.g. at the sample application zone).
  • the further binding molecules may be present in the test sample (e.g. added to the sample as part of the reagent buffer prior to application to the solid support).
  • the provided method may be performed in lateral flow or vertical flow devices in certain embodiments. Generally, therefore, the provided methods or re detection devices may rely upon some form of solid support.
  • the solid support may define a liquid flow path for the sample.
  • the solid support comprises a chromatographic medium or a capillary flow device.
  • the invention may be provided in a test strip format in some embodiments.
  • the solid support may comprise a detection zone upstream or downstream of the capture zone.
  • the detection zone may be downstream of the sample application zone (if present). In some embodiments, the detection zone may be the same as the capture zone (i.e. only one zone is present which functions as both the capture zone and the detection zone).
  • the detection zone may sense changes to electric potential (e.g. it may sense or detect charge transfer).
  • the solid support may further comprise a detection reagent zone.
  • the detection reagent zone may be upstream of the capture zone and/or downstream of the sample application zone.
  • the detection reagent may be pre-deposited, for example it may be sprayed onto or embedded in or on the solid support at the detection zone.
  • the detection reagent may be pre-deposited in or on the solid support, wherein the solid support is an individual pad of a lateral flow strip or device.
  • the application of the test sample to the solid support e.g. at the sample application zone, if present, may solubilise or otherwise mobilise the detection reagent so that it flows to the capture zone.
  • the detection reagent may be applied onto the solid support, for example it may be present in a fluid that is applied onto the solid support, for example at the sample application zone, if present,
  • the detection reagent may, for example, be present in a reagent buffer.
  • the detection reagent may be contacted with the sample prior to, simultaneously with, or after application to the solid support. In embodiments where the detection reagent is not pre-deposited on the solid support, it is preferred that the detection reagent is contacted with the sample prior to application to the solid support.
  • the capture zone may be formed on a solid support. Any support to which the capture molecules may be attached to form a capture zone is intended to be encompassed.
  • the solid support may take the form of a bead (e.g. a sepharose or agarose bead) or a well (e.g. in a microplate), for example.
  • the solid support may be a pad.
  • the solid support may form part of a lateral flow test strip.
  • Figure 2 One example of a solid support is visualised in Figure 2, in which the capture molecule immobilized at the capture zone of the solid support binds to the further binding molecule which is bound to the analytedetection reagent complex, thus, immobilizing the analyte-detection reagent complex at the capture zone.
  • the capture zone may be defined by the immobilization therein or thereon of capture molecules capable of binding to the analyte or to the further binding molecules. Immobilization of capture molecules may be achieved by any suitable means.
  • the capture molecules may be immobilized by directly binding to the solid support or immobilized indirectly via binding to a carrier molecule, such as a protein, associated with, or bound to, the solid support.
  • the capture molecule is not pre-immobilized at the capture zone. Instead, the capture molecule becomes immobilized once the test sample and/or reagent buffer have been added to the solid support.
  • the capture molecule may be comprised in the test sample and/or reagent buffer added in step a) of the method described herein. However, embodiments in which the capture molecule is pre-immobilized at the capture zone are preferred.
  • TMB is an example of an indicator molecule and any suitable indicator molecule as defined herein may be used instead of TMB.
  • TMB is of high purity.
  • the purity of TMB is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.
  • the high purity of TMB ensures that the TMB does not react with the electron carrier before the reagents are in the proximity of the catalytic agent.
  • a solid support may comprise a detection reagent (“Pt-Ab2”), a capture molecule (e.g. PSA), and a further binding molecule (“Biotin-Ab1”) pre-deposited on a solid support (e.g. a lateral flow strip).
  • the detection reagent and the further binding molecule may be predeposited on separate pads.
  • the capture molecule may be pre-deposited at the capture zone (i.e. the black area in the middle of the nitrocellulose membrane).
  • the test sample is applied to the sample application zone (e.g. the “Biotin-Ab1” pad).
  • the sample solution flows towards the right side of the strip, it solubilizes and/or mobilizes the detection reagent and the further binding molecule, allowing for the formation of a further binding molecule- analyte-detection reagent complex.
  • Any order and/or orientation of the detection reagent pad and the further binding molecule pad is envisaged.
  • the binding order to analyte may be such that first the detection reagent binds to the analyte, or such that first the further binding molecule binds to the analyte.
  • the detection reagent and the further binding molecule may bind to the analyte at the same time.
  • the further binding molecule may bind to the analyte at a side that is different to the side of binding of the detection reagent.
  • the complex is immobilized at the capture zone by binding of the further binding molecule part of the complex to the capture molecule which is immobilized at the capture zone.
  • a solution comprising the indicator molecule is applied directly to the pad (e.g. to the same sample application zone as the test sample). Once at the capture zone, the indicator molecule is oxidized by hydrogen peroxide with the help of the catalytic agent immobilized at the capture zone.
  • hydrogen peroxide may be pre-deposited on the strip (ideally in the proximity of the capture zone, and downstream of the sample application zone and upstream of the capture zone); or hydrogen peroxide may be generated at the capture zone or upstream of the capture zone using one of the methods described herein.
  • an electron carrier may form part of the solution comprising the indicator molecule.
  • the electron carrier may be pre-deposited on the strip upstream of the capture zone or at the capture zone.
  • the indicator molecule e.g. TMB
  • the steps are similar as the steps performed on the strip of Figure 1 , however; here, after immobilization of the final complex at the capture zone, a solution is applied directly to the pad comprising the indicator molecule to solubilize and/or mobilize this reagent so that it is allowed to flow towards the capture zone (i.e. the right side of the strip).
  • the pad comprising the indicator molecule may be physically separated from the detection reagent and/or the further binding molecule pad, e.g. by the use of a suitable barrier, such as double-sided tape.
  • the solution added to the pad may comprise the indicator molecule.
  • the analyte-detection reagent complex may be captured via a capture molecule immobilized at a capture zone.
  • the capture zone may be a capture zone of a solid support.
  • the invention provides a method for detecting an analyte in a test sample, the method may comprise the steps: a) bringing the test sample into contact with a detection reagent to form an analytedetection reagent complex; b) capturing the analyte-detection reagent complex via a capture molecule immobilized at a capture zone of a solid support; c) generating or mobilising hydrogen peroxide; d) generating an oxidized indicator molecule through oxidation of an indicator molecule by the hydrogen peroxide; e) reducing the oxidised indicator molecule to produce a charge transfer; and f) measuring the charge transfer to detect the presence or absence of the analyte in the sample.
  • the method may comprise the steps: a) bringing the test sample into contact with a detection reagent to form an analytedetection reagent complex; b) binding of a further binding molecule to the analyte-detection reagent complex; c) capturing the analyte-detection reagent complex via the further binding molecule which binds to the capture molecule immobilized at a capture zone of a solid support; d) generating or mobilising hydrogen peroxide; e) generating an oxidized indicator molecule through oxidation of an indicator molecule by the hydrogen peroxide; f) reducing the oxidised indicator molecule to produce a charge transfer; and g) measuring the charge transfer to detect the presence or absence of the analyte in the sample.
  • the further binding molecule may bind to the analyte before, at the same time or after the detection reagents binds to the analyte.
  • the method may comprise: a) bringing the test sample into contact with a further binding molecule and a detection reagent to form a further binding molecule-analyte-detection reagent complex; b) capturing the further binding molecule-analyte-detection reagent complex via the further binding molecule portion of the complex at a capture zone of a solid support, optionally via the capture molecules immobilized at the capture zone; c) generating or mobilising hydrogen peroxide; d) generating an oxidized indicator molecule through oxidation of an indicator molecule by the hydrogen peroxide; e) reducing the oxidised indicator molecule to produce a charge transfer; and f) measuring the charge transfer to detect the presence or absence of the analyte in the sample.
  • the further binding molecule and the detection reagent interact with and/or bind to different binding sites of an analyte.
  • a “sandwich” structure is formed in which the analyte is “sandwiched” between the further binding molecule and the detection reagent.
  • the binding molecule of the detection reagent may be capable of binding to a first binding site of the analyte and the capture molecule may be capable of binding to a second binding site of the analyte.
  • the first and second binding sites of the analyte are different.
  • a pre-deposited reagent is allowed to enter a solution. This makes it available to react with one or more of the assay components.
  • the hydrogen peroxide becomes available to oxidise the indicator molecule in the presence of the catalytic agent.
  • hydrogen peroxide may be pre-deposited on a solid support upstream of the capture zone, at the capture zone or in a close proximity of the capture zone.
  • generation of hydrogen peroxide is preferred over mobilisation of hydrogen peroxide.
  • generating hydrogen peroxide is meant that hydrogen peroxide is formed de novo. Thus, this excludes the application of hydrogen peroxide. That is to say that in the step of generating hydrogen peroxide, the user is not required to manually add hydrogen peroxide.
  • the hydrogen peroxide generation may be referred to as “in situ” generation, as the hydrogen peroxide is generated where it is needed, for example in or on the solid support.
  • the step of generating hydrogen peroxide may be performed at ,or in the close proximity of, the capture zone, for example upstream of the capture zone.
  • the step of generating hydrogen peroxide may be performed with the use of at least one electrode.
  • hydrogen peroxide may be generated electrochemically.
  • at least two, at least three, at least four, at least five, at least six or at least seven electrodes are used.
  • the electrode may apply or adjust the electric potential to a specific value to generate hydrogen peroxide.
  • the specific value may be between -300mV and -1000mV, between -400mV and -900mV, or between -500mV and - 700mV.
  • the electrode may apply the electric potential of between -300mV and -1000mV, between -400mV and -900mV, or between -500mV and -700mV.
  • the electric potential is about -600mV.
  • at least two electrodes are used.
  • the electric potential between the two electrodes may between -300mV and - 1000mV, between -400mV and -900mV, or between -500mV and -700mV, preferably about -600mV to generate hydrogen peroxide.
  • the electric potential may be applied for between 0.1-60 seconds, 1- 45 seconds, 5-30 seconds or 10-25 seconds to generate hydrogen peroxide, preferably the electric potential is applied for about 20 seconds. If the method described herein is performed on a solid support (e.g.
  • the solid support may comprise at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 electrodes.
  • the solid support comprises at least 4 electrodes.
  • the electrode(s) may be distributed across the solid support.
  • the electrode(s) may be located on the top of the solid support.
  • the electrode(s) may be located on the bottom of the solid support.
  • the electrode(s) are in fluid communication with the solid support.
  • the electrode may be printed, preferably screen-printed.
  • the electrode may be located and the capture zone and/or the detection zone (if present).
  • the capture zone may also function as a detection zone.
  • at least two electrodes are located at the capture zone and/or a detection zone (if present).
  • the electrode(s) may be a carbon electrode (e.g. a carbon-coated electrode) and/or a silver electrode (e.g. a silver-coated electrode).
  • a carbon electrode e.g. a carbon-coated electrode
  • a silver electrode e.g. a silver-coated electrode
  • at least one carbon electrode is present. More preferably still, at least one carbon electrode and at least one silver electrode is present in or on the solid support.
  • hydrogen peroxide is generated on a carbon electrode. For example, dissolved oxygen may be reduced to hydrogen peroxide directly at a carbon electrode.
  • the hydrogen peroxide may be generated in the presence of an electron carrier and the application of reduction potential as described herein.
  • the electron carrier e.g. quinone
  • hydrogen peroxide may be produced according to the following equations:
  • Step 1
  • the production of hydrogen peroxide may be precisely timed by applying the electric potential to the electrode after the test sample has been brought into contact with the detection reagent.
  • the production of hydrogen peroxide may be precisely timed by applying the electric potential to the electrode after the test sample has been applied to the application zone of the solid support (e.g. a lateral flow strip or device).
  • the electric potential may be applied to the electrode about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, or about 20 minutes after the test sample has been brought into contact with the detection reagent.
  • a delay in the application of the electric potential allows for the formation of the analyte-detection reagent complex and capturing of said complex at the capture zone.
  • the electron carrier may be in the proximity of the capture zone.
  • the electron carrier may be in a fluid communication with an electrode (or at least one of the two, three, four, five, six or seven electrodes used) in the proximity of the capture zone.
  • the electron carrier may be coated on at least one electrode (i.e. the electron carrier (e.g. quinone) may be mixed with the electrode material when the electrode is prepared).
  • the electron carrier e.g. quinone
  • the term “proximity” as used herein refers to a distance which allows for the reaction to take place.
  • the electron carrier may be close enough to the capture zone and in a fluid communication with the electrode(s) so that the electric potential generated by the electrode (or at least two, three, four, five, six, or seven electrodes) allows for the electron carrier to carry electron(s) to generate hydrogen peroxide by 2-electron electrochemical reduction.
  • the electrode(s) may be located on a solid support.
  • the electron carrier may be added directly onto the electrode(s) of the solid support.
  • the electron carrier may be comprised in a test sample (or a buffer added to the test sample).
  • the electron carrier may be pre-deposited on or in the solid support upstream of the capture zone. In this example, the test sample (and any test sample buffer, is present) solubilizes the pre-deposited electron carrier so that the electron carrier flows towards the capture zone together with the test sample (and is in a fluid communication with the electrode(s)).
  • the electrode(s) may be a carbon electrode and/or a silver electrode.
  • hydrogen peroxide is generated on a carbon electrode.
  • the silver electrode is used as a counter electrode.
  • the electron carrier is 2-hydroxy-1 ,4-naphthoquinone.
  • the electric potential between two electrodes is set to about -600mV (called herein a “sensing mode”) which allows the electron carrier (e.g. 2-hydroxy-1 ,4-naphthoquinone) to carry electrons to generate hydrogen peroxide by a 2-electron electrochemical reduction reaction.
  • the step of generating hydrogen peroxide may be performed with the use of photogeneration.
  • light illumination e.g. blue LED illumination
  • the source of light might be applied to the capture zone.
  • the source of light may be applied for between 0.1-60 seconds, 1-45 seconds, 3-25 seconds or 5-20 seconds to generate hydrogen peroxide, preferably the source of light is applied for about 10 seconds.
  • a method relying on photogeneration of hydrogen peroxide is shown in Figure 13 (bottom) in which blue LED light is applied to a capture zone of a test strip.
  • hydrogen peroxide is generated in the presence of an electron carrier as described herein.
  • the method described herein relies on photogeneration of hydrogen peroxide in the presence of FMN.
  • FMN is slight sensitive which makes it particularly suitable for use in the method.
  • the production of hydrogen peroxide may be precisely timed by applying the source of light after the test sample has been brought into contact with the detection reagent.
  • the source of light may be applied about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, or about 20 minutes after the test sample has been brought into contact with the detection reagent.
  • a delay in the application of the source of light allows for the formation of the analyte-detection reagent complex and capturing of said complex at the capture zone.
  • the step of photogeneration may be automated (i.e. require no or minimal input from the user).
  • the indicator molecule may be any molecule which can be oxidised by hydrogen peroxide to form an oxidised form (i.e. an oxidised indicator molecule).
  • the indicator molecule may be a hydrogen donor for the reduction of hydrogen peroxide to water.
  • the indicator molecule may be a hydrogen donor for the reduction of hydrogen peroxide in the presence of a catalytic agent (details of which are discussed elsewhere herein).
  • the indicator molecule may be any molecule which can form a reduced form (i.e. a reduced indicator molecule) upon application of a particular electric potential.
  • the indicator molecule may be any molecule which can be oxidised by hydrogen peroxide to form an oxidised form and form a reduced form upon application of a particular electric potential.
  • the indicator molecule may have a colour or may be colourless (or substantially colourless).
  • the indicator molecule is colourless (or substantially colourless) at least in its reduced form.
  • the indicator molecule may have a colour in its oxidised form.
  • the indicator molecule may have a pale bluegreen colour in its reduced form and blue colour in its oxidised form.
  • the change of colour between the two forms is measured.
  • the change of colour may be measured using a spectrophotometer.
  • the indicator molecule may be present in a reaction buffer and may, for example, be brought into contact with the test sample prior to the step of bringing the test sample into contact with a detection reagent and/or a solid support.
  • the indicator molecule may be pre-deposited (for example sprayed or embedded) in or on a zone which is upstream of the capture zone.
  • a zone comprising a pre-deposited indicator molecule is referred to herein as an “indicator zone”.
  • the indicator zone may be downstream of the application zone (if present).
  • the location of the indicator zone is such that the test sample, once applied to the solid support, can mobilise the indicator molecule and allow it to flow to the capture zone.
  • the indicator molecule is pre-deposited, for example sprayed or embedded, in or on a zone which is located between the sample application zone and the capture zone.
  • the indicator molecule may, for example, be a chromogenic substance.
  • the indicator molecule may, for example, be 3, 3', 5,5'- Tetramethylbenzidine (TMB).
  • TMB can act as a hydrogen donor for the reduction of hydrogen peroxide to water by a catalytic agent (e.g. a platinum nanoparticle or a peroxidase enzyme, such as horseradish peroxidase).
  • the resulting one-electron oxidation product is a diimine-diamine complex, which causes the solution to take on a blue colour, and this colour change can be read on a spectrophotometer at the wavelengths of 370 and 650 nm.
  • the oxidation of TMB to 3, 3', 5,5'- tetramethylbenzidine diamine is shown below:
  • the step of generating an oxidized indicator molecule may be performed at (or in the close proximity of) the capture zone.
  • the oxidation of an indicator molecule by hydrogen peroxide may be catalysed by a catalytic agent (which forms part of the detection reagent).
  • a catalytic agent which forms part of the detection reagent.
  • the oxidised indicator molecule Box may be generated according to the following equation:
  • the reduced form of the indicator molecule is indicated by “BRD“ and the oxidised form of the indicator molecule is indicated by “Box”.
  • methods and devices described herein may rely on (or comprise) a barrier configured to dissolve after a pre-determined time period of being in contact with a solution, such that the solution is delivered via the dissolution of the barrier at a pre-determined time.
  • the solution may comprise the indicator molecules, such that the production of hydrogen peroxide may be more precisely timed as a result of the indicator molecules reaching the capture zone at a predetermined time after the solution was deposited.
  • the barrier may, for example, be a gelatin film barrier.
  • the test sample and/or a solution comprising the reaction reagent(s) may be prevented from entering the solid support or travelling along the solid support by the gelatin film barrier.
  • the solid support may comprise a pad comprising gelatinase, glucose and indicator molecules (such as TMB) in a dried state.
  • the pad e.g. a porous pad
  • the pad comprising gelatinase, glucose and indicator molecules is preferably located upstream of the gelatin film barrier.
  • Depositing the test sample and/or the solution comprising the reaction reagent(s) may rehydrate the gelatinase. The rehydrated gelatinase may begin to dissolve the gelatin film barrier.
  • the gelatin film barrier may dissolve after a pre-determined time period (controlled, for example, by the thickness of the gelatin film barrier). After the pre-determined time period, the gelatin film barrier may rupture and the test sample and/or the solution may pass along the solid support, carrying glucose and the indicator molecules.
  • the capture zone may comprise glucose oxidase (GOx) in an immobilised state. When the glucose reaches the GOx, hydrogen peroxide may be generated.
  • GOx glucose oxidase
  • the catalytic agent is present in the proximity of the capture zone.
  • the analyte-detection reagent complex may be immobilized at the capture zone, thereby bringing the catalytic agent portion of the detection reagent in the proximity of the indicator molecule and hydrogen peroxide.
  • the indicator molecule is oxidised if both hydrogen peroxide and the catalytic agent are co-localized to the capture zone.
  • the catalytic agent may improve generation of an oxidized indicator molecule by a 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 100-fold, 500-fold, 1000-fold as compared to the generation of an oxidized indicator molecule in the absence of the catalytic agent (e.g. at all in the reaction sample, or at the capture zone). That is to say that the catalytic agent is, preferably, essentially required for any, or any significant, oxidation of an indicator molecule.
  • the step of generating an oxidized indicator molecule may preferably only take place in the presence of an analyte in the test sample.
  • the step of generating an oxidized indicator molecule may generate significantly more oxidized indicator molecule in the presence of the analyte in the test sample as compared to the absence of the analyte in the test sample.
  • the step of generating an oxidized indicator molecule may generate at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 75 times, at least 100 times, at least 500 times, or at least 1000 times more oxidized indicator molecule in the presence of the analyte in the test sample as compared to the absence of the analyte in the test sample.
  • the step of reducing the oxidized indicator molecule to produce a charge transfer may require no or minimal input from the user. That is to say that, preferably, the step of reducing the oxidized indicator molecule is automated. This step may be performed about 1 second, about 5 seconds, about 10 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, or about 20 minutes after the step of generating hydrogen peroxide. Preferably, the step of reducing the oxidized indicator molecule is performed about 1 minute after the step of generating hydrogen peroxide.
  • the step of reducing the oxidized indicator molecule may be performed using at least one, at least two, at least three, at least four, at least five, at least six, or at least seven electrode(s).
  • the electric potential may be applied by at least one electrode.
  • the electric potential may be applied between at least two, at least three, at least four, at least five, at least six or at least seven electrodes.
  • the electric potential is applied between two electrodes.
  • the electric potential may be sufficient for the oxidised indicator molecule to be reduced, generating the indicator molecule and a charge transfer. The reduction may occur according to the following equation:
  • the step of reducing the oxidized indicator molecule may be performed by switching the electric potential of an electrode from 0 mV to between -10mV to -150mV.
  • the electric potential is switched from 0 mV to between -75 mV and -125 mV. More preferably still, the electric potential is switched from OmV to -100 mV. If at least two electrodes are used, initially, there may be an absence of an electric potential (i.e. an open circuit potential).
  • the electric potential may be switched on between the at least two electrodes to a value of between 0 mV and -150 mV, or a value of between 0 mV and -100 mV.
  • the electric potential may be switched (on) for between 0.1-60 seconds, 0.3-30 seconds, 0.5-10 seconds, or 0.75-1.5 seconds. Preferably, the electric potential is switched (on) for 1 second.
  • the indicator molecule may be a chromogenic substance.
  • the indicator molecule may be 3,3',5,5'-Tetramethylbenzidine (TMB).
  • the charge transfer may be indicative of a presence or absence of an analyte in the test sample.
  • the step of measuring the charge transfer may be used to detect the presence or absence of the analyte in the test sample.
  • the charge transfer may be measured by any means known to the skilled person. For example, the charge transfer may be measured by chronoamperometry.
  • the charge transfer may be measured on at least one electrode.
  • the charge transfer may be measured on at least two, at least three, at least four, at least five, at least six, or at least seven electrodes.
  • the measurement may be corrected for blank (background) values.
  • the method may further comprise a step of calibration (e.g.
  • the step of measuring the charge transfer may include subtracting the background level of the charge transfer to arrive at corrected values for the charge transfer.
  • a potential may be applied to the working electrode and the counter electrode such that the working electrode potential at the working electrode is controlled to be a constant specific potential value measured against a third reference electrode (and the reference electrode has a constant half-cell potential in the solution). This process may be, for example, performed by a potentiostat circuit.
  • the counter and reference electrode can be the same electrode. The potential may be held at that specific potential for a fixed time and the current flowing through the working and counter electrodes measured.
  • the analyte (if present in the test sample) may be detected at a concentration of less than 350 pg/mL by the method described herein.
  • the analyte may be detected at a concentration of less than 300 pg/mL, less than 250 pg/mL, less than 200 pg/mL, less than 150 pg/mL, less than 100 pg/mL, less than 50 pg/mL, less than 25 pg/mL, less than 12.5 pg/mL or less than 10 pg/mL.
  • the method described herein is very sensitive.
  • the method described herein is able to detect an analyte at a concentration of as little as 10 pg/mL.
  • the method described herein is at least about 8 times more sensitive than the conventional lateral flow test which relies on visual detection of an indicator molecule (e.g. a blue form of TMB).
  • the method described herein requires little or no input from the user beyond providing the test sample. Though the step of providing the sample may also be automated.
  • the automated nature of the method, devices, systems and kits described herein relies, in part, on the pre-deposition of various reagents on the solid support.
  • the automated nature of the method, devices, systems and kits described herein may rely on the presence of a battery which may be activated by the liquid from the test sample and/or reaction buffer.
  • the battery provides a sufficient source of power to active a timer which may initiate various steps of the method. For example, after a first period of time from activation of the battery, a first potential is applied to the at least one electrode to generate hydrogen peroxide.
  • the first period of time may be 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 12 minutes, 15 minutes, or 20 minutes.
  • a second potential is applied to the at least one electrode such that the oxidised indicator molecule is reduced, generating the indicator molecule and a charge transfer.
  • the second period of time may be greater than the first period of time by 1 second, 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 12 minutes, 15 minutes, or 20 minutes.
  • the battery may provide a sufficient source of power to send the measurements of the charge transfer to an external medium (e.g. a computer storage medium or a computer application).
  • the step of collecting the test sample may be performed before the step of bringing the test sample into contact with the detection reagent.
  • the sample may, for example, be collected by collecting a swab sample from a suitable area as discussed elsewhere herein; by a biopsy or by phlebotomy; or by collecting a urine, amniotic fluid, peritoneal fluid, or faecal sample.
  • test sample may further comprise reagents useful in the method described herein (e.g. the indicator molecule, the electron carrier, or water).
  • reagents useful in the method described herein e.g. the indicator molecule, the electron carrier, or water.
  • At least one step of the method described herein may be performed on a solid support.
  • at least steps b)-d) of the method are performed on a solid support.
  • Steps e) and/or f) may also be performed on a solid support.
  • the presence of a solid support e.g. which comprises the capture zone
  • the co-localization of some of the reagents used in the methods described herein allows to perform various method steps.
  • the step of generating hydrogen peroxide may efficiently be performed if an electron carrier (e.g.
  • a quinone is located in the proximity of at least one electrode, or a source of light, at the capture zone or upstream of the capture zone.
  • the step of generating an oxidized indicator molecule may efficiently be performed if hydrogen peroxide and a catalytic agent are in close proximity.
  • the analyte-detection reagent complex is immobilized at the capture zone so that the catalytic agent (which forms part of the detection reagent) is placed in the close proximity to hydrogen peroxide which is generated at the capture zone and/or upstream of the capture zone. This close proximity of the reagents allows for oxidation of an indicator molecule to generate an oxidized indicator molecule.
  • FIG. 2 This example is visualised in Figure 2, in which the capture molecule at the capture zone of the solid support indirectly captures the analyte-detection reagent complex by binding to the (optional) further binding molecule which is bound to the analyte-detection reagent complex, thus, immobilizing the analyte-detection reagent complex at the capture zone.
  • the step of reducing the oxidised indicator molecule may also be performed at the capture zone.
  • the resulting charge transfer may be detected and/or measured at the capture zone to determine whether an analyte is present in the test sample.
  • the analytedetection complex In the absence of an analyte in the test sample, the analytedetection complex is not formed and therefore the step of capturing the analyte-detection reagent complex at a capture zone does not take place.
  • the absence of the analyte-detection reagent complex (and especially the catalytic agent portion of the detection reagent) at the capture zone prevents the catalytic reaction of oxidation of an indicator molecule. Since the oxidised indicator molecule is not formed, the step of reducing the oxidized indicator molecule to produce a charge transfer does not take place. This is reflected when the charge transfer is measured in step f) to detect presence, or, in this case, absence of an analyte in the sample.
  • the invention provides a method for detecting an analyte in a test sample, the method may comprise the steps: a) bringing the test sample into contact with a detection reagent to form an analyte-detection reagent complex, the detection reagent comprising: (i) a binding molecule; and (ii) a catalytic agent; b) capturing the analyte-detection reagent complex via a capture molecule immobilized at a capture zone of a solid support; c) generating or mobilising hydrogen peroxide; d) generating an oxidized indicator molecule through oxidation of an indicator molecule by the hydrogen peroxide in the presence of the catalytic agent; e) reducing the oxidised indicator molecule to produce a charge transfer; and f) measuring the charge transfer to detect the presence or absence of the analyte in the sample.
  • the method described herein may further comprise a wash step to remove unwanted reagents.
  • the wash step may be performed after step b) to remove uncaptured analyte and/or detection reagent.
  • the wash step may remove an analyte and/or detection reagent which have not formed a complex.
  • the wash step generally reduces background reading when the charge transfer is measured. This is because a detection reagent which has not formed a complex with an analyte may catalyse (via the catalytic agent portion) the reaction of oxidation of an indicator molecule if it is in a close proximity of the capture zone even in the absence of an analyte.
  • the wash step may be performed with a buffer.
  • the buffer used in the wash step would typically not contain any reagents required for the method.
  • the buffer may optionally contain an indicator molecule and/or a charge donor to ensure the reaction mixture comprises the required concentration of reagents.
  • the methods provided herein require several reagents, including a detection reagent and an indicator molecule, and an electron carrier. Various suitable manners for providing each of these reagents are discussed herein.
  • one or more of any of these reagents may be either pre-deposited, e.g. sprayed or embedded, in or on a solid support, or they may be added to the solid support as part of the test sample (or the reaction buffer).
  • one or more of any of these reagents may be present in a reagent buffer.
  • the detection reagent, the indicator molecule, and the electron carrier may all be present in a reagent buffer.
  • the reagent buffer may in some embodiments be brought into contact with the test sample.
  • one or more of any of these reagents may be predeposited on the solid support.
  • the detection reagent, the indicator molecule, and the electron carrier may all be pre-deposited on the solid support. Any combinations are contemplated, thus it is also contemplated that at least one of the reagents is present in a reagent buffer and at least one of the reagents is pre-deposited on the solid support.
  • the reagents may be added separately from the test sample (or the reaction buffer).
  • various reagents may be added at various steps directly to the solid support.
  • the reagents used in the method are pre-deposited on the solid support.
  • the solid support e.g. a lateral flow strip
  • the detection reagent may be deposited on one pad near the sample application zone (dark blue pad in Figure 2)
  • the indicator molecule pad may be deposited on a second pad near the sample application zone
  • the indicator molecule may be added to the second pad directly.
  • the further binding molecule may also be deposited on a pad near the sample application zone (yellow pad in Figure 2).
  • the pad comprising the indicator molecule may not be in a fluid communication with other pads of the solid support (e.g. a lateral flow test strip). If the method relies on the presence of electrode(s), the electrode may also be pre-deposited on the solid support (e.g. in the capture zone - where the capture molecules are immobilized) or the electrode may be on the top or bottom of the solid support and in fluid communication with the solid support. For example, the electrode(s) may be clipped or otherwise attached to the solid support.
  • the invention also provides a device configured to perform the method of the invention.
  • the device may comprise:
  • (c) means for detecting a charge transfer.
  • the device also comprises a battery.
  • the battery may be activated by the liquid from the test sample and/or reaction buffer.
  • the device may comprise: (a) a solid support;
  • the invention may provide a device comprising:
  • a solid support comprising: i. a sample application zone for receiving a test sample; ii. a capture zone comprising an immobilized capture molecule, wherein the capture molecule is capable of capturing an analyte which is bound to a detection reagent;
  • the sample application zone and the capture zone may be the same zone. That is to say that the sample may be applied directly to the capture zone.
  • the solid support may comprise a sample application zone to which the sample is applied.
  • the sample application zone may be pre-loaded with the detection reagent, the indicator molecule and/or the further binding molecule, such that when the test sample is applied the detection reagent, the indicator molecule and/or the further binding molecule travel downstream towards the capture zone.
  • the sample may contain a pre-mixed solution of the analyte and the detection reagent.
  • the solution may further comprise the indicator molecule and/or the further binding molecules.
  • the pre-mixed solution may be added to the sample application zone (or directly to the capture zone). Where the test sample and indicator molecule may be pre-mixed or pre-incubated it is possible to omit the sample application zone.
  • the sample application zone may contain a barrier, which holds the sample in the sample application zone for a pre-determined period of time. This permits the analyte to interact with the detection reagent and, optionally the further binding molecules for a sufficient period to form an analytedetection reagent complex (or a further binding molecule-analyte-detection reagent complex). This may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 60 minutes.
  • the barrier may be degraded by the sample, or otherwise removed, after this period of time thus allowing the sample to continue to flow through the device.
  • the invention may provide a device comprising:
  • a solid support comprising: i. a sample application zone for receiving a test sample; ii. a capture zone comprising an immobilized capture molecule, wherein the capture molecule is capable of capturing an analyte which is bound to a detection reagent;
  • the capture molecule may also bind to the analyte which is not bound (or has not bound) to the detection reagent. However, if the analyte is not bound to the detection reagent (i.e. the analytedetection reagent complex is not formed) at the capture zone, the charge transfer will be indicative of an absence of the analyte in the test sample. This is because the catalytic agent portion of the detection reagent (as described herein) will not be in the proximity of the indicator molecule.
  • the invention may provide a device comprising:
  • a sample application zone for receiving a test sample i. a capture zone comprising an immobilized capture molecule, wherein the capture molecule is capable of capturing an analyte which is bound to a detection reagent;
  • the device may comprise:
  • a solid support comprising: i. a sample application zone for receiving a test sample; ii. a capture zone downstream of the sample application zone and comprising an immobilized capture molecule, wherein the capture molecule is capable of capturing an analyte which is bound to a detection reagent;
  • the solid support may further comprise a reagent detection zone, a control zone, and/or a detection zone as described herein.
  • the detection zone may be the same as the capture zone.
  • the detection zone may comprise at least one electrode (preferably, at least two, at least three, at least four, at least five, at least six, at least seven electrodes).
  • the capture zone may be configured to bind and immobilize the capture molecule.
  • the battery may be configured to be activated by contact with a liquid.
  • 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(s) may be in fluidic contact with at least some of the reagents used in the methods so the electrode potential is in a solution.
  • the reference electrode may have a constant half-cell potential in the solution.
  • the working electrode potential may be 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 region.
  • 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.
  • Fluidic contact between the electrode(s) and the support may be achieved in any appropriate way.
  • an electrode sheet comprising the electrode(s) may be brought into contact with the support such that when the support is wet, the electrode(s) is in fluidic contact with the support.
  • the electrode(s) 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(s) may be connected to a potentiostat circuit in a similar way.
  • the electrode sheet may comprise electrical contacts. When the electrode sheet is brought into contact with the support, the electrical contacts may align with conductive tracks on the support. The pressure applied to keep the electrode(s) in fluidic contact with the support may also be used to maintain an electrical connection between the electrical contacts and the conductive tracks.
  • the electrode(s) may be printed onto a support.
  • a test sample (which is a solution, optionally, comprising an analyte and a reaction buffer) is deposited on (or added to) the sample application zone.
  • the test sample may comprise an electron carrier and an indicator molecule.
  • the test sample may be incident on the battery, activating the battery and triggering a sequence of potentials applied to the at least one electrode.
  • the analyte 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.
  • the interaction with the capture molecule is indirect, via the further binding molecule.
  • a first potential is applied to the at least one electrode in the presence of the electrode donor to produce hydrogen peroxide in the capture zone.
  • An oxidised indicator molecule is generated at the capture zone, wherein the oxidised indicator molecule is produced via hydrogen peroxide oxidation of the indicator molecule in the presence of the catalytic agent.
  • the first period of time may be 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 12 minutes, 15 minutes, or 20 minutes.
  • a second potential is applied to the at least one electrode such that the oxidised indicator molecule is reduced, generating the indicator molecule and a charge transfer.
  • the second period of time may be greater than the first period of time by 1 second, 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 12 minutes, 15 minutes, or 20 minutes.
  • 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 device may rely on at least one, at least two, at least three, at least four, at least five, at least six, at least seven electrodes.
  • the exemplary arrangement of electrodes in the device is shown in Figure 4.
  • the device may comprise at least one carbon electrode.
  • the device may comprise at least one silver electrode.
  • the device may comprise at least one carbon electrode and at least one silver electrode.
  • the device may comprise at least 2, at least 3, at least 4, or at least 5 carbon electrodes.
  • the device may comprise at least 2, or at least 3 silver electrodes.
  • the exemplary arrangement of electrodes in Figure 4 contains 5 carbon electrodes and 2 silver electrodes.
  • the device comprises a solid support in the form of a lateral flow strip (D), an upper cassette (A) and a lower cassette (F) together forming a protective housing, an arrangement of seven electrodes (B) and means for holding a sensor and a strip (C).
  • the battery may be activated for long enough to detect and/or record the charge transfer.
  • the battery may be activated for long enough to power at least one electrode.
  • the battery may be activated for long enough to power a source of light.
  • the data from the detection and/or recordal may be stored on a computer medium or in a computer application.
  • the invention also relates to a corresponding computer application for use with the method, device or kit described herein.
  • the computer application when executed by the processor, is configured to access, measure and/or analyse the charge transfer.
  • the computer application may determine the presence or absence of an analyte in the sample based on the recorded charge transfer values.
  • the computer application may output from the processor whether the analyte is present, and, if so, optionally output a treatment to be administered to the subject from which the sample was collected.
  • the invention provides a system for detecting an analyte in the test sample, wherein the system comprises:
  • a storage medium comprising a computer application that, when executed by the processor, is configured to: a. access, measure and/or analyse the charge transfer values; b. determine whether the analyte is present in the test sample; and c. output from the processor the presence or absence of the analyte, and optionally output a treatment to be administered to the subject from which the sample was collected.
  • the computer application may optionally perform correction of charge transfer values to subtract the background charge transfer values as described herein.
  • the invention also relates to the computer applications used in the device, kit and system described herein.
  • the computer-implemented method, system, and computer program product may be embodied in a computer application, for example, that operates and executes on a processor, such as in the context of a computing machine.
  • the application When executed, the application performs the relevant analyses to output the presence or absence of the analyte, and optionally a treatment to be administered to the subject from which the sample was collected.
  • the processor may be comprised within any computer, server, embedded system, or computing system.
  • the computer may include various internal or attached components such as a system bus, system memory, storage media, input/output interface, and a network interface for communicating with a network, for example.
  • the computer may be implemented as a conventional computer system, an embedded controller, a laptop, a server, a customized machine, any other hardware platform, such as a laboratory computer or device, for example, or any combination thereof.
  • the computing machine may be a distributed system configured to function using multiple computing machines interconnected via a data network or bus system, for example.
  • the processor may be configured to execute code or instructions to perform the operations and functionality described herein, manage request flow and address mappings, and to perform calculations and generate commands.
  • the processor may be configured to monitor and control the operation of the components in the computing machine.
  • the processor may be a general purpose processor, a processor core, a multiprocessor, a reconfigurable processor, a microcontroller, a digital signal processor (“DSP”), an application specific integrated circuit (“ASIC”), a graphics processing unit (“GPU”), a field programmable gate array (“FPGA”), a programmable logic device (“PLD”), a controller, a state machine, gated logic, discrete hardware components, any other processing unit, or any combination or multiplicity thereof.
  • DSP digital signal processor
  • ASIC application specific integrated circuit
  • GPU graphics processing unit
  • FPGA field programmable gate array
  • PLD programmable logic device
  • the processor may be a single processing unit, multiple processing units, a single processing core, multiple processing cores, special purpose processing cores, co-processors, or any combination thereof.
  • the processor along with other components of the computing machine, may be a virtualized computing machine executing within one or more other computing machines.
  • the storage medium may be selected from a hard disk, a floppy disk, a compact disc read only memory (“CD-ROM”), a digital versatile disc (“DVD”), a Blu-ray disc, a magnetic tape, a flash memory, other non-volatile memory device, a solid-state drive (“SSD”), any magnetic storage device, any optical storage device, any electrical storage device, any semiconductor storage device, any physicalbased storage device, any other data storage device, or any combination or multiplicity thereof.
  • the storage media may store one or more operating systems, application programs and program modules such as module, data, or any other information.
  • the storage media may be part of, or connected to, the computing machine.
  • the storage media may also be part of one or more other computing machines that are in communication with the computing machine, such as servers, database servers, cloud storage, network attached storage, and so forth.
  • the storage media may therefore represent examples of machine or computer readable media on which instructions or code may be stored for execution by the processor.
  • Machine or computer readable media may generally refer to any medium or media used to provide instructions to the processor.
  • Such machine or computer readable media associated with the module may comprise a computer software product.
  • the input/output (“I/O”) interface may be configured to couple to one or more external devices, to receive data from the one or more external devices, and to send data to the one or more external devices. Such external devices along with the various internal devices may also be known as peripheral devices.
  • the I/O interface may include both electrical and physical connections for operably coupling the various peripheral devices to the computing machine or the processor.
  • the I/O interface may be configured to communicate data, addresses, and control signals between the peripheral devices, the computing machine, or the processor.
  • the I/O interface may be configured to implement any standard interface, such as small computer system interface (“SCSI”), serial-attached SCSI (“SAS”), fiber channel, peripheral component interconnect (“PCI”), PCI express (PCIe), serial bus, parallel bus, advanced technology attached (“ATA”), serial ATA (“SATA”), universal serial bus (“USB”), Thunderbolt, FireWire, various video buses, and the like.
  • SCSI small computer system interface
  • SAS serial-attached SCSI
  • PCIe peripheral component interconnect
  • PCIe PCI express
  • serial bus parallel bus
  • ATA advanced technology attached
  • SATA serial ATA
  • USB universal serial bus
  • Thunderbolt FireWire
  • the I/O interface may be configured to implement only one interface or bus technology.
  • the I/O interface may be configured to implement multiple interfaces or bus technologies.
  • the I/O interface may be configured as part of, all of, or to operate in conjunction with, the system bus.
  • the I/O interface may include one or more buffers for buffer
  • the I/O interface may couple the computing machine to various input devices including mice, touchscreens, scanners, electronic digitizers, sensors, receivers, touchpads, trackballs, cameras, microphones, keyboards, any other pointing devices, or any combinations thereof.
  • the I/O interface may couple the computing machine to various output devices including video displays, speakers, printers, projectors, tactile feedback devices, automation control, robotic components, actuators, motors, fans, solenoids, valves, pumps, transmitters, signal emitters, lights, and so forth.
  • the computing machine may operate in a networked environment using logical connections through the network interface to one or more other systems or computing machines across the network.
  • the network may include wide area networks (WAN), local area networks (LAN), intranets, the Internet, wireless access networks, wired networks, mobile networks, telephone networks, optical networks, or combinations thereof.
  • the network may be packet switched, circuit switched, of any topology, and may use any communication protocol. Communication links within the network may involve various digital or an analog communication media such as fiber optic cables, free-space optics, waveguides, electrical conductors, wireless links, antennas, radio-frequency communications, and so forth.
  • the processor may be connected to the other elements of the computing machine or the various peripherals discussed herein through the system bus. It should be appreciated that the system bus may be within the processor, outside the processor, or both. According to some embodiments, any of the processor, the other elements of the computing machine, or the various peripherals discussed herein may be integrated into a single device such as a system on chip (“SOC”), system on package (“SOP”), or ASIC device.
  • SOC system on chip
  • SOP system on package
  • ASIC application specific integrated circuit
  • Embodiments may comprise a computer program that embodies the functions described and illustrated herein, wherein the computer program is implemented in a computer system that comprises instructions stored in a machine-readable medium and a processor that executes the instructions.
  • the embodiments should not be construed as limited to any one set of computer program instructions.
  • a skilled programmer would be able to write such a computer program to implement one or more of the disclosed embodiments described herein. Therefore, disclosure of a particular set of program code instructions is not considered necessary for an adequate understanding of how to make and use embodiments.
  • the example embodiments described herein can be used with computer hardware and software that perform the methods and processing functions described previously.
  • the systems, methods, and procedures described herein can be embodied in a programmable computer, computer-executable software, or digital circuitry.
  • the software can be stored on computer-readable media.
  • computer-readable media can include a floppy disk, RAM, ROM, hard disk, removable media, flash memory, memory stick, optical media, magneto-optical media, CD-ROM, etc.
  • Digital circuitry can include integrated circuits, gate arrays, building block logic, field programmable gate arrays (FPGA), etc.
  • the methods, systems and test kits may incorporate means for Automatic Identification and Data Capture (AIDC), such as a Radio-frequency identification tag or card (RIF).
  • AIDC Automatic Identification and Data Capture
  • REF Radio-frequency identification tag or card
  • the device, system or kit described herein may further comprises a display for the output from the processor. This is intended to give a simple visual and/or audible read-out of the assays performed on the sample.
  • the display may be operably connected to the processor running the computer application.
  • the output or read-out may be an instruction to the subject in some embodiments. Depending upon the algorithm employed suitable read-outs may be selected from “increase/decrease frequency of testing”, which may be to a specified level or frequency for example or equivalent wordings.
  • the outputs may be displayed by a suitable display module on a remote computing device (e.g. tablet, phone or computer), which is in operable connection with the processor/computer application housed in the analyser.
  • a remote computing device e.g. tablet, phone or computer
  • the outputs may be displayed on a suitable display module (e.g. tablet, phone or computer) which is in remote connection (e.g. Wireless Internet connection, Bluetooth or any other Near Field Communication connection) with the processor/computer application.
  • a suitable display module e.g. tablet, phone or computer
  • remote connection e.g. Wireless Internet connection, Bluetooth or any other Near Field Communication connection
  • LumiraDx Connect LumiraDx Connect
  • the processor and storage medium housed in the analyser may be configured to measure or analyse the charge transfer on the one or more devices and transmit the data to a remote computing device (e.g. tablet, phone or computer) which is configured to analyse the determined levels of the charge transfer and output whether the analyte is present in the sample, and, optionally the treatment to be administered to the subject from which the sample was collected.
  • a remote computing device e.g. tablet, phone or computer
  • the data may be transmitted to a remote computing device (e.g. tablet, phone or computer) via a cloud-based computing service which is configured to analyse the determined levels of the charge transfer and output whether the analyte is present in the sample, and, optionally the treatment to be administered to the subject from which the sample was collected.
  • the remote computing device is configured to display the output calculated by the cloud-based computing service.
  • the devices may also include a control zone to confirm sample has passed through the device satisfactorily. In the absence of confirmation by the control zone, the system, device or kit may indicate an invalid result to the user, for example via a display.
  • the invention provides an analyte detection kit.
  • the analyte detection kit may comprise:
  • the analyte detection kit may comprise:
  • a solid support comprising: i. a sample application zone for receiving a test sample; ii. a capture zone downstream of the sample application zone and comprising an immobilized capture molecule, wherein the capture molecule is capable of capturing an analyte which is bound to a detection reagent;
  • the solid support may be provided without the capture molecules attached.
  • the user of the kit may immobilize the capture molecules on the solid support to form the capture zone prior to use of the device with a test sample.
  • the kit may, therefore, also comprise means for immobilizing the capture molecules on the solid support.
  • the solid support may comprise a nitrocellulose membrane.
  • the invention provides use of the device of the invention or the analyte detection kit of the invention for detecting an analyte in a test sample.
  • the analyte may be a viral biomarker.
  • the virus may be a respiratory virus, preferably a coronavirus.
  • Figure 1 illustrates an exemplary test strip.
  • Figure 2 illustrates an exemplary test strip.
  • Figure 3 shows an exploded view of an exemplary detection device.
  • PCB printed circuit board.
  • Figure 4 shows an exemplary arrangement of the electrodes in the detection device.
  • Figure 5 shows cyclic voltammetry readings for four samples: 1) a control sample comprising citrate/saline buffer; 2) a sample comprising 2-hydroxy-1 ,4-napthoquinone (Q2), 3) a sample comprising tetramethyl benzidine (TMB); and 4) a sample comprising both Q2 and TMB.
  • Figure 6 shows a charge of TMB reduction using hydrogen peroxide on working electrodes.
  • Figure 7 shows a relationship between TMB conversion and Pt70 number.
  • Figure 8 illustrates limit of detection (LoD) of Nucleoprotein in dry assay. Top - conventional lateral flow test. Bottom - lateral flow test with TMB-peroxide conversion.
  • Figure 9 shows the digital readings in Cube units for data in Figure 8.
  • Figure 10 illustrates the limit of detection of Nucleoprotein using electrode detection and TMB- peroxide system.
  • Figure 11 illustrates the limit of detection of Nucleoprotein using electrode detection and TMB- peroxide system (log values).
  • Figure 12 shows the results for electrochemical generation of hydrogen peroxide using horseradish peroxidase (HRP) as catalyst.
  • HRP horseradish peroxidase
  • Figure 13 shows an experimental step for photogeneration of hydrogen peroxide.
  • a method for detecting an analyte in a test sample comprising the steps: a) bringing the test sample into contact with a detection reagent to form an analyte-detection reagent complex, the detection reagent comprising: (i) a binding molecule; and (ii) a catalytic agent; b) capturing the analyte-detection reagent complex via a capture molecule immobilized at a capture zone of a solid support; c) generating or mobilising hydrogen peroxide; d) generating an oxidized indicator molecule through oxidation of an indicator molecule by the hydrogen peroxide in the presence of the catalytic agent; e) reducing the oxidised indicator molecule to produce a charge transfer; and f) measuring the charge transfer to detect the presence or absence of the analyte in the sample.
  • test sample comprises a saliva sample, a nasal swab sample, a blood sample, a wound sample, a urine sample, an amniotic fluid sample, a peritoneal fluid sample, an ascites sample, a breast milk sample.
  • test sample comprises a reagent buffer
  • the reagent buffer comprises the indicator molecule
  • ii. the reagent buffer comprises chloride
  • the reagent buffer comprises water, iv. the reagent buffer comprises an electron carrier, and/or v. the reagent buffer comprises the detection reagent.
  • the solid support comprises a control zone downstream of the capture zone, optionally downstream of the detection zone.
  • the solid support comprises a detection reagent zone upstream of the capture zone, optionally wherein the detection reagent zone is downstream of the sample application zone.
  • the detection reagent is pre-deposited, such as sprayed onto or embedded in or on the solid support at the detection reagent zone.
  • the detection reagent becomes solubilised and/or mobilised.
  • the method of any one of clauses 1 -20 wherein the method is performed on a lateral flow strip or a lateral flow device.
  • the method of any one of clauses 1 -21 wherein the method further comprises a wash step after step b) to remove uncaptured analyte and/or detection reagent.
  • the method of any one of clauses 1-22 wherein hydrogen peroxide is generated at the capture zone or upstream of the capture zone.
  • the solid support comprises at least one electrode.
  • the method of clause 25, wherein the at least one electrode is located at the capture zone and/or the detection zone.
  • the solid support comprises at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 electrodes, preferably, wherein the solid support comprises at least 4 electrodes.
  • the method of clause 27 or clause 28, wherein 2 of the at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 electrodes are located at the capture zone and/or detection zone.
  • any one of clauses 27-29 wherein the at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 electrode(s) is a carbon-coated and/or a silver-coated electrode.
  • the method of any one of clauses 1-30 wherein hydrogen peroxide is generated by applying an electric potential by the at least one electrode, optionally wherein hydrogen peroxide is generated by applying an electric potential by the at least one electrode to an electron carrier.
  • the method of clause 31 wherein the electric potential is between -300mV and -1000 mV, preferably is about -600 mV.
  • the electron carrier is a flavin mononucleotide, or a quinone, such as 2-hydroxy-1 ,4-naphthoquinone, 2-methoxy-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, or 1 ,4-dihydroxyanthraquinone.
  • the electron carrier is a flavin mononucleotide, or a quinone, such as 2-hydroxy-1 ,4-naphthoquinone, 2-methoxy-1 ,4-naphthoquinone, 9,10- phenanthrenequinone, 9,10-anthraquinon
  • the indicator molecule is tetramethylbenzidine (TMB).
  • TMB tetramethylbenzidine
  • the indicator molecule is: (a) pre-deposited, e.g. sprayed onto or embedded in or on the solid support downstream of the sample application zone; (b) present in the reagent buffer of the test sample; and/or (c) added to the solid support downstream of the sample application zone and upstream of the capture zone.
  • the electron carrier is: (a) pre-deposited, e.g.
  • any one of clauses 1-48 wherein the analyte, if present, is detected at a concentration of less than 350 pg/ml, less than 300 pg/ml, less than 250 pg/ml, less than 200 pg/ml, less than 150 pg/ml, less than 100 pg/ml, less than 50 pg/ml, less than 25 pg/ml, less than 12.5 pg/ml, or less than 10 pg/ml.
  • a device configured to perform the method of any one of clauses 1-49 comprising:
  • a solid support comprising: i. a sample application zone for receiving the test sample; ii. a capture zone downstream of the sample application zone and comprising an immobilized capture molecule, wherein the capture molecule is capable of binding to an analyte which is bound to a detection reagent;
  • An analyte detection kit comprising:
  • a solid support comprising: i. a sample application zone for receiving the test sample; ii. a capture zone downstream of the sample application zone and comprising an immobilized capture molecule, wherein the capture molecule is capable of binding to an analyte which is bound to a detection reagent;
  • Example 1 In-situ generation of hydrogen peroxide by electrochemical means compatible with a lateral flow immunoassay environment
  • Example 2 Electrochemical detection of SARS CoV2 Nucleoprotein Antigen at 1 ng/ml in a double antibody sandwich lateral flow immunoassay, driven by localised H2O2 generation within the strip.
  • H2O2 was generated in the lateral flow strip, in the presence of TMB and 2-hydroxy-1 ,4-naphthoquinone.
  • the nitrocellulose test strips were placed in contact with a screen- printed array of carbon electrodes in which there was also a silver reference electrode.
  • the test (capture) line comprised immobilised polystreptavidin (PSA).
  • PSA immobilised polystreptavidin
  • a proven pair of antibodies against SARS-CoV-2 NP antigen was used as the immunoreagents - one was conjugated to biotin and the other to platinum nanoparticles. These two antibodies bound to the NP molecules (to form immune complexes) when they were all mixed together in running buffer (PBS - 1%BSA -0.1%Tween20).
  • the biotin-labelled antibody bound to the immobilised PSA, bringing with it any NP and platinum nanoparticle label.
  • a chase fluid (50 pl) was then applied containing 0.4 mM TMB and 1 mM quinone. This chase fluid was allowed to flow for 5 minutes before the electrode array was activated by applying -0.6V for 20 seconds to the first working electrode WE1 (with respect to a silver reference nearby). During this chronoamperometry the Q2 quinone was reduced, leading to the formation of H2O2 (oxygen dependent) which, in turn, oxidised TMB. All of the reactants diffused and flowed downstream toward the other electrodes.
  • Platinum nanoparticle-Antibody-Analyte (Pt-Ab-Ag) complex on the test line helps the oxidation of TMB.
  • Example 3 Generation of hydrogen peroxide with TMB-FMN-Pt70 drop on screen-printed electrodes
  • Example 3 50pl drop containing TMB, FMN and 70nm Pt nanoparticles.
  • the test in Example 3 is a test on sensor only without a test strip involved. Pt particles are added in the mixture with TMB and FMN.
  • TMB can be oxidised by peroxide when catalytic agent Pt particles are present
  • Pt-Ab-Ag can act as catalytic agent as free Pt nanoparticles in TMB conversion.
  • FMN and naphthoquinone can both be used to generate peroxide, particularly can be done in a later flow setup in a cassette.
  • TMB causes a visual increase in signal compared to without TMB.
  • the visual test can be quantified by digitally recording the intensity of line on a Cube data Reader.
  • the sensitivity of nucleoprotein is about eight times better with TMB-peroxide application.
  • Example 5 Limit of detection (LoD) of Nucleoprotein (NP) using electrode detection and TMB- peroxide system.
  • the following method of assay was used to test the assay strips in the cassette. 50 pl sample was added through sample port onto the biotin pad followed by a 5 minute wait. 25 pl of wash buffer was then added through the same sample port followed by a further 5 minutes wait. 45 pl TMB solution was then added through the TMB port to the top empty pad. The assay was left for 5 minutes before 85 pl of wash solution was added to the sink pad through the stop solution port to stop the flow in the strip. A further 5 minutes was then allowed for the oxidised TMB to concentrate on the lines before the electrochemical measurement was run. Apply -100mV for 1 second to each working electrode in sequence. Calculate the total charge in the time 200milliseconds to 800 milliseconds for each electrode.
  • the limit of detection of nucleoprotein is ⁇ 10 pg/ml (see Figure 11).
  • Example 6 Electrochemical generation of hydrogen peroxide using horseradish peroxidase (HRP) as catalyst
  • the TMB solution was prepared with 0.1 mM FMN but excluding the hydrogen peroxide.
  • Conjugated HRP was used to enzymatically produce the oxidised TMB.
  • Biotin-5B1 and Pt-11A7 are sprayed on 8951 (10mm wide) conjugation pads, respectively.
  • the spray rate is 0.8pl/mm.
  • NO and conjugate bands are laminated with sink pads as shown in Figure 2. Strips are cut into 4 mm that can fit into the Strip Clip as in Figure 3.
  • test strip is shown in Figure 2.
  • Equipment used in Examples 1-8 includes:

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Abstract

L'invention concerne des procédés de détection de la présence ou de l'absence d'un analyte dans un échantillon. L'invention concerne également des dispositifs de détection de la présence ou de l'absence d'un analyte dans un échantillon. De plus, l'invention concerne des kits et des systèmes de détection d'analyte.
PCT/EP2024/066352 2023-06-13 2024-06-13 Procédé et dispositif de détection d'un analyte dans un échantillon Pending WO2024256533A1 (fr)

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US20060057578A1 (en) * 2002-05-08 2006-03-16 Yissum Research Development Company Of The Hebrew University Of Jerusalem Determination of an analyte in a liquid medium
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
WO2022080994A1 (fr) * 2020-10-16 2022-04-21 주식회사 마라나노텍코리아 Trousse de test de diagnostic covid
CA3204808A1 (fr) * 2021-01-13 2022-07-21 Mark William Grinstaff Methodes, essais et systemes de detection d'un analyte cible

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US20060057578A1 (en) * 2002-05-08 2006-03-16 Yissum Research Development Company Of The Hebrew University Of Jerusalem Determination of an analyte in a liquid medium
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
WO2022080994A1 (fr) * 2020-10-16 2022-04-21 주식회사 마라나노텍코리아 Trousse de test de diagnostic covid
CA3204808A1 (fr) * 2021-01-13 2022-07-21 Mark William Grinstaff Methodes, essais et systemes de detection d'un analyte cible

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DEMPSEY EITHNE ET AL: "Disposable Printed Lateral Flow Electrochemical Immunosensors for Human Cardiac Troponin T", IEEE SENSORS JOURNAL, IEEE, USA, vol. 18, no. 5, 1 March 2018 (2018-03-01), pages 1828 - 1834, XP011676501, ISSN: 1530-437X, [retrieved on 20180130], DOI: 10.1109/JSEN.2018.2789436 *
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