Method and device for detection of an analyte in a sample

Abstract

The invention relates to methods for detecting the presence or absence of an analyte in a sample. The invention also relates to devices for detecting the presence or absence of an analyte in a sample. In addition, the invention relates to analyte detection kits and systems.

Description

[0001] METHOD AND DEVICE FOR DETECTION OF AN ANALYTE IN A SAMPLE

[0002] FIELD

[0003] 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. In addition, the invention relates to analyte detection kits.

[0004] BACKGROUND

[0005] 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. In addition, 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.

[0006] Furthermore, the results of the assay procedure are typically in the form of a visual output that requires interpretation by a user. For example, 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.

[0007] Thus, a need exists for a method and device which improves the accuracy and reproducibility of analyte detection. In addition, a need exists for an analyte detection method and device which reduces a manual input required by a user.

[0008] SUMMARY OF THE INVENTION

[0009] In a first aspect, 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.

[0010] 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.

[0011] Thus, 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.

[0012] For example, 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.

[0013] In all aspects described herein, instead of being immobilized at the capture zone, 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. In another aspect, the invention provides a device configured to perform the method of the invention.

[0014] The device may comprise:

[0015] (a) a solid support;

[0016] (b) means for generating or mobilising hydrogen peroxide;

[0017] (c) means for detecting a charge transfer; and

[0018] (d) a battery configured to be activated by contact with a liquid.

[0019] For example, the invention may provide a device comprising:

[0020] (a) a solid support, the 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;

[0021] (b) means for generating or mobilising hydrogen peroxide at the capture zone or upstream of the capture zone;

[0022] (c) means for detecting a charge transfer; and

[0023] (d) a battery configured to be activated by contact with a liquid.

[0024] For example, the device may comprise:

[0025] (a) a solid support, the 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;

[0026] (b) means for generating or mobilising hydrogen peroxide at the capture zone or upstream of the capture zone;

[0027] (c) means for detecting a charge transfer; and

[0028] (d) a battery configured to be activated by contact with a liquid.

[0029] As used herein, 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.

[0030] In a further aspect, the invention provides an analyte detection kit. The analyte detection kit may comprise:

[0031] (a) a solid support comprising a capture zone;

[0032] (b) means for generating hydrogen peroxide at the capture zone or upstream of the capture zone;

[0033] (c) means for detecting a charge transfer; and

[0034] (d) a battery configured to be activated by contact with a liquid. The analyte detection kit may comprise:

[0035] (a) a solid support, the 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;

[0036] (b) means for generating hydrogen peroxide at the capture zone or upstream of the capture zone;

[0037] (c) means for detecting a charge transfer; and

[0038] (d) a battery configured to be activated by contact with a liquid.

[0039] In a further aspect, 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.

[0040] Each aspect or embodiment as defined herein may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0041] Other preferred embodiments of the methods, devices, analyte detection kits and uses according to the invention appear throughout the specification and in particular in the examples.

[0042] 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.

[0043] 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.

[0044] DETAILED DESCRIPTION

[0045] Unless otherwise defined herein, scientific, and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition.

[0046] It should be understood that singular prepositions such as “a,” “an,” and “the,” are often used for convenience, however, all instances of the singular are intended to encompass the plural unless otherwise indicated either explicitly or from context. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Further, it should be understood that all references, including journal articles, books, patents, technical documents, and the like, mentioned in this disclosure are hereby incorporated by reference in their entirety and for all purposes.

[0047] The term “about” as used herein for numerical data refers to a value within 10% of the underlying parameter (i.e., plus or minus 10%), and use of the term “about” at the beginning of a string of values modifies each of the values (i.e., “about 1 , 2 and 3” refers to about 1 , about 2 and about 3). For example, an electric potential that is “about” -600mV can include values between (and including) - 540mV and -660mV.

[0048] The term “analyte” as used herein 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. 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. For example, 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. For example, 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. Alternatively, the method may detect / determine / measure the concentration of the analyte in a quantitative manner.

[0049] The term “test sample” or “sample” as used herein refers to any sample, including environmental and clinical samples. For example, 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. Thus, the 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.

[0050] The sample may be, or comprise, a fluid and / or a solid. For example, it may be a solution, emulsion, or suspension. It is preferable that the test sample used in the methods provided herein or used with the devices or kits provided herein is or comprises a fluid. Thus, in some embodiments, 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.

[0051] Thus, the test sample may further comprise 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.

[0052] 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.

[0053] In some embodiments, the methods provided herein may include a step of obtaining a suitable sample from a subject. However, in other embodiments the methods do not include such a step and instead employ a “provided” sample that was previously obtained from a subject. Thus, the sample may be a provided sample.

[0054] The term “charge transfer” (or a “reduction charge”) as used herein 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. Typically, the indicator molecule is reduced by application of a specific electric potential. For example, 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. For example, the charge transfer may be measured by chronoamperometry. The charge transfer may be measured on at least one working electrode. Preferably, 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.

[0055] The term “electron carrier” as used herein 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. In one embodiment, 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.

[0056] The electron carrier may, for example, be a quinone, flavin mononucleotide (FMN), or nicotinamide adenine dinucleotide. For example, 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. Preferably, 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.

[0057] The term “electron donor” as used herein refers to any chemical entity that donates electrons to another compound or molecule. In the context of the present invention, 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. In one embodiment, the electron donor is used to generate hydrogen peroxide by 2-electron electrochemical reduction.

[0058] The electron donor may, for example, be a reduced form of a quinone, of a flavin mononucleotide (FMN), or of a nicotinamide adenine dinucleotide. For example, 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. Preferably, the electron donor is a reduced form of 2-hydroxy-1 ,4- naphthoquinone. The electron carrier (abbreviated as “Q”) may generate hydrogen peroxide by 2-eletron electrochemical reduction as follows:

[0059] Step 1 : wherein Kc is the rate constant for the chemical step (step 2).

[0060] The above reaction is further described with reference to Hossain et al. (1989) “The electrochemistry of graphite and modified graphite surfaces: the reduction of O2”, Tammeveski et al. (2001) “Surface redox catalysis for O2 reduction on quinone-modified glassy carbon electrodes”, and Kullapere et al. (2009) “Electroreduction of oxygen on glassy carbon electrodes modified with in situ generated anthraquinone diazonium cations”. Step 1 is a fast electron transfer reaction, and the chemical step (step 2) is a rate-determining reaction. In step 1 , 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). In step 2, the electron donor is further reduced or disproportionated to generate peroxide.

[0061] In a specific embodiment, 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.

[0062] The term “at least one” (e.g. “at least one electrode”) as used herein 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.

[0063] 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.

[0064] The terms “bind”, “binding” and the like should be understood to mean that the binding is specific unless otherwise specified. Thus, for example, the binding molecule of the detection reagent should be capable of specifically binding to the analyte.

[0065] In any of the methods provided herein, the order of the method steps may typically be initiated in alphabetical order, with step a) preceding step b), which in turn precedes step c) and so on. However, deviations from this alphabetical order are also contemplated, in particular it is envisaged that 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.

[0066] 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).

[0067] 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. Preferably, 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. For example, if the analyte is a SARS-CoV-2 nucleocapsid protein antigen, the antibody or fragment thereof may have high specificity and / or selectivity for SARS-CoV-2 nucleocapsid protein antigen.

[0068] 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. For example, 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. For example, the catalytic agent may be a horseradish peroxidase or a platinum nanoparticle (such as a catalytic platinum nanoparticle). Preferably, the catalytic agent is a platinum nanoparticle.

[0069] Preferably, the binding molecule is an antibody and the catalytic agent is a platinum nanoparticle. Thus, preferably, 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.

[0070] The step of bringing the test sample into contact with a detection reagent should typically be performed before step b). In some embodiments, 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. Thus, 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.

[0071] In other embodiments, as discussed below, the step of bringing the test sample into contact with a detection reagent may be performed on a solid support.

[0072] 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.

[0073] 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.

[0074] In some embodiments (direct capture embodiments), the capture molecule (of the capture zone) is capable of binding to the analyte part of the analyte-detection complex. In these embodiments, the analyte-detection reagent complex is captured at the capture zone directly. In these direct capture embodiments, the capture molecule is capable of binding to a binding site of the analyte. As mentioned elsewhere herein, the detection reagent is also capable of binding to a binding site of the analyte. Thus, in these embodiments, 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.

[0075] In other embodiments (indirect capture embodiments), the capture molecule (of the capture zone) is capable of binding to a further binding molecule. In these embodiments, 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.

[0076] In indirect capture embodiments, 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. As mentioned elsewhere herein, the detection reagent is also capable of binding to a binding site of the analyte. Thus, in these embodiments, 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.

[0077] In indirect capture embodiments, 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. Preferably, the further binding molecule comprises a biotin molecule. More preferably still, the further binding molecule comprises a biotin molecule and an antibody or a fragment therefore. In this example, 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. Of course, other binding pair interactions may be used to immobilize the analyte-detection reagent complex in the capture zone. The further binding molecule may be specific and / or selective for the analyte part of the analyte-detection reagent complex.

[0078] At least one step of the method described herein may be performed on a solid support. Preferably, 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)) may, in some embodiments, be performed before the sample is applied to a solid support as described above. Alternatively, step (a) may be performed on the solid support, in which case the solid support may comprise the detection reagent in pre-deposited form. In embodiments where the solid support comprises the detection reagent, the zone comprising the detection reagent may be at or downstream of the sample application zone, provided it is upstream of the capture zone.

[0079] 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. Alternatively, the solid support may comprise a detection zone downstream of the capture zone.

[0080] 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. For example, 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.

[0081] In some embodiments of any of the aspects provided herein that involve a solid support, 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. Such a pre-deposit may, for example, be achieved by spraying a reagent onto or embedding a reagent on or in the solid support. For example, 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). For example, 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. For example, the electron carrier may be predeposited on or in the solid support at (or near) the capture zone. The pre-deposition of the reagents on the pad reduces the user’s error as the user is no longer required to manually add reagents to the solid support.

[0082] Any of the reagents used in the method may be added directly to or onto the solid support. For example, 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. Preferably, however, 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.

[0083] In some embodiments of any of the aspects provided herein that involve a 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. By “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.

[0084] Preferably, the method described herein is performed on a lateral flow strip or in a lateral flow device.

[0085] In some embodiments of any of the aspects provided herein that involve a solid support, the further binding molecule may be pre-deposited in or on the solid support. In the context of a lateral flow strip (or device), the further binding molecule may be pre-deposited in or on the solid support downstream of the sample application zone. For example, 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). Alternatively (or additionally), 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).

[0086] 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. In specific embodiments, 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.

[0087] 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).

[0088] 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. In an embodiment which relies on the presence 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. Alternatively, 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, Thus, 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.

[0089] 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. 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.

[0090] 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.

[0091] In some embodiments, 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.

[0092] Embodiments of the invention are also described with references to the Figures. It should be understood that the Figures are exemplary and that whilst specific agents are referenced, they are exemplary and can be replaced with any suitable alternative. Thus, for example, TMB is an example of an indicator molecule and any suitable indicator molecule as defined herein may be used instead of TMB. Preferably, TMB is of high purity. For example, 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.

[0093] With reference to Figure 1 , 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). In the method performed on the strip of Figure 1 , the test sample is applied to the sample application zone (e.g. the “Biotin-Ab1” pad). As 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. Once created, 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. Then, 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. In the method of Figure 1 , 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. In the embodiment in which hydrogen peroxide is generated, an electron carrier may form part of the solution comprising the indicator molecule. Alternatively, the electron carrier may be pre-deposited on the strip upstream of the capture zone or at the capture zone.

[0094] With reference to Figure 2, an embodiment in which the indicator molecule (e.g. TMB) is either predeposited on a different pad or is added to a different pad of the strip is also envisaged. In the method performed on the strip of Figure 2, 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). In this embodiment, 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. Alternatively, if the indicator molecule is not pre-deposited, the solution added to the pad may comprise the indicator molecule.

[0095] Thus, 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. Accordingly, 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.

[0096] 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.

[0097] The further binding molecule may bind to the analyte before, at the same time or after the detection reagents binds to the analyte. Thus, 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.

[0098] Preferably, the further binding molecule and the detection reagent interact with and / or bind to different binding sites of an analyte. Preferably, a “sandwich” structure is formed in which the analyte is “sandwiched” between the further binding molecule and the detection reagent. Thus, 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. Thus, preferably, the first and second binding sites of the analyte are different.

[0099] By “mobilising” is meant that a pre-deposited reagent is allowed to enter a solution. This makes it available to react with one or more of the assay components. Thus, by mobilising hydrogen peroxide, the hydrogen peroxide becomes available to oxidise the indicator molecule in the presence of the catalytic agent. For example, 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.

[0100] In all aspects and embodiments, generation of hydrogen peroxide is preferred over mobilisation of hydrogen peroxide.

[0101] By “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.

[0102] The step of generating hydrogen peroxide may be performed with the use of at least one electrode. Thus, hydrogen peroxide may be generated electrochemically. Optionally, 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. Thus, the electrode may apply the electric potential of between -300mV and -1000mV, between -400mV and -900mV, or between -500mV and -700mV. Preferably, the electric potential is about -600mV. Preferably, at least two electrodes are used. In the embodiments where 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. a later flow strip or device), 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. Preferably, 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. Preferably, 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. Preferably, 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). Preferably, 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. Preferably, hydrogen peroxide is generated on a carbon electrode. For example, dissolved oxygen may be reduced to hydrogen peroxide directly at a carbon electrode.

[0103] 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) may act as a mediator to lower the potential required for the generation of hydrogen peroxide. Thus, for an electron carrier Q, hydrogen peroxide may be produced according to the following equations:

[0104] Step 1 :

[0105] 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). For example, 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.

[0106] 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. In one embodiment, 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 term “proximity” as used herein refers to a distance which allows for the reaction to take place. Here, 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. Thus, the electron carrier may be added directly onto the electrode(s) of the solid support. Alternatively, the electron carrier may be comprised in a test sample (or a buffer added to the test sample). Alternatively, for example, if the method is carried out in a lateral flow device or strip, 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)).

[0107] The electrode(s) may be a carbon electrode and / or a silver electrode. Preferably, hydrogen peroxide is generated on a carbon electrode. Preferably, the silver electrode is used as a counter electrode. As mentioned elsewhere herein, preferably, the electron carrier is 2-hydroxy-1 ,4-naphthoquinone. Thus, in a specific embodiment, 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.

[0108] The step of generating hydrogen peroxide may be performed with the use of photogeneration. For example, light illumination (e.g. blue LED illumination) may be used. If hydrogen peroxide is generated with the use of photogeneration, 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. Preferably, hydrogen peroxide is generated in the presence of an electron carrier as described herein. In an embodiment, 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. For example, 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).

[0109] 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). Thus, 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). Preferably, 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. For example, the indicator molecule may have a pale bluegreen colour in its reduced form and blue colour in its oxidised form. In one embodiment, the change of colour between the two forms is measured. For example, the change of colour may be measured using a spectrophotometer.

[0110] In some embodiments, 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. Alternatively or additionally, in embodiments where the method is performed on a solid support (e.g. a later flow strip), the indicator molecule may be pre-deposited (for example sprayed or embedded) in or on a zone which is upstream of the capture zone. Such a zone comprising a pre-deposited indicator molecule is referred to herein as an “indicator zone”. For example, 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. Thus, preferably, 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:

[0111] 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). For example, for an indicator molecule B and a catalytic agent C (wherein the detection reagent comprises the catalytic agent), the oxidised indicator molecule Box may be generated according to the following equation:

[0112] BRD + H O2> Box

[0113] The reduced form of the indicator molecule is indicated by “BRD“ and the oxidised form of the indicator molecule is indicated by “Box”.

[0114] In certain embodiments, 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. For example, 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.

[0115] 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) 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. The skilled person will appreciate that these are exemplary components and that other suitable components may be used, for example any polymeric films together with appropriate enzymes.

[0116] Preferably, 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. In a preferred embodiment, 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.

[0117] As the capture of the analyte-detection reagent complex (which comprises a catalytic agent) at the capture zone depends on the presence of an analyte in the test sample, the step of generating an oxidized indicator molecule may preferably only take place in the presence of an analyte in the test sample. The skilled person will appreciate that some background reactions may occur in the absence of one or more relevant components, e.g. in the absence of the analyte. Thus, 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. For example, 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.

[0118] 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. Preferably, 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:

[0119] Box +e* BRDwhere Box denotes an oxidized indicator molecule and BRD denotes a reduced indicator molecule.

[0120] 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. Preferably, 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).

[0121] The charge transfer may be indicative of a presence or absence of an analyte in the test sample. Thus, 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. When the charge transfer is measured and / or analysed on electrode(s), the measurement may be corrected for blank (background) values. Thus, the method may further comprise a step of calibration (e.g. before step a)) to establish a background level of the charge transfer. Thus, 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. In one example of measuring the charge transfer using chronoamperometry, 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. In some examples, 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. In these examples, no current flows through the reference electrode as the circuit only measures its potential. The value of the current(amps) is multiplied by the time(seconds) giving the total charge(coulombs) used in the reduction reaction. This value is proportional to the concentration of the indicator molecules (e.g. TMB(ox)) used in the method.

[0122] 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. Thus, the method described herein is very sensitive. As shown in Example 5, the method described herein is able to detect an analyte at a concentration of as little as 10 pg / mL. As shown in Example 4, 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).

[0123] As will be appreciated by a person skilled in the art, 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. In addition, 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. After a second period of time from activation of the battery, 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).

[0124] 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.

[0125] The test sample may further comprise reagents useful in the method described herein (e.g. the indicator molecule, the electron carrier, or water).

[0126] It will be appreciated by a person skilled in the art that at least one step of the method described herein may be performed on a solid support. Preferably, 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) allows for co-localization of the reagents used in the methods described herein. The co-localization of some of the reagents used in the methods described herein allows to perform various method steps. For example, 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. For example, the step of generating an oxidized indicator molecule may efficiently be performed if hydrogen peroxide and a catalytic agent are in close proximity. Thus, if the analyte is present in the test sample, 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. 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. Thus, 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. 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.

[0127] Thus, 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.

[0128] The method described herein may further comprise a wash step to remove unwanted reagents. For example, 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. However, 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.

[0129] It would be understood to the skilled person that 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). For example, one or more of any of these reagents may be present in a reagent buffer. For example, 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. In embodiments where at least part of the method is performed on a solid support, one or more of any of these reagents may be predeposited on the solid support. For example, 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.

[0130] Thus, the reagents may be added separately from the test sample (or the reaction buffer). For example, various reagents may be added at various steps directly to the solid support. Preferably, however, the reagents used in the method are pre-deposited on the solid support. As shown in Figure 2, the solid support (e.g. a lateral flow strip) may contain separate pads where different reagents are deposited. For example, 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, or 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.

[0131] The invention also provides a device configured to perform the method of the invention. The device may comprise:

[0132] (a) a solid support;

[0133] (b) means for generating or mobilising hydrogen peroxide; and

[0134] (c) means for detecting a charge transfer.

[0135] Preferably, the device also comprises a battery. The battery may be activated by the liquid from the test sample and / or reaction buffer.

[0136] The device may comprise: (a) a solid support;

[0137] (b) means for generating or mobilising hydrogen peroxide;

[0138] (c) means for detecting a charge transfer; and

[0139] (d) a battery configured to be activated by contact with a liquid.

[0140] The features, reagents and steps described herein in the context of the method are applicable in the context of the device and kit aspects of the invention. Further description of certain features, reagents and steps is provided below to better visualize the device and kit aspects of the invention. However, it is to be understood that the features, reagents and steps described in the context of a device are also applicable mutatis mutandis to the method and kit aspects of the invention described herein. The same consideration is made in respect of described uses.

[0141] The invention may provide a device comprising:

[0142] (a) a solid support, the 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;

[0143] (b) means for generating or mobilising hydrogen peroxide at the capture zone or upstream of the capture zone; and

[0144] (c) means for detecting a charge transfer; and

[0145] (d) a battery configured to be activated by contact with a liquid.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] The invention may provide a device comprising:

[0150] (a) a solid support, the 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;

[0151] (b) means for generating or mobilising hydrogen peroxide at the capture zone or upstream of the capture zone;

[0152] (c) means for detecting a charge transfer; and

[0153] (d) a battery configured to be activated by contact with a liquid.

[0154] 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.

[0155] The invention may provide a device comprising:

[0156] (a) a solid support, the solid support comprising:

[0157] Hi. 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;

[0158] (b) means for generating or mobilising hydrogen peroxide at the capture zone or upstream of the capture zone;

[0159] (c) means for detecting a charge transfer; and

[0160] (d) a battery configured to be activated by contact with a liquid.

[0161] The device may comprise:

[0162] (a) a solid support, the 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;

[0163] (b) means for generating or mobilising hydrogen peroxide at the capture zone or upstream of the capture zone;

[0164] (c) means for detecting a charge transfer; and

[0165] (d) a battery configured to be activated by contact with a liquid. 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.

[0166] 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.

[0167] 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.

[0168] Fluidic contact between the electrode(s) and the support may be achieved in any appropriate way. In certain embodiments, 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. In certain embodiments, pressure may be applied to maintain fluidic contact. For example, spring fingers may be used to hold the electrode sheet in place. In certain embodiments, the electrode(s) may be connected to a potentiostat circuit in a similar way. For example, 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. In other embodiments, the electrode(s) may be printed onto a support.

[0169] In use, 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. In an event that the test sample contains an analyte, 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. Optionally, the interaction with the capture molecule is indirect, via the further binding molecule.

[0170] After a first period of time from activation of the battery, 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.

[0171] After a second period of time from activation of the battery, 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.

[0172] As described herein, 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.

[0173] An example of an assembled device is shown in Figure 3. In the figure, 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).

[0174] 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.

[0175] Thus, 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.

[0176] Thus, the invention provides a system for detecting an analyte in the test sample, wherein the system comprises:

[0177] (a) one or more devices described herein;

[0178] (b) a processor; and

[0179] (c) 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.

[0180] The computer application may optionally perform correction of charge transfer values to subtract the background charge transfer values as described herein.

[0181] The invention also relates to the computer applications used in the device, kit and system described herein. Thus, in certain embodiments, 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. 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.

[0182] As used herein, 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.

[0183] 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.

[0184] 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. 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. According to certain example embodiments, the processor, along with other components of the computing machine, may be a virtualized computing machine executing within one or more other computing machines.

[0185] 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.

[0186] 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.

[0187] 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. The I / O interface may be configured to implement only one interface or bus technology. Alternatively, 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 buffering transmissions between one or more external devices, internal devices, the computing machine, or the processor.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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. However, it should be apparent that there could be many different ways of implementing embodiments in computer programming, and the embodiments should not be construed as limited to any one set of computer program instructions. Further, 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. Further, those skilled in the art will appreciate that one or more aspects of embodiments described herein may be performed by hardware, software, or a combination thereof, as may be embodied in one or more computing systems. Moreover, any reference to an act being performed by a computer should not be construed as being performed by a single computer as more than one computer may perform the act.

[0192] 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. For example, 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.

[0193] 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).

[0194] 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.

[0195] In some embodiments, 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. This may take the form of a connectivity platform based, for instance, on cloud-based computing services. Thus, 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. One example of such is LumiraDx Connect (LumiraDx). In particular embodiments, 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. In other embodiments, 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. Thus, 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.

[0196] In a further aspect, the invention provides an analyte detection kit. The analyte detection kit may comprise:

[0197] (a) a solid support;

[0198] (b) means for generating or mobilising hydrogen peroxide at the capture zone or upstream of the capture zone;

[0199] (c) means for detecting a charge transfer; and

[0200] (d) a battery configured to be activated by contact with a liquid.

[0201] The analyte detection kit may comprise:

[0202] (a) a solid support, the 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;

[0203] (b) means for generating or mobilising hydrogen peroxide at the capture zone or upstream of the capture zone;

[0204] (c) means for detecting a charge transfer, which is indicative of the presence of an analyte in the test sample; and

[0205] (d) a battery configured to be activated by contact with a liquid.

[0206] In the case of the kits of the invention, the solid support may be provided without the capture molecules attached. In those embodiments, 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.

[0207] In certain embodiments, the solid support may comprise a nitrocellulose membrane.

[0208] In a further aspect, 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.

[0209] Each aspect or embodiment as defined herein may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. Other preferred embodiments of the methods, devices, analyte detection kits and uses according to the invention appear throughout the specification and in particular in the examples.

[0210] These and other aspects of the invention will now be described with reference to the accompanying Figures, in which:

[0211] Figure 1 illustrates an exemplary test strip.

[0212] Figure 2 illustrates an exemplary test strip.

[0213] Figure 3 shows an exploded view of an exemplary detection device. PCB, printed circuit board.

[0214] Figure 4 shows an exemplary arrangement of the electrodes in the detection device.

[0215] 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.

[0216] Figure 6 shows a charge of TMB reduction using hydrogen peroxide on working electrodes.

[0217] Figure 7 shows a relationship between TMB conversion and Pt70 number.

[0218] 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.

[0219] Figure 9 shows the digital readings in Cube units for data in Figure 8.

[0220] Figure 10 illustrates the limit of detection of Nucleoprotein using electrode detection and TMB- peroxide system.

[0221] Figure 11 illustrates the limit of detection of Nucleoprotein using electrode detection and TMB- peroxide system (log values).

[0222] Figure 12 shows the results for electrochemical generation of hydrogen peroxide using horseradish peroxidase (HRP) as catalyst.

[0223] Figure 13 shows an experimental step for photogeneration of hydrogen peroxide. The foregoing detailed description has been provided by way of explanation and illustration, and is not intended to limit the scope of the appended claims. Many variations in the presently preferred embodiments illustrated herein will be apparent to one of ordinary skill in the art, and remain within the scope of the appended claims and their equivalents.

[0224] The invention is further disclosed in the following clauses:

[0225] 1 . A method for detecting an analyte in a test sample, the method 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.

[0226] 2. The method of clause 1 , wherein the binding molecule of the detection reagent is capable of binding to a first binding site of the analyte and wherein the capture molecule is capable of binding to a second binding site of the analyte.

[0227] 3. The method of clause 2, wherein the first and second binding sites are different.

[0228] 4. The method of any one of clauses 1-3, wherein the 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.

[0229] 5. The method of any one of clauses 1-4, wherein the test sample comprises a reagent buffer, optionally wherein: i. the reagent buffer comprises the indicator molecule, ii. the reagent buffer comprises chloride,

[0230] Hi. the reagent buffer comprises water, iv. the reagent buffer comprises an electron carrier, and / or v. the reagent buffer comprises the detection reagent.

[0231] 6. The method of any one of clauses 1-5, wherein the binding molecule comprises or essentially consist of an antibody, or a fragment thereof, or a biotin molecule.

[0232] 7. The method of any one of clauses 1-6, wherein the catalytic agent comprises or essentially consist of a platinum nanoparticle or a horseradish peroxidase.

[0233] 8. The method of any one of clauses 1-7, wherein the binding molecule is conjugated to the catalytic agent.

[0234] 9. The method of any one of clauses 1-8, wherein the capture molecule binds to the analyte via a further binding molecule. The method of clause 9, wherein the further binding molecule comprises an antibody or a fragment thereof, or a biotin molecule. The method of clause 9 or clause 10, wherein the further binding molecule is: (i) pre-deposited, such as sprayed onto or embedded in or on the solid support at the sample application zone or downstream of the sample application zone; (ii) present in the reagent buffer of the test sample; and / or (iii) added to the solid support at the sample application zone; or downstream of the sample application zone and upstream of the capture zone. The method of any one of clauses 1-11 , wherein the capture molecule is an antibody or a fragment thereof, or a streptavidin molecule. The method of any one of clauses 1-12, wherein steps b) to d) of the method are carried out on the solid support. The method of any one of clauses 1-13, wherein the solid support defines a liquid flow path of the test sample, optionally wherein the solid support forms part of a lateral flow device. The method of any one of clauses 1-14, wherein the solid support comprises a sample application zone upstream of the capture zone. The method of any one of clauses 1-15, wherein: (i) the capture zone is also a detection zone, or (ii) the solid support comprises a detection zone downstream of the capture zone. The method of any one of clauses 1-16, wherein the solid support comprises a control zone downstream of the capture zone, optionally downstream of the detection zone. The method of any one of clauses 1-17, wherein 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 method of clause 18, wherein the detection reagent is pre-deposited, such as sprayed onto or embedded in or on the solid support at the detection reagent zone. The method of clause 18 or 19, wherein after the application of the test sample to the sample application 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 method of any one of clauses 1-23, wherein hydrogen peroxide is generated electrochemically. The method of any one of clauses 1-24, wherein 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 method of any one of clauses 1 -26, wherein 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, wherein one of the at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 electrodes is located at a control zone of the solid support. 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. The method of 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 method of any one of clauses 1-30, wherein the hydrogen peroxide is generated by light illumination of an electron carrier. The method of any one of clauses 1-33, wherein the step of generating hydrogen peroxide is performed 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 after the application of the test sample to the solid support. The method of any one of clauses 31-33, wherein 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 method of any one of clauses 1-35, wherein the indicator molecule is tetramethylbenzidine (TMB). The method of any one of clauses 1-36, wherein 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 method of any one of clauses 31-33 or 35, wherein the electron carrier 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 method of any one of clauses 1-38, wherein the step of generating an oxidized indicator molecule is performed at the capture zone or in the proximity of the capture zone. The method of any one of clauses 1-39, wherein in the step of generating an oxidized indicator molecule, the oxidation is catalysed by the catalytic agent. The method of any one of clauses 1 -40, wherein the step of reducing the oxidised indicator molecule is performed using at least one electrode. The method of any one of clauses 1 -41 , wherein the step of reducing the oxidised indicator molecule is performed by switching the electric potential to between 0 mV and -100 mV. The method of any one of clauses 1 -42, wherein the step of reducing the oxidised indicator molecule is performed for 0.1-60 seconds, 0.3-30 seconds, 0.5-10 seconds or 0.75-1.5 seconds. The method of any one of clauses 1 -43, wherein the step of measuring the charge transfer takes place at least 1 second, at least 2 seconds, at least 3 seconds, at least 4 seconds, at least 5 seconds, at least 10 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 10 minutes, after the hydrogen peroxide is generated. The method of any one of clauses 1-44, wherein the step of measuring the charge transfer is performed by chronoamperometry. The method of any one of clauses 1-45, wherein the analyte is a SARS-CoV-2 nucleocapsid protein antigen. The method of any one of clauses 1-46, wherein the method further comprises a step of calibration, optionally before step a), to establish a background level of the charge transfer. The method of any one of clauses 1-47, wherein the step of measuring the charge transfer includes subtracting the background level of the charge transfer. The method of 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:

[0235] (a) a solid support, the 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;

[0236] (b) means for generating or mobilising hydrogen peroxide at the capture zone or upstream of the capture zone;

[0237] (c) means for detecting a charge transfer, which is indicative of the presence of an analyte in the test sample; and

[0238] (d) a battery configured to be activated by contact with a liquid. An analyte detection kit, the kit comprising:

[0239] (a) a solid support, the 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;

[0240] (b) means for generating or mobilising hydrogen peroxide at the capture zone or upstream of the capture zone; (c) means for detecting a charge transfer, which is indicative of the presence of an analyte in the test sample; and

[0241] (d) a battery configured to be activated by contact with a liquid.

[0242] 52. Use of the device of clause 50 or the analyte detection kit of clause 51 for detecting a virus in a test sample.

[0243] 53. The use of clause 52, wherein the virus is a respiratory virus, preferably a coronavirus.

[0244] EXAMPLES

[0245] Example 1 : In-situ generation of hydrogen peroxide by electrochemical means compatible with a lateral flow immunoassay environment

[0246] Cyclic voltammetry was carried out on separate solutions of tetramethyl benzidine (TMB) and 2- hydroxy-1 ,4-napthoquinone (Q2), as well as Q2 and TMB together, and citrate saline buffer as the control. The following solutions were prepared:

[0247] 1) pH 5 citrate / saline buffer (50mM citrate and 50mM sodium chloride)

[0248] 2) Q2 at 1 mM in pH 5 citrate / saline buffer

[0249] 3) TMB at 0.4mM in pH 5 citrate / saline buffer

[0250] 4) Q2 at 1 mM and TMB at 0.4mM in pH 5 citrate / saline buffer

[0251] Each solution was subjected to cyclic voltammetry (CV) in a commercially available instrument, following the manufacturer’s instructions. The results are shown in Figure 5.

[0252] From Figure 5, it can be seen that the peaks of Q2 and TMB do not overlap, proving that Q2 can be used at a reduction potential of -0.6V in the presence of TMB, without interference. From this CV analysis the reduction potentials for Q2 and TMBox, which defines the parameters for in-situ H2O2 production and for detecting the presence of labelled antibodies (and hence analyte) on the test line, were chosen. These reduction potential for Q2 and TMBox are less or equal -0.6 V and less or equal 0.1 V, respectively.

[0253] 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.

[0254] Rationale: In this example, 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). 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). When they passed through the PSA line, the biotin-labelled antibody bound to the immobilised PSA, bringing with it any NP and platinum nanoparticle label.

[0255] For this experiment, it was necessary to determine the natural background electrochemical signal present in the running buffer alone, as detected by the working electrodes when in close contact with a saturated test strip. This was achieved by repeatedly using identical electrode arrays with identical test strips in the experiment. The first run was with a blank sample (comprising running buffer and everything else except the NP antigen). The readings from working electrodes 1 , 3 and 4 were recorded. Then a second strip was placed in exactly the same position in the cassette, complete with a new but equivalent electrode array, but this time the running buffer was spiked with 1 ng / ml of NP. The chronoamperometric readings from the blank sample were subtracted from the readings of the NP-containing sample to give the results displayed in the bar chart (Figure 6). This procedure was carried out in duplicate for the blank readings and the positive readings.

[0256] Procedure: The liquid samples (70pl) were applied to their respective lateral flow strip in which the streptavidin test line captured biotin-antibody. Of course, the platinum NP-antibodies could only be captured on the back of the biotin -antibodies when the NP was also present to form the immune complex.

[0257] 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.

[0258] One minute after the 20-second H2O2 surge (allowing oxidation of the TMB), the potential was re-set to -0.1V for just 1 second on all of the working electrodes by chronoamperometry, resulting in the reduction of oxidised TMBox to TMBRd. The reduction charges measured on working electrodes WE1 , WE3 and WE4 are presented in Figure 6, where the blank (background) values have been subtracted. Blank values were recorded as:

[0259] WE1 : -32

[0260] WE3: -36

[0261] WE4: -100

[0262] The blank-adjusted charge values obtained from 1 ng / ml Nucleocapsid Protein samples demonstrate the successful transduction of the immunoassay binding events into electronic signals using the method of the invention.

[0263] In this example, the multiple working electrode design enabled the generation of H2O2 reduction charge measurements at chosen locations around the test line. Figure 6 demonstrates:

[0264] 1 . Electrochemically generation of hydrogen peroxide by reducing naphthoquinone in situ.

[0265] 2. Platinum nanoparticle-Antibody-Analyte (Pt-Ab-Ag) complex on the test line helps the oxidation of TMB.

[0266] 3. Reduction of [TMB]ox can be detected on the downstream Working electrode 1 , 3 and 4 with similar level. The charge is linked to the number of Pt-Ab-Ag complex on the test line, thus the antigen level in sample.

[0267] In Figure 6, both sensor and test strip are in position, i.e., inside the cassette, while the operation on the electrodes are the same as in Figure 7 (see Example 3). Pt-Ab is dried on the test strip and would be released in test and captured on the test line when nucleoprotein is in the sample. Pt-Ab-Ag complex helps TMB’s oxidation after hydrogen peroxide is formed by reduction of naphthoquinone.

[0268] Example 3: Generation of hydrogen peroxide with TMB-FMN-Pt70 drop on screen-printed electrodes

[0269] 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.

[0270] Protocol:

[0271] 1) Mix 50 pl of 0.38mM TMB, 1 mM FMN, 50mM NaCI, pH 5.0 citrate-PBS buffer, and Pt nanoparticles. Drop on the screen-printed electrode

[0272] 2) Apply i-t scan at -0.65 V on WE1 vs silver electrode to reduce FMN; generate H2O2 and then oxidise TMB

[0273] 3) i-t scan at -0.1 V at WE1 vs counter electrode to reduce [TMB]Ox. Collect the change of charge for analysis on working electrode 1 .

[0274] The results shown in Figure 7 demonstrate:

[0275] 1 . Hydrogen peroxide is generated by reducing FMN;

[0276] 2. TMB can be oxidised by peroxide when catalytic agent Pt particles are present

[0277] 3. Linear relation between Pt70 number and reduced charge of TMB

[0278] Conclusion of Figures 6 (Example 2) and 7 (Example 3):

[0279] 1 . Pt-Ab-Ag can act as catalytic agent as free Pt nanoparticles in TMB conversion.

[0280] 2. FMN and naphthoquinone can both be used to generate peroxide, particularly can be done in a later flow setup in a cassette.

[0281] 3. Reduction charge of the oxidised TMB is proportional to Pt numbers, thus the antigen level in sample. Example 4: Limit of detection (LoD) of Nucleoprotein (NP) in dry assay with visual TMB conversion (Visual detection)

[0282] Demonstrated: The use of TMB causes a visual increase in signal compared to without TMB.

[0283] Dry test steps:

[0284] 1) Run strip with sample 50 pl NP in PBS-1% BSA-0.1% Tween20 for 12 min

[0285] 2) Rinse strip with 2x35 pl of running buffer PBS-BSA-Tween20

[0286] 3) Apply 40 pl TMB (contains peroxide)-0.1% Tween20, 10 min

[0287] 4) Record by a Cube Data Reader

[0288] Method:

[0289] Firstly, apply sample and run a conventional lateral flow test. Rinse the strip and apply TMB-peroxide mixture to amply the signal on the test line.

[0290] The visual test can be quantified by digitally recording the intensity of line on a Cube data Reader.

[0291] Results:

[0292] The results are shown in Figure 8. The limit of detection of nucleoprotein is 100 pg / ml after conventional lateral flow test (black lines in top picture) and improved to 12.5 pg / ml with TMB application (blue lines in the bottom picture). In Figure 9, the digital readings in Cube units are provided, which are just for comparison in this test and are not comparable to electrochemical results.

[0293] Conclusion:

[0294] The sensitivity of nucleoprotein is about eight times better with TMB-peroxide application.

[0295] Example 5: Limit of detection (LoD) of Nucleoprotein (NP) using electrode detection and TMB- peroxide system.

[0296] 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.

[0297] For each concentration of NP, the mean and standard deviation of the charge of the sum of working electrode 2 (WE2) and working electrode 3 (WE3) was calculated (Table 1). These electrodes were aligned at the positions of the two test lines. The plot of charge versus NP concentration showed a linear relationship with a R2 value of 0.95 (Figure 10). A plot of working electrode 1 (WE1) shows a background level of charge from the unoxidized TMB in contact with that electrode.

[0298] Table 1. Calculations of charge on working electrodes.

[0299] Log Total charge is plotted against Log NP concentration (background offset) is shown in Figure 11.

[0300] The limit of detection of nucleoprotein is <10 pg / ml (see Figure 11).

[0301] Example 6: Electrochemical generation of hydrogen peroxide using horseradish peroxidase (HRP) as catalyst

[0302] The TMB solution was prepared with 0.1 mM FMN but excluding the hydrogen peroxide. An extra silver electrode, positioned nearer to the sample pads, was used in the device of the invention as the counter while the carbon working electrode was used to generate the peroxide at a potential of -750 mV for 30 seconds. Conjugated HRP was used to enzymatically produce the oxidised TMB.

[0303] Concentrations of HRP do not directly correspond to NP concentrations, however, the results indicate that peroxide can be generated in-situ. Results are shown in Figure 12.

[0304] Example 7: Photogeneration of hydrogen peroxide

[0305] Initial experiments on the generation of hydrogen peroxide using FMN and blue LED illumination were performed.

[0306] Method:

[0307] Run the test strip with reagents as above. Cut the test line to stop flow and then add 5ul of FMN / TMB and expose for 10 secs to generate hydrogen peroxide. Measure blue dye produced optically. Method shown in Figure 13. Example 8: Preparation of exemplary test strip

[0308] Preparation of Conjugate Pt70-11A7

[0309] 1) Mix 1.2 pl of 1 mg / ml 11A7 with 50 pl of pH 7.5, 20mM TES conjugation buffer in a protein low bind Eppendorf tube.

[0310] 2) Ultrasonicate the bottle of Pt70 solution for 2-3 seconds prior to use. Take 1 ml of PtNP-70 (ODO.91-0.97) and mix with 11A7 in tube. This makes 1.2pg / ml 11A7 in conjugation with Pt particles. Incubate in a 37oC heat block for 30 min.

[0311] 3) Add 10 pl of 300mg / ml BSA for blocking at room temperature for 30 min on a rotator.

[0312] 4) Spin at 4000g for 20 min in a microcentrifuge. Discard the supernatant and re-suspend in 1 mM BOR buffer supplemented with 5% sucrose-3% BSA-1% Tween20 to obtain OD7.5 of Pt70-11 A7. Ultrasonicate and vortex the tube for 2-3 seconds.

[0313] Preparation of conjugates for dry assay

[0314] 2) Prepare 8 pg / ml Biotin-5B1 in 1 mM BOR drying buffer.

[0315] 3) Biotin-5B1 and Pt-11A7 are sprayed on 8951 (10mm wide) conjugation pads, respectively. The spray rate is 0.8pl / mm. Spray head of 8mm high with the spray pressure 4psi. Dry at 60°C at 5mm / s in Hedinair thermal tunnel.

[0316] 4) Sealed in Al pouches with desiccant and dry for at least overnight.

[0317] Preparation of NO

[0318] 1) Prepare 1 mg / ml polystreptavidin in pH 7.2 PBS buffer with 1% sucrose

[0319] 2) Plot PSA as test lines on nitrocellulose membrane, such as 95NC, at a rate of 0.1 pl / mm on Isoflow Dispensor. Dry at 60oC.

[0320] Assemble strip and prepare device for testing

[0321] 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.

[0322] Exemplary test strip is shown in Figure 2.

[0323] Table 2. Make up of Wash buffer solution Materials used in Examples 1-8

[0324] Reagents used in Examples 1-8 are shown in Table 3.

[0325] Table 3. Reagents used in examples 1-8.

[0326] Equipment used in Examples 1-8 includes:

[0327] • IviumSoft and Electrochemical Analyzer (Ivium Technologies)

[0328] • Cube Data Reader (opTricon GmbH)

Claims

CLAIMS1 . A method for detecting an analyte in a test sample, the method 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.

2. The method of claim 1 , wherein the binding molecule of the detection reagent is capable of binding to a first binding site of the analyte and wherein the capture molecule is capable of binding to a second binding site of the analyte.

3. The method of claim 1 or claim 2, wherein the 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.

4. The method of any one of claims 1-3, wherein the test sample comprises a reagent buffer, optionally wherein: i. the reagent buffer comprises the indicator molecule, ii. the reagent buffer comprises chloride,Hi. the reagent buffer comprises water, iv. the reagent buffer comprises an electron carrier, and / or v. the reagent buffer comprises a detection reagent.

5. The method of any one of claims 1-4, wherein the binding molecule comprises or essentially consists of an antibody, or a fragment thereof, or a biotin molecule.

6. The method of any one of claims 1-5, wherein the catalytic agent comprises or essentially consist of a platinum nanoparticle or a horseradish peroxidase.

7. The method of any one of claims 1-6, wherein the binding molecule is conjugated to the catalytic agent.

8. The method of any one of claims 1-7, wherein the capture molecule binds to the analyte via a further binding molecule.

9. The method of claim 8, wherein the further binding molecule comprises an antibody or a fragment thereof, or a biotin molecule.

10. The method of any one of claims 1-9, wherein the capture molecule is an antibody or a fragment thereof, or a streptavidin molecule.

11. The method of any one of claims 1-10, wherein steps b) to d) of the method are carried out on the solid support, preferably a lateral flow strip or a lateral flow device.

12. The method of any one of claims 1-11 , wherein the solid support comprises a sample application zone upstream of the capture zone.

13. The method of any one of claims 1-12, wherein: (i) the capture zone is also a detection zone, or (ii) the solid support comprises a detection zone downstream of the capture zone.

14. The method of any one of claims 1-13, wherein the method further comprises a wash step after step b) to remove uncaptured analyte and / or detection reagent.

15. The method of any one of claims 1-14, wherein hydrogen peroxide is generated at the capture zone or upstream of the capture zone.

16. The method of any one of claims 1-15, wherein hydrogen peroxide is generated electrochemically.

17. The method of any one of claims 1-16, wherein the solid support comprises at least one electrode, optionally wherein: (i) the at least one electrode is located at the capture zone and / or the detection zone, and / or (ii) 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.

18. The method of any one of claims 1-17, wherein hydrogen peroxide is generated by applying an electric potential by the at least one electrode, optionally wherein the electric potential is between -300mV and -1000 mV, preferably is about -600 mV.

19. The method of claim 18, wherein hydrogen peroxide is generated by applying an electric potential by the at least one electrode to an electron carrier.

20. The method of any one of claims 1-15, wherein the hydrogen peroxide is generated by light illumination of an electron carrier.

21. The method of any one of claims 1-20, wherein the step of generating hydrogen peroxide is performed 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 after the application of the test sample to the solid support.

22. The method of claim 20 or claim 21 , wherein 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, preferably wherein the electron carrier is 2-hydroxy-1 ,4-naphthoquinone.

23. The method of any one of claims 1-22, wherein the indicator molecule is tetramethylbenzidine (TMB).

24. The method of any one of claims 1-23, wherein the step of generating an oxidized indicator molecule is performed at the capture zone or in the proximity of the capture zone.

25. The method of any one of claims 1-24, wherein the step of reducing the oxidised indicator molecule is performed (i) using at least one electrode, and / or (ii) by switching the electric potential to between 0 mV and -100 mV.

26. The method of any one of claims 1-25, wherein the step of reducing the oxidised indicator molecule is performed for 0.1-60 seconds, 0.3-30 seconds, 0.5-10 seconds or 0.75-1.5 seconds.

27. The method of any one of claims 1-26, wherein the step of measuring the charge transfer takes place at least 1 second, at least 2 seconds, at least 3 seconds, at least 4 seconds, at least 5 seconds, at least 10 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 10 minutes after hydrogen peroxide is generated, optionally wherein the step of measuring the charge transfer is performed by chronoamperometry.

28. The method of any one of claims 1-27, wherein the analyte is a SARS-CoV-2 nucleocapsid protein antigen.

29. The method of any one of claims 1-28, 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 200pg / 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.

30. A device configured to perform the method of any one of claims 1-29 comprising:(a) a solid support, the 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 capturing an analyte which is bound to a detection reagent;(b) means for generating hydrogen peroxide at the capture zone or upstream of the capture zone;(c) means for detecting a charge transfer, which is indicative of the presence of an analyte in the test sample; and(d) a battery configured to be activated by contact with a liquid.31 . An analyte detection kit, the kit comprising:(a) a solid support, the 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 capturing an analyte which is bound to a detection reagent;(b) means for generating hydrogen peroxide at the capture zone or upstream of the capture zone;(c) means for detecting a charge transfer, which is indicative of the presence of an analyte in the test sample; and(d) a battery configured to be activated by contact with a liquid.

32. Use of the device of claim 30 or the analyte detection kit of claim 30 for detecting a virus in a test sample.

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