Electronic assay device

Abstract

An electronic assay device and a method of detecting the presence or absence of an analyte in a sample using an electronic assay device are provided. The electronic assay device is configured to output an electronic signal indicative of the presence of an analyte in a sample. The electronic assay device comprises a sample receiving region, a detection reagent comprising a catalytic agent, capture molecules, a test region, and a battery configured to be activated by contact with a liquid. The test region comprises an electrode assembly and a test line configured to bind to the capture molecules. A sequence of potentials is applied to the electrode assembly upon activation of the battery. A first potential is applied to the electrode assembly to produce hydrogen peroxide in the test region. A second potential is applied to the electrode assembly to reduce oxidised indicator molecules, generating a charge transfer.

Description

[0001] Electronic assay device

[0002] Field of the disclosure

[0003] The disclosure relates to the field of assay devices.

[0004] The Background

[0005] It is known to provide assay devices for detecting 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. This procedure 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.

[0006] Furthermore, the results of the assay procedure typically require 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. Interpretation of the results of the assay procedure may introduce further variation into the measurements.

[0007] Summary of the disclosure

[0008] According to an embodiment of the present disclosure there is provided a method of detecting the presence or absence of an analyte in a sample using an electronic assay device, wherein the electronic assay device is configured to output an electronic signal indicative of the presence of an analyte in a sample. The electronic assay device comprises a sample receiving region. The electronic assay device further comprises a detection reagent comprising a catalytic agent. The electronic assay device further comprises capture molecules. The electronic assay device further comprises a test region comprising an electrode assembly and a test line configured to bind to the capture molecules. The electronic assay device further comprises a battery configured to be activated by contact with a liquid. The method comprises depositing a sample and a solution on the sample receiving region, such that the analyte, where present, binds to the detection reagent via a first binding site of the analyte and binds to the capture molecule via a second binding site of the analyte; and the solution is incident on the battery, activating the battery and triggering a sequence of potentials applied to the electrode assembly; and the bound analyte, where present, and the solution are incident on the test region, wherein the solution carries indicator molecules to the test region. After a first period of time from activation of the battery, the method further comprises applying a first potential to the electrode assembly to produce hydrogen peroxide in the test region. The method further comprises generating oxidised indicator molecules at the test region, wherein the oxidised indicator molecules are produced via hydrogen peroxide oxidation of the indicator molecules in the presence of the catalytic agent. After a second period of time from activation of the battery, the method further comprises applying a second potential to the electrode assembly such that the oxidised indicator molecules are reduced, generating the indicator molecules and a charge transfer. The method further comprises measuring the charge transfer, wherein the charge transfer is indicative of the presence or absence of the analyte. The method further comprises outputting data indicative of the charge transfer.

[0009] In this way, the presence or absence of an analyte in a sample may be accurately detected. Once the sample has been deposited, the presence or absence of an analyte in a sample may be accurately detected without further intervention from a user, meaning that the accuracy of the detection is not vulnerable to variation in timing by a user or other variations between assay procedures. Furthermore, the result of the assay may be measured electronically without the need for user interpretation.

[0010] The solution may comprise the indicator molecules.

[0011] Advantageously, the indicator molecules may be added in a solution such that the indicator molecules are carried to the test region. The indicator molecules may be in the same solution as the sample, reducing the number of steps performed by the user.

[0012] The first potential may be applied to the electrode assembly in the presence of an electron donor. An electron donor or redox mediator may can lower the size of the potential required for the reaction to produce hydrogen peroxide to take place.

[0013] Either the solution may comprise the electron donor; or the electrode assembly may comprise the electron donor.

[0014] In this way, the electron donor may be present at the test region. In an event that the solution comprises the electron donor, the electron donor may be carried to the test region by the solution. In an event that the electrode assembly comprises the electron donor, the electron donor is present at the test region.

[0015] The detection reagent may be in a dried state on the electronic assay device and depositing the solution may release the detection reagent.

[0016] In this way, the detection reagent may be present without the user needing to add it. Advantageously, this reduces the number of steps carried out by the user and so reduced variation between assays.

[0017] The capture molecule may be in a dried state on the electronic assay device and depositing the solution may release the capture molecule.

[0018] In this way, the capture molecules may be present without the user needing to add them. Advantageously, this reduces the number of steps carried out by the user and so reduced variation between assays.

[0019] The capture molecule may bind to the test line via an intermediate molecule.

[0020] Advantageously, the capture molecules may be carried to the test line by a solution and may be fixed at the test line by the intermediate molecule.

[0021] The sample receiving region may comprises a first region configured to receive the sample and a buffer solution; and a second region, configured to receive a reagent solution comprising the electron donor and the indicator molecule. In this way, the sample and a buffer solution may be added to the device separately to the reagent solution. Advantageously, any reaction between the solutions before it is required by the method may be avoided.

[0022] The method may comprise depositing the sample and a buffer solution on the first region and depositing the reagent solution on the second region.

[0023] In this way, the sample and a buffer solution may be added to the device separately to the reagent solution. Advantageously, any reaction between the solutions before it is required by the method may be avoided.

[0024] The first region may comprise the capture molecule and depositing the buffer onto the first region may release the capture molecule.

[0025] In this way, the capture molecule may be added to the solution to bind with the analyte, where present. The capture molecule and the analyte may be carried by the buffer solution to the test region, providing enough time for all the analyte present to bind to capture molecules.

[0026] The first region may comprise the detection reagent and depositing the buffer onto the first region may release the detection reagent.

[0027] In this way, the detection reagent may be added to the solution to bind with the analyte, where present. The detection reagent and the analyte may be carried by the buffer solution to the test region, providing enough time for all the analyte present to bind to the detection reagent.

[0028] The first region may comprise a first pad comprising the capture molecule and a second pad comprising the detection reagent, wherein the method may comprise depositing the sample and solution on the first pad and wherein the sample and solution may subsequently flow through the second pad.

[0029] In this way, both the capture molecules and the detection reagent may be released by the solution and may bind to analyte in the sample. The electrode assembly may comprise a first working electrode and a counter electrode, wherein the second potential may be applied between the first working electrode and the counter electrode.

[0030] The first potential may be applied between the first working electrode and the counter electrode.

[0031] The electrode assembly may further comprise a second working electrode, wherein the first potential may be applied between the second working electrode and the counter electrode.

[0032] The electrode assembly may further comprise a second working electrode and a third working electrode, wherein the first potential may be applied between the second working electrode and the third working electrode.

[0033] The first working electrode may be adjacent to the counter electrode, and the second working electrode may be adjacent to the third working electrode.

[0034] The electrode assembly may further comprise a first reference electrode.

[0035] Advantageously, a reference value for the measurement may be obtained using the first reference electrode.

[0036] The first potential may be applied between the first working electrode and the counter electrode, and the second potential may be referenced to the reference electrode.

[0037] The first working potential may be applied between the first working electrode and the second working electrode and the second potential may be applied: between the first working electrode and the counter electrode; and subsequently between the reference electrode and the counter electrode.

[0038] In this way, a control value may be obtained by subsequently applying the second potential between the reference electrode and the counter electrode.

[0039] The electrode assembly may further comprise a second reference electrode, wherein the first potential may be applied between the first working electrode and the counter electrode and wherein the second potential may be applied: between the first working electrode and the counter electrode; subsequently between the first reference electrode and the counter electrode; and subsequently between the second reference electrode and the counter electrode.

[0040] In this way, a first and second control value may be obtained by subsequently applying the second potential between the first reference electrode and the counter electrode and subsequently applying the second potential between the second reference electrode and the counter electrode.

[0041] The first working electrode may be between the first reference electrode and the second reference electrode.

[0042] The test region may be aligned with the first working electrode.

[0043] In this way, the hydrogen peroxide may be produced at the test region.

[0044] The test region may intersect with the counter electrode.

[0045] In this way, the analyte is bound at the region where the second potential is applied.

[0046] The counter electrode may be shaped such that the counter electrode is adjacent to each other electrode of the electrode assembly.

[0047] In this way, potentials may be applied between the counter electrode and each other electrode of the electrode assembly.

[0048] In an event that an analyte is present, the data indicative of charge transfer may be further indicative of a concentration of the analyte such that the method further comprises determining the concentration of the analyte.

[0049] Advantageously, the amount of analyte present in a sample may be quantified without need for user interpretation. The detection reagent may comprise a molecular binding reagent labelled with the catalytic agent.

[0050] The catalytic agent may comprise catalytic platinum nanoparticles.

[0051] The catalytic agent may comprise horse-radish peroxidase.

[0052] The capture molecule may comprise Biotin-Ab1.

[0053] The electron donor may comprise a quinone.

[0054] The electron donor may comprise flavin mononucleotide.

[0055] The indicator molecule may comprise 3,3',5,5'-Tetramethylbenzidine, TMB.

[0056] The intermediate molecule may comprise Polystreptavidin, PSA.

[0057] According to an embodiment of the present disclosure, there is provided: an electronic assay device configured to output an electronic signal indicative of the presence of an analyte in a sample. The electronic assay device comprises a detection reagent comprising a catalytic agent configured to bind to a first binding site of the analyte, where present. The electronic assay device further comprises capture molecules configured to bind to a second binding site of the analyte, where present. The electronic assay device further comprises a test region comprising an electrode assembly and a test line configured to bind to the capture molecule. The electronic assay device further comprises a battery configured to be activated by contact with a liquid. The electronic assay device further comprises a sample receiving region configured to receive a sample and a solution such that the solution is incident on the test region and on the battery. The electronic assay device further comprises a microprocessor configured to initiate a sequence of potentials applied to the electrode assembly upon activation of the battery. After a first period of time from activation of the battery, the microprocessor is further configured to apply a first potential to the electrode assembly to produce hydrogen peroxide in the test region, wherein the hydrogen peroxide oxidises indicator molecules in the presence of the catalytic agent to produce oxidised indicator molecules, wherein the indicator molecules are carried to the test region by the solution. After a second period of time from activation of the battery, microprocessor is further configured to apply a second potential to the electrode assembly such that the oxidised indicator molecules are reduced, generating the indicator molecules and a charge transfer. The microprocessor is further configured to measure the charge transfer, wherein the charge transfer is indicative of the presence or absence of the analyte. The microprocessor is further configured to output data indicative of the charge transfer.

[0058] Brief description of the drawings

[0059] A specific embodiment of the disclosure will now be described, by way of example only, with reference to the accompanying drawings in which:

[0060] Figure 1 shows a schematic diagram of an electronic assay device according to an embodiment of the present disclosure.

[0061] Figure 2 shows a schematic diagram of an electronic assay device according to an embodiment of the present disclosure.

[0062] Figure 3 shows a schematic diagram of an electronic assay device according to an embodiment of the present disclosure.

[0063] Figure 4 shows a schematic diagram of an electronic assay device according to an embodiment of the present disclosure.

[0064] Figures 5A to 5C show flowcharts illustrating methods according to embodiments of the present disclosure.

[0065] Figure 6 shows a schematic diagram of an electronic assay device according to an embodiment of the present disclosure.

[0066] Figures 7A to 7D show schematic diagrams of electrode assemblies for use in an assay device according to an embodiment of the present disclosure.

[0067] Figures 8A and 8B show schematic diagrams of electrode assemblies for use in an assay device according to an embodiment of the present disclosure. Figure 9 shows a schematic diagram of a top view of an electronic assay device according to an embodiment of the present disclosure.

[0068] Figure 10 shows a schematic diagram of an electronic assay device according to an embodiment of the present disclosure.

[0069] Figure 11 shows a schematic diagram of an electronic assay device according to an embodiment of the present disclosure.

[0070] Detailed description

[0071] An electronic assay device is provided according to an aspect of the disclosure, wherein the electronic assay device is configured to output an electronic signal indicative of the presence of an analyte in a sample. The electronic assay device allows detection of the presence or absence of analyte in a sample.

[0072] The electronic assay device comprises a sample receiving region, a detection reagent, capture molecules, a test region and a battery. The detection reagent comprises a catalytic agent. The test region comprises an electrode assembly and a test line configured to bind to the capture molecules. The battery is configured to be activated by contact with a liquid. With reference to Figure 1 , a simplified schematic diagram of a side view of an electronic assay device 100 according to an embodiment of the disclosure is shown, showing the sample receiving region 110 and the test line 120. The test line 120 may be located on a substrate 140. The test line 120 is shown with a schematic of a capture molecule 130 bound to the test line of the test region. The capture molecule may be carried to the test region by the solution. The capture molecule may, if an analyte is present, be bound to an analyte before reaching the test region. In other embodiments, the capture molecule may be fixed at the test region and the analyte, where present, may bind to the capture molecule at the test region. This arrangement is exemplary, and other arrangements of the sample receiving region and test region are possible. The electrode assembly 150 may be above or below the substrate 140. The test region may comprise a region of the substrate and the electrode assembly. For example, the test line of the test region may be located on an upper surface of the substrate, while the electrode assembly may be located adjacent to a lower surface of the substrate. In other embodiments, the electrode assembly may be located adjacent to the upper surface of the substrate.

[0073] In use, a sample and a solution are deposited on the sample receiving region 110. The solution is incident on the battery, activating the battery and triggering a sequence of potentials applied to the electrode assembly. The solution may be deposited as a single solution, or as more than one solution. In an event that the solution is deposited as more than one solution, the more than one solution may be deposited in different regions or in the same region. In use, the solution is incident on the test region, and therefore on the electrode assembly. With reference to Figure 2, in an event that the sample contains an analyte, the analyte 210 binds to the detection reagent 220 via a first binding site of the analyte and binds to the capture molecule 130 via a second binding site of the analyte.

[0074] After a first period of time from activation of the battery, a first potential is applied to the electrode assembly to produce hydrogen peroxide in the test region. Indicator molecules are present at the test region, having been carried to the test region by the solution. In certain embodiments, the deposited solution comprises the indicator molecules. An oxidised indicator molecule is generated at the test region, wherein the oxidised indicator molecule is produced via hydrogen peroxide oxidation of the indicator molecule in the presence of the catalytic agent. The first period of time may be any time period that is long enough that the solution has reached the test region, but short enough that the solution has not dried on the test region. For example, it may take between 4 and 10 minutes for a sample to and the solution to travel from the sample receiving region 110 to the test region 120.

[0075] After a second period of time from activation of the battery, a second potential is applied to the electrode assembly such that the oxidised indicator molecule is reduced, generating the indicator molecule and a charge transfer. The charge transfer is measured, wherein the charge transfer is indicative of the presence or absence of the analyte. Data indicative of the charge transfer is output.

[0076] In certain embodiments, the first potential may be applied to the electrode assembly to produce hydrogen peroxide, without the presence of an electron donor. In other embodiments, the first potential may be applied to the electrode assembly in the presence of an electron donor to produce hydrogen peroxide. In certain embodiments, the solution may comprise the electron donor. In other embodiments, the electrode assembly may comprise the electron donor. For example, the electron donor may be mixed with the electrode material when the electrodes of the electrode assembly are prepared.

[0077] In certain embodiments, such as that shown in Figures 1 and 2, the sample receiving region may be in a different lateral position on the assay device than the test region. The sample and solution may flow from the sample receiving region to the test region via substrate 140. For example, the substrate 140 may comprise or support a lateral flow strip, membrane or other means for allowing fluid flow. In embodiments where the sample and solution may flow from the sample receiving region to the test region via substrate 140, the electrode assembly is configured to be in fluidic contact with the substrate when the solution reaches the test region. The distance of the test line 120 from the sample receiving region 110 may affect the time taken to carry out an assay and the sensitivity of the assay. A longer distance between the test line 120 and the sample receiving region 110 increases the time taken for the sample and the solution to travel from the sample receiving region 110 to the test line 120, and so increases the time taken to carry out an assay. The analyte, where present, and the capture molecules and detection reagent may be present in the solution as it travels from the sample receiving region 110 to the test line 120. A longer distance between the test line 120 and the sample receiving region 110 provides more time for the sample molecules to bind to the capture molecules, increasing the sensitivity of the assay.

[0078] In certain embodiments, the detection reagent may be in a dried state on the electronic assay device. Depositing the solution onto the sample receiving region releases the detection reagent. The detection reagent may flow with the solution to the test region. The analyte may bind to the detection reagent at the location of release of the detection reagent, or as the solution flows to the test region, or at the test region.

[0079] In embodiments, the capture molecules may be in a dried state on the electronic assay device. Depositing the solution may release the capture molecules, such that the capture molecules flow to the test region with the solution. The capture molecules may bind to the test line via intermediate molecules, wherein the intermediate molecules are located on the test line. In certain embodiments, the deposited solution may comprise the indicator molecules, such that the indicator molecules flow to the test region with the solution. In other embodiments, the indicator molecules may be in a dried state on the electronic assay device. Depositing the solution may release the capture molecules, such that the capture molecules flow to the test region with the solution.

[0080] With reference to Figures 1 and 2, in use of certain embodiments, a sample and a solution are deposited on the sample receiving region 110. The solution comprises the indicator molecules. The solution is incident on the battery, activating the battery and triggering a sequence of potentials applied to the electrode assembly. The solution may be deposited as a single solution, or as more than one solution. In an event that the solution is deposited as more than one solution, the more than one solution may be deposited in different regions or in the same region. The capture molecules and the detection reagent may be in a dried state on the sample receiving region 110. The capture molecules and the detection reagent may be released by the solution. With reference to Figure 2, in an event that the sample contains an analyte, the analyte 210 binds to the detection reagent 220 via a first binding site of the analyte and binds to the capture molecule 130 via a second binding site of the analyte. The solution is further incident on the test region, and therefore on the electrode assembly. The bound analyte, where present, binds to the test line.

[0081] After a first period of time from activation of the battery, a first potential is applied to the electrode assembly to produce hydrogen peroxide in the test region. Indicator molecules are present at the test region, having been carried to the test region by the solution. An oxidised indicator molecule is generated at the test region, wherein the oxidised indicator molecule is produced via hydrogen peroxide oxidation of the indicator molecule in the presence of the catalytic agent. After a second period of time from activation of the battery, a second potential is applied to the electrode assembly such that the oxidised indicator molecule is reduced, generating the indicator molecule and a charge transfer. The charge transfer is measured, wherein the charge transfer is indicative of the presence or absence of the analyte. Data indicative of the charge transfer is output.

[0082] With reference to Figures 1 and 2, in use of certain embodiments, a sample and a solution are deposited on the sample receiving region 110. The solution does not comprise the indicator molecules. The solution is incident on the battery, activating the battery and triggering a sequence of potentials applied to the electrode assembly. The solution may be deposited as a single solution, or as more than one solution. In an event that the solution is deposited as more than one solution, the more than one solution may be deposited in different regions or in the same region. The capture molecules, the detection reagent and the indicator molecules may be in a dried state on the sample receiving region 110. The capture molecules, the detection reagent and the indicator molecules may be released by the solution. As above, the solution travelling from the sample receiving region to the test region therefore comprises the capture molecules, detection reagent and indicator molecules. With reference to Figure 2, in an event that the sample contains an analyte, the analyte 210 binds to the detection reagent 220 via a first binding site of the analyte and binds to the capture molecule 130 via a second binding site of the analyte. The solution is further incident on the test region, and therefore on the electrode assembly. The bound analyte, where present, binds to the rest region.

[0083] After a first period of time from activation of the battery, a first potential is applied to the electrode assembly to produce hydrogen peroxide in the test region. Indicator molecules are present at the test region, having been carried to the test region by the solution. An oxidised indicator molecule is generated at the test region, wherein the oxidised indicator molecule is produced via hydrogen peroxide oxidation of the indicator molecule in the presence of the catalytic agent. After a second period of time from activation of the battery, a second potential is applied to the electrode assembly such that the oxidised indicator molecule is reduced, generating the indicator molecule and a charge transfer. The charge transfer is measured, wherein the charge transfer is indicative of the presence or absence of the analyte. Data indicative of the charge transfer is output.

[0084] The sample receiving region 110 may comprise one region configured to receive both the sample and solution. The sample and solution may be received by the same region, at the same time or at different times. For example, the sample receiving region 110 may comprise a sample pad. The solution may comprise a buffer solution. The solution may comprise a buffer solution and a reagent solution. In other embodiments, the sample receiving region 110 may comprise more than one sub-region. The more than one subregion may each be configured to receive one or more of the sample and the solution. In certain embodiments, the solution may comprise a buffer solution and a reagent solution. The buffer solution may be deposited with the sample. The reagent solution may be deposited separately to the sample and buffer solution. The reagent solution may comprise the indicator molecules. A first sub-region of the sample receiving region 110 may be configured to receive the sample and buffer solution and a second sub-region of the sample receiving region 110 may be configured to receive the reagent solution. For example, the sample receiving region 110 may comprise more than one pad, each pad comprising a sub-region of the sample receiving region 110. In certain embodiments, the battery may be configured to be activated by the buffer solution. In certain embodiments, the battery may be configured to be activated by the reagent solution. In certain embodiment, the solution may comprise an electrolyte solution configured to activate the battery.

[0085] With reference to Figure 3, the sample receiving region 310 of an assay device 300 may comprise a first sub-region 320 and a second sub-region 330, wherein the first sub-region 320 is configured to receive the sample and a buffer solution (indicated by arrow 321) and wherein the second sub-region 330 is configured to receive a reagent solution (indicated by arrow 331). In certain embodiments, the reagent solution may comprise the indicator molecules. In other embodiments, the indicator molecules may be in a dried state on the second sub-region 330, such that the indicator molecules are released when the reagent solution is deposited on the second sub-region 330. In embodiments where the solution comprises an electron donor, the reagent solution may further comprise the electron donor. The method may comprise depositing the sample and a buffer solution on the first subregion 320 and depositing the reagent solution on the second sub-region 320. The first sub-region 320 and the second sub-region 330 may be separated such that the reagent solution added to the second sub-region 330 is not incident on the first sub-region 320. For example, a barrier 340 may be located between the first sub-region 320 and the second sub-region 330 such that fluid cannot be transferred between the first sub-region 320 and the second sub-region 330 or vice versa, but such that fluid can still flow from the first subregion 320 to the test line 120 and from the second sub-region 330 to the test line 120.

[0086] The first sub-region 320 may comprise the capture molecule in a dried state, wherein depositing the buffer onto the first region releases the capture molecule. The first subregion 320 may comprise the detection reagent in a dried state, wherein depositing the buffer onto the first sub-region 320 releases the detection reagent.

[0087] In certain embodiments, with reference to Figure 4, the sample receiving region 410 of an assay device 400 may comprise a first sub-region 420 configured to receive the sample and the buffer solution (indicated by arrow 421). The first sub-region 420 may comprise the capture molecules in a dried state. The sample receiving region 410 may further comprise a second sub-region 430 comprising the detection reagent in a dried state, wherein the sample and solution are deposited on the first sub-region 420 and wherein the sample and solution subsequently flow through the second sub-region 430. The first sub-region 420 and the second sub-region 430 may each comprise a pad. The sample receiving region 410 may further comprise a third sub-region 440, wherein the third sub-region 440 is configured to receive a reagent solution (indicated by arrow 441). In certain embodiments, the reagent solution may comprise the indicator molecules. In other embodiments, the indicator molecules may be in a dried state on the third sub-region 440, such that the indicator molecules are released when the reagent solution is deposited on the third subregion 440. In embodiments where the solution comprises an electron donor, the reagent solution may further comprise the electron donor. The method may comprise depositing the sample and a buffer solution on the first sub-region 420 and depositing the reagent solution on the third sub-region 440. The third sub-region 440 may be separated from the first subregion 420 and the second sub-region 430 such that the reagent solution added to the third sub-region 440 is not incident on the first sub-region 420 or the second sub-region 430. For example, a barrier 450 may be located between the third sub-region 440 and the first and second sub-regions 420, 430 such that fluid cannot be transferred between the third subregion 440 and the first and second sub-regions 420, 430 or vice versa. The barrier 450 may be located such that fluid can still flow from the first sub-region 420 to the second subregion 430 and then to the test line 120 and from the third sub-region 440 to the test line 120.

[0088] In certain embodiments, the first and second sub-regions 420, 430 may comprise separate pads. The pads may be located as shown in Figure 4, with the pad of the first sub-region 420 above and partially overlapping the pad of the second sub-region 430. The pads may be arranged in different configurations. For example, the pad of the first sub-region 420 may be directly above and substantially overlapping the pad of the second sub-region 430. In another example, the pad of the first sub-region 420 may be adjacent to the pad of the second sub-region 430. In other embodiments, the first and second sub-regions 420, 430 may be on the same pad.

[0089] In certain embodiments, the sample and buffer solution flow from the first sub-region 320 or

[0090] 420 to the test line 120 without being incident on the battery. The reagent solution may be incident on the battery and may flow from the second sub-region 330 or third sub-region

[0091] 440 to the test line 120.

[0092] In certain embodiments, an absorbent pad or sink pad may be placed at the opposite end of the substrate to the sample receiving region. This may be used to absorb excess solution or wash solutions. In certain embodiments, a wash solution may be added to the sink pad after the indicator molecule reaches the test region, such that the oxidised indicator molecule is concentrated around the test region and does not flow away.

[0093] With reference to Figure 5, a method of detecting the presence or absence of an analyte using any electronic assay device of the present disclosure is illustrated. Various embodiments of the method are illustrated in Figures 5A, 5B and 5C. With reference to Figure 5A, the method comprises depositing a sample and a solution on the sample receiving region at step 510. At step 520 the analyte, where present, binds to the detection reagent via a first binding site of the analyte and binds to the capture molecule via a second binding site of the analyte. At step 530, indicator molecules are incident on the test region. The solution is incident on the battery, activating the battery at step 540 and triggering a sequence of potentials applied to the electrode assembly. After a first period of time from activation of the battery, step 550 of the method comprises applying a first potential to the electrode assembly to produce hydrogen peroxide in the test region. The method further comprises generating an oxidised indicator molecule at the test region at step 560, wherein the oxidised indicator molecule is produced via hydrogen peroxide oxidation of the indicator molecule in the presence of the catalytic agent. After a second period of time from activation of the battery, step 570 of the method comprises applying a second potential to the electrode assembly such that the oxidised indicator molecule is reduced, generating the indicator molecule and a charge transfer. The method further comprises measuring the charge transfer at step 580, wherein the charge transfer is indicative of the presence or absence of the analyte. At step 590 the method comprises outputting data indicative of the charge transfer.

[0094] In certain embodiments, the solution deposited at step 510 may comprise the indicator molecules.

[0095] In certain embodiments, the solution deposited at step 510 may comprise an electron donor. The electron donor may be incident on the test region at step 530. At step 550, the first potential may be applied to the electrode assembly in the presence of the electron donor. In other embodiments, at step 550 the first potential may be applied to the electrode assembly in the presence of the electron donor, wherein the electrode assembly comprises the electron donor. In other embodiments, at step 550 first potential may be applied to the electrode assembly without an electron donor being present.

[0096] In certain embodiment, steps 530 and 540 may occur in the order shown in Figure 5A, or in the opposite order, or at the same time.

[0097] In certain embodiments, step 510 may comprise a single step of depositing a sample and a solution on the sample receiving region, such that the analyte, where present, binds to the detection reagent via a first binding site of the analyte and binds to the capture molecule via a second binding site of the analyte at step 520, and the solution is incident on the battery at step 540. The solution may follow one path, which allows the analyte to bind to the detection reagent and capture molecule and be incident on the test region and also allows the solution to be incident on the battery. The solution may be split and follow more than one path, such that part of the solution follows a first path that allows the analyte to bind to the detection reagent and capture molecule and be incident on the test region, and another part of the solution follows a second path that allows the solution to be incident on the battery. For example, the second path may follow a tube or channel to the battery.

[0098] In other embodiments, with reference to Figure 5B, step 510 may comprise more than one stage of depositing a sample and a solution on the sample receiving region. In a first stage 511 of step 510, the sample may be deposited on the sample receiving region such that at step 520 the analyte, where present, binds to the detection reagent via a first binding site of the analyte and binds to the capture molecule via a second binding site of the analyte. The solution deposited at stage 511 may comprise one or both of the capture molecules and detection reagent. The solution deposited at stage 511 may release one or both of the capture molecules and detection reagent from a dried state. The sample may be deposited with a buffer solution, either separately from the sample or mixed together with the sample. In a second stage 512 of step 510, the solution may be deposited on the sample receiving region such that at step 530 indicator molecules (and electron donor, if present in the solution) are incident on the test region, and at step 540 the solution is incident on the battery. Alternatively, the solution deposited at first stage 511 may be incident on the battery at step 540. The solution deposited at stage 512 may comprise the indicator molecules, or the solution deposited at stage 512 may release the indicator molecules from a dried state.

[0099] With reference to Figure 5C, in certain embodiments step 510 may comprise a first stage 511, a second stage 512 and a third stage 513. In the first stage 511 of step 510, the sample may be deposited on the sample receiving region such that at step 520 the analyte, where present, binds to the detection reagent via a first binding site of the analyte and binds to the capture molecule via a second binding site of the analyte. The solution deposited at stage 511 may comprise one or both of the capture molecules and detection reagent. The solution deposited at stage 511 may release one or both of the capture molecules and detection reagent from a dried state. The sample may be deposited with a buffer solution, either separately from the sample or mixed together with the sample. In the second stage 512 of step 510, the solution may be deposited on the sample receiving region such that at step 530 indicator molecules (and electron donor, if present in the solution) are incident on the test region. The solution deposited at stage 512 may comprise the indicator molecules, or the solution deposited at stage 512 may release the indicator molecules from a dried state. In the third stage 513 of step 510, a solution may be deposited such that the solution is incident on the battery at step 540, wherein the solution comprises an electrolyte configured to activate the battery at step 540.

[0100] A method according to an embodiment of the disclosure will now be described in more detail by reference to a specific example, which is not considered limiting. The method will be described with reference to the electronic assay device 600 illustrated in Figure 6.

[0101] In this example, step 510 of depositing the sample and solution on the sample receiving region 610 comprises a first stage 511 and a second stage 512. The first stage 511 of step 510 comprises depositing a sample 621 and a buffer solution 622 onto a first sub-region 630 of the sample receiving region 610, as indicated by arrow 620. The first sub-region 630 may comprise a first pad. The first sub-region 630 comprises capture molecules 631 , wherein the capture molecules 631 are in a dried form on or in the first sub-region 630. The buffer solution 622 releases the capture molecules 631 from the first sub-region 630, and the sample 621, buffer solution 622 and capture molecules 631 subsequently flow to a second sub-region 640. The second sub-region 640 may comprise a second pad. The second sub-region 640 comprises the detection reagent 641, wherein the detection reagent 641 is in a dried form on or in the second sub-region 640. The buffer solution 622 releases the detection reagent 641 from the second sub-region 640. The sample 621 , buffer solution 622, capture molecules 631 and detection reagent 641 subsequently flow along a substrate 680 to a test region 690. The test region may comprise intermediate molecules 691, wherein the intermediate molecules 691 are configured to bind to the capture molecules 631. Figure 6 indicates the molecules at the test region after step 520 in an event that an analyte is present in the sample 621. Each analyte of the sample 621 binds to a capture molecule 631 and to a detection reagent 641. The capture molecule 631 binds to the intermediate molecule 691, such that the analyte and detection reagent 641 are captured at the test region. In certain embodiments, the test region may change colour upon incidence of a liquid, so test region 690 may change colour when the sample 621, buffer solution 622, capture molecules 631 and detection reagent 641 reach the test region 690 after step 510. Electrode assembly 692 is shown below the substrate 680, but may be located above the substrate 680.

[0102] The detection reagent comprises a catalytic agent configured to catalyse the reaction in which hydrogen peroxide oxidises an indicator molecule. In order for an accurate detection of the presence or absence of the analyte, this reaction must only be catalysed by the detection reagents bound to the test line. Therefore, the hydrogen peroxide must be absent from the test device until after step 520, when the complex including the analyte and detection reagent is captured at the test line. Any unbound detection reagent should be washed away before the hydrogen peroxide is generated. The method may comprise an additional step after step 520 of depositing a wash solution on the assay device such that any surplus, unbound detection reagent is washed from test region and surrounding area of the substrate.

[0103] The second stage 512 of step 510 comprises depositing a reagent solution 651 onto a third sub-region 660 of the sample receiving region 610, as indicated by arrow 650. In embodiments where the test region changes colour at step 520 the second stage of step 510 may be carried out when or after the test region changes colour. The reagent solution 651 comprises the indicator molecules. In certain embodiments, the reagent solution 651 may further comprise an electron donor. The reagent solution 651 may be prevented from being incident on the first sub-region 630 or the second sub-region 640 by a barrier 670. The reagent solution 651 is incident on the test region. In an example, the reagent solution 651 may be incident on the battery at step 540, activating the battery and triggering a sequence of potentials applied to the electrode assembly. In another example, the second stage of step 510 comprises depositing a reagent solution 651 onto a third sub-region 660 of the sample receiving region 610, as indicated by arrow 650. The reagent solution 651 comprises indicator molecules. In certain embodiments, the reagent solution 651 may further comprise an electron donor. The reagent solution 651 is prevented from being incident on the first sub-region 630 or the second sub-region 640 of by a barrier 670. The reagent solution 651 is incident on the test region, but is not incident on the battery. A third stage 513 of step 510 comprises depositing an electrolyte solution on the battery, such that the battery is activated at step 540 and a sequence of potentials applied to the electrode assembly is triggered.

[0104] In certain embodiments, the detection reagent 641 may be in a dried form on the first subregion 630 of the sample receiving region 610, such that the buffer solution 622 releases the detection reagent 641 from the first sub-region 630, and the sample 621, buffer solution 622 and detection reagent 641 subsequently flow to the second sub-region 640. The capture molecules 631 may be in a dried form on the second sub-region 640. The buffer solution 622 releases the capture molecules 631 from the second sub-region 640. In other embodiments, the capture molecules and the detection reagent may be in dried form on the same sub-region of the sample receiving region. For example, the capture molecules and the detection reagent may be dried onto the same pad. The capture molecules and the detection reagent may be interspersed with one another, or may be dried onto adjacent sections of the pad.

[0105] In other embodiments, the reagent solution may be deposited onto the same sub-region of the sample receiving region as the sample. The reagent solution may be deposited with the sample. For example, the reagent solution may be mixed with the sample. The reagent solution may be deposited separately to the sample but onto the same sub-region of the sample receiving region as the sample.

[0106] At step 540, a solution may be deposited such that the solution is incident on the battery, wherein the solution comprises an electrolyte configured to activate the battery. In certain embodiments, the solution may be deposited on the battery. In other embodiments, a solution received by the sample receiving region may be diverted or split such that the solution is incident on the battery. The buffer solution or the reagent solution may be used to activate the battery. Steps 510 to 540 may occur in a different manner, depending on the architecture of the assay device. The result of steps 510 to 540 is that a complex including the analyte (if present) and the detection reagent is captured at the test line, the battery has been activated and the reagent solution 651 has reached the test region.

[0107] After a first period from activation of the battery, step 550 comprises applying a first potential to the electrode assembly. In certain embodiments, the first potential is applied in the presence of the electrode donor to produce hydrogen peroxide in the test region. For an electron acceptor A, hydrogen peroxide may be produced according to the following equations:

[0108] 2e“

[0109] Step 1) A + 2H+- > H2A

[0110] Step 2)

[0111] Kc is the rate constant for chemical step 2. The production of hydrogen peroxide may be precisely timed by applying the first potential to the electrode assembly after a first period from activation of the battery and for a particular duration.

[0112] Step 560 comprises generating an oxidised indicator molecule at the test region at step 560. The oxidised indicator molecule is produced via hydrogen peroxide oxidation of the indicator molecule in the presence of the catalytic agent. For an indicator molecule B and a catalytic agent C (wherein the detection reagent comprises the catalytic agent), the oxidised generator molecule Box may be generated according to the following equation: c Equation 3: BRD + B2O2>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. This reaction is carried out in the presence of the catalytic agent, and the quantity of Box produced is proportional to the quantity of catalytic agent present. If an analyte is present, the detection reagent comprising the catalytic agent is bound at the test line, as part of the complex including the analyte and the capture molecule. The quantity of detection reagent is directly proportional to the quantity of analyte (for example, one detection reagent molecule or conjugate may be bound to one analyte). The quantity of Box produced is therefore proportional to the quantity of analyte bound at the test line.

[0114] At step 570 a second potential is applied to the electrode assembly such that the oxidised indicator molecule is reduced, generating the indicator molecule and a charge transfer. The reduction may occur according to the following equation: e-1

[0115] Equation 4: Box- > BRD

[0116] The charge transfer is measured at step 580, wherein the charge transfer is indicative of the presence or absence of the analyte. The charge transfer is proportional to the of BRD produced according to Equation 4, which is proportional to the quantity of Box produced according to Equation 3 and is therefore proportional to the quantity of analyte bound at the test line. The measurement of charge transfer may therefore be used to detect the presence or absence of an analyte (for example by determining whether the charge transfer is above or below a threshold) and / or to quantify the analyte. As will be described, this may be carried out using the electrode assembly. In certain embodiment, steps 570 and 580 may occur simultaneously. Electrodes of the electrode assembly to which the second potential is applied may also be used to measure the charge transfer, such that the second potential is used for generating and measuring the charge transfer. At step 590 the method comprises outputting data indicative of the charge transfer.

[0117] 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. As described below, the electrode assembly may be in fluidic contact with the substrate so the electrode assembly is in a solution. The reference electrode may have a constant half-cell potential in the solution. The working electrode potential is held at that constant potential for a fixed time period, and the current flowing through the working electrode and counter electrode is measured. The measured current value (in Amps) may be multiplied by the time period (in seconds) to provide the total charge value in Coulombs used in the reduction reaction. The charge value is proportional to the amount of oxidised indicator molecule present during the reduction. The charge value is therefore proportional to the quantity of analyte bound at the test line. The reference electrode and the counter electrode may be the same electrode or different electrodes. In an event that the reference electrode is a separate electrode to the counter electrode, no current flows through the reference electrode as the circuit only measures its potential.

[0118] The assay device comprises a battery that is activated by liquid. In certain embodiments, once activated the battery is configured to provide power such that voltages can be applied to the electrode assembly, such as in steps 550 and 570. In certain embodiments, the battery is further configured to provide power such that the data indicative of charge transfer (output in step 590) is written to memory.

[0119] The electrode assembly may be positioned adjacent to the substrate. The electrode assembly may be positioned such that when the substrate is wet, the electrode assembly is in fluidic contact with the substrate. For example, the electrode assembly may be above or below the substrate. The electrode assembly may be planar, such that the electrode assembly is parallel to the substrate. Examples of electrode assemblies will be described with reference to Figures 7 and 8, although other electrode assemblies are also possible. In Figures 7 and 8 the position of the test line is indicated by a white rectangle overlaid on the electrode assembly. This is merely indicative of the position of the test line relative to the electrodes, and is not intended to be precise or to scale. The shapes of the electrodes in Figures 7 and 8 are examples only.

[0120] Figure 7A shows an electrode assembly 710 with a position of the test line indicated by rectangle 711. The electrode assembly 710 comprises a counter electrode 712 and a first working electrode 713. The test line 711 is aligned with the first working electrode 713. The first potential Vi is applied between the first working electrode 713 and the counter electrode 712. The second potential V2 is applied between the first working electrode 713 and the counter electrode 712.

[0121] Figure 7B shows an electrode assembly 720 with a position of the test line indicated by rectangle 721. The electrode assembly 720 comprises a counter electrode 722, a first working electrode 723 and a second working electrode 724. The test line 721 is aligned with the first working electrode 723. The first potential Vi is applied between the second working electrode 724 and the counter electrode 722. The second potential V2 is applied between the first working electrode 723 and the counter electrode 722. The first working electrode 723 may be positioned adjacent to the counter electrode 722. As shown in Figure 7B, the counter electrode 722 may be L-shaped such that the counter electrode 722 is adjacent to both the first working electrode 723 and to the second working electrode 724. The first working electrode 723, and therefore the position of the test line, may be between the counter electrode 722 and the second working electrode 724.

[0122] Figure 7C shows an electrode assembly 730 with a position of the test line indicated by rectangle 731. The electrode assembly 730 comprises a counter electrode 732, a first working electrode 733, a second working electrode 734 and a third working electrode 735. The test line 731 is aligned with the first working electrode 733. The first potential Vi is applied between the second working electrode 734 and the third working electrode 735. The second potential V2 is applied between the first working electrode 733 and the counter electrode 732. The first working electrode 733 may be positioned adjacent to the counter electrode 732. The second working electrode 734 may be positioned adjacent to the third working electrode 735. The first working electrode 733, and therefore the position of the test line, may be between the counter electrode 732 and the second and third working electrodes 734, 735.

[0123] Figure 7D shows an electrode assembly 740 with a position of the test line indicated by rectangle 741. The electrode assembly 740 comprises a counter electrode 742, a first working electrode 743 and a reference electrode 744. The test line 741 is aligned with the first working electrode 743. The first potential Vi is applied between the first working electrode 743 and the counter electrode 742. The second potential V2 is applied between the first working electrode 743 and the counter electrode 742, referenced to the second working electrode 744. The first working electrode 743 may be positioned adjacent to the counter electrode 742. The first working electrode 743, and the test line, may be between the counter electrode 742 and the reference electrode 744.

[0124] Figure 8A shows an electrode assembly 810 with a position of the test line indicated by rectangle 811. The electrode assembly 810 comprises a counter electrode 812, a first working electrode 813 and a reference electrode 814. The test line 811 is aligned with the first working electrode 813. The first potential Vi is applied between the first working electrode 813 and the counter electrode 812. The second potential V2A is applied between the first working electrode 813 and the counter electrode 812. The second potential V2B is subsequently also applied between the reference electrode 814 and the counter electrode 812 to provide a control value. The first working electrode 813 may be positioned adjacent to the counter electrode 812. As shown in Figure 8A, the counter electrode 812 may be L- shaped such that the reference electrode 814 is adjacent to the counter electrode 812 and the first working electrode 813 is between the counter electrode 812 and the reference electrode 814.

[0125] Figure 8B shows an electrode assembly 820 with a position of the test line indicated by rectangle 821. The electrode assembly 820 comprises a counter electrode 822, a first working electrode 823, a first reference electrode 824 and a second reference electrode 825. The test line 821 is aligned with the first working electrode 823. The first potential Vi is applied between the first working electrode 823 and the counter electrode 822. The second potential V2A is applied between the first working electrode 823 and the counter electrode 822. The second potential V2B is subsequently also applied between the first reference electrode 824 and the counter electrode 822 to provide a first control value. The second potential V2C is subsequently also applied between the second reference electrode 825 and the counter electrode 822 to provide a second control value. The first working electrode 823 may be adjacent to the counter electrode 822. As shown in Figure 8B, the first working electrode may also be between the first reference electrode 824 and the second reference electrode 825. The first reference electrode 824 and the second reference electrode 825 may be adjacent to the counter electrode 822.

[0126] As shown in Figures 7 to 8, the test line at which the analyte is captured may be aligned with the first working electrode. The test line may also overlap the counter electrode.

[0127] The hydrogen peroxide generation may occur upstream of the test line at which the analyte, where present, is captured, such that the generated hydrogen peroxide flows to the bound analyte.

[0128] Fluidic contact between the electrode assembly and the substrate may be achieved in any appropriate way. In certain embodiments, an electrode sheet comprising the electrode assembly may be brought into contact with the substrate such that when the substrate is wet, the electrode assembly is in fluidic contact with the substrate. The electrode assembly may be held in place to maintain fluidic contact. 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 assembly may be connected to the 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 substrate, the electrical contacts may align with conductive tracks on the substrate or a base board. The pressure applied to keep the electrode assembly in fluidic contact with the substrate may also be used to maintain an electrical connection between the electrical contacts and the conductive tracks.

[0129] In other embodiments, the electrode assembly may be printed onto a base board. The base board may be coated with the substrate material, such that good fluidic contact is achieved. The electrode assembly may be connected permanently to conductive tracks on the base board.

[0130] In certain embodiments, the electrode assembly may be screen printed onto a base, such as a polymer base. In certain embodiments, the electrode assembly may be fabricated on a PCB. The electrodes of the electrode assembly may be composed of one or more of carbon, silver, platinum and gold. For example, the counter electrode may be silver. The working electrode(s) may be carbon. A gap between adjacent electrodes may be large enough to ensure that the adjacent electrodes do not short together during the manufacturing process. The gap between the adjacent electrodes may be small enough such that a potential applied at the counter electrode to maintain a potential at the working electrode is below a threshold potential. The gap between the adjacent electrodes may be small enough such that noise is below a threshold. In certain embodiments, a gap between two electrodes between which a potential is applied may be between 0.1 mm and 1 mm.

[0131] In certain embodiments, the solution deposited at the sample receiving region may be split, with part of the solution being diverted to activate the battery. With reference to Figure 9, the solution may be deposited via sample port 910 of an electronica assay device 900. The solution and the sample may travel down the assay strip 920, comprising the test region. Part of the solution may be diverted down an activation fluid channel 930 such that that part of the solution is incident on the battery 940. The activation fluid channel 930 may be filled with a porous material.

[0132] In certain embodiments, the detection reagent may comprise a molecular binding agent labelled with redox active molecules or structures. The redox active molecules or structures are the catalytic agent for the reaction in which the hydrogen peroxide (H2O2) oxidises the indicator molecule. The redox active molecules or structures may comprise the enzyme horse-radish peroxidase, or catalytic platinum nanoparticles. The molecular binding reagent is configured to bind to the analyte, and may comprise an antibody. For example, the molecular binding reagent may comprise Biotin. In certain embodiments, the test region may comprise polystreptavidin configured to bind to Biotin.

[0133] In certain embodiments, the electron donor may comprise flavin mononucleotide, a quinone or a derivative of a quinone. Flavin mononucleotide is photosensitive. Quinones (Q) have the further advantage of being cheaper and produce hydrogen peroxide efficiently. The production of hydrogen peroxide follows a 2 electron electrochemical reduction, as follows:

[0134] Examples of suitable quinones are 2-hydroxy-1 ,4-naphthoquinone, 2- meth oxy- 1,4- naphthoquinone, 9,10-phenanthrenequinone, 9,10-anthraquinone, 1,4-naphthoquinone, 1 ,2-Naphthoquinone, 2-methoxy-1 ,4-naphthoquinone, 5,8-Dihydroxy-1 ,4-naphthoquinone and 1 ,4-dihydroxyanthraquinone.

[0135] A redox mediator such as quinone or other electron donor can lower the size of the potential required for the reaction to take place (i.e. a less negative potential is required). However, it is also possible to use a larger (more negative) potential to directly reduce dissolved oxygen at the electrode to produce hydrogen peroxide, without the presence of a redox mediator such as quinone. This may be advantageous, as excess quinone may reduce the oxidised indicator molecule. However, the higher potentials required to produce hydrogen peroxide directly may further reduce the hydrogen peroxide to water. Precise control of the potential may be required.

[0136] In certain embodiments, the indicator molecule may comprise one of glucose oxidase and 3,3’,5,5’-tetramethyl benzidine (TMB).

[0137] In certain embodiments, the indicator molecule comprises 3,3’,5,5’-tetramethyl benzidine.

[0138] The oxidation of the indicator molecule by the catalytic agent (in this example Pt nanoparticles) may occur according to the following equation: pt

[0139] TMB + H2O2- > TMB0X

[0140] TMBox is blue, so there may be a visual indication that this oxidation has occurred. The reduction of the oxidised indicator molecule, which takes place when the second potential is applied, may occur as follows:

[0141] The indicator molecule may already be present when the production of hydrogen peroxide takes place, so it is important that no unwanted side reactions occur when the production of hydrogen peroxide takes place in the present of the indicator molecule. When TMB is used as an indicator molecule, using 2-hydroxy-1,4-naphthoquinone (Q2) for the electron donor provides a good current density, without overlapping peaks of TMB during signal generation (when the second potential is applied).

[0142] In a specific example, where the catalytic agent comprises Platinum nanoparticles, the electron donor comprises a Quinone and the indicator molecule comprises TMB, the first potential may be -1.0 V. The second potential may be -0.1 V.

[0143] Platinum nanoparticles are beneficial for use as the catalytic agent. Platinum nanoparticles have peroxidase-like properties and catalyze the oxidation of TMB by H2O2, yielding a blue product. Platinum nanoparticles may have higher catalytic efficiency than horseradish peroxidase. Platinum nanoparticles may be more stable than natural enzymes, and can be made of high purity with controlled sizes. The surfaces of the nanoparticles can be easily modified with functional groups (such as COOH and -NH2) for conventional labelling of biomolecules and ligands.

[0144] In certain embodiments, the substrate may comprise a nitrocellulose membrane.

[0145] As described above, the battery is liquid activated and may be required to power the electrodes and to write data to memory. The battery may be single use. In certain embodiments, the battery may comprise a magnesium (Mg) anode and a silver chloride (AgCI) cathode, separated by a porous separator. The anode and cathode may be planar. When electrolyte is added to the separator, Mg is consumed and AgCI is converted to Ag and a voltage is generated. The Mg anode may be a foil cathode, and the AgCI anode may be screen printed. Copper(l) chloride may be used instead of AgCl. In certain embodiments, the battery may be provided between two layers of Kapton.

[0146] In certain embodiments, the output of the data indicative of the charge transfer may comprise writing the data to memory. In certain embodiment, data indicative of the charge transfer may be communicated to a user. For example, the data may be transmitted to a device, such as via near-field communication components or antenna. The data may be transmitted to a smart-phone or other device. The assay device may comprise an LED or other light or electronic visual indicator to indicate presence or absence of the analyte.

[0147] In certain embodiments, a potentiostat may be used to control the electrode assembly and measure the charge transfer. The assay device may comprise a micro-chip capable of running key assay functions such as the potentiostat, voltage switching, and signal amplification. The potentiostat may be configured to use a specific current measurement range. The potentiostat may be fabricated on a PCB board. In certain embodiments, the potentiostat may comprise a microcontroller with analogue capability, a quad operational amplifier (op-amp) and an NFC chip. The data indicative of the charge transfer may be transferred to the NFC chip and stored in non-volatile memory. After this has occurred, the battery is no longer needed. The NFC chip may then be powered up using an antenna, such as a mobile phone NFC antenna, and the data may be transferred to an external memory such as the phone memory.

[0148] The charge transfer may be measured using chronoamperometry. The process may be performed by a potentiostat circuit. A potential may be applied to a working electrode and a counter electrode such that the working electrode potential at the working electrode is controlled to be a constant specific potential value as measured against a reference electrode. The reference electrode may have a constant half-cell potential in the solution. The potential is held at that specific potential value for a fixed time and the current flowing through the working and counter electrodes is measured. No current may flow through the reference electrode as the circuit may only measure its potential. The value of the current in Amperes is multiplied by the time in seconds to give the total charge in Coulombs used in the reduction reaction. This charge value is proportional to the concentration of the oxidised indicator molecule present. In certain embodiments, the counter electrode may be used as the reference electrode. In certain embodiments, an electronic assay device may 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 dissolved 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 test region at a pre-determined time after the solution was deposited. The solution may be configured to activate the battery at a pre-determined time after the solution was deposited.

[0149] With reference to Figure 10, an electronic assay device 1000 may comprise a sample receiving region 1010 comprising a first sub-region 1020, configured to receive the sample as indicated by arrow 1021. The first sub-region 1020 is further configured to receive a buffer solution, indicated by arrow 1022. The buffer solution may release capture molecules and detection reagent present in the sample receiving region (in a pad 1030, for example). In use, the buffer solution and sample travel via the substrate 1060 to the test region 1070. The sample receiving region 1010 may further comprise a second sub-region 1040, configured to receive a signal activation fluid, as indicated by arrow 1041. In use, the signal activation fluid may be to be incident on the battery. The second sub-region 1040 may comprise a porous pad, positioned above a gelatin film barrier 1050. The signal activation fluid may be prevented from entering the substrate 1060 or travelling along the substrate 1060 by the gelatin film barrier 1050. The porous pad of the second sub-region 1040 may comprise gelatinase, glucose and indicator molecules (such as TMB) in a dried state. In use, depositing the signal activation fluid rehydrates the gelatinase. The rehydrated gelatinase begins to dissolve the gelatin film barrier 1050. The gelatin film barrier 1050 dissolves after a pre-determined time period (controlled, for example, by the thickness of the gelatin film barrier 1050). After the pre-determined time period, the gelatin film barrier 1050 ruptures and the signal activation fluid passes along the substrate 1060, carrying glucose and the indicator molecules. The test region may comprise glucose oxidase (GOx) in an immobilised state. When the glucose reaches the GOx, hydrogen peroxide is generated. The electrode assembly 1090 is shown below the substrate 1060, but may be located above the substrate 1060. The electronic assay device 1000 may further comprise a sump or sink pad 1080.

[0150] With reference to Figure 11, the test region 1070 of the electronic assay device 1000 shown in Figure 10 may comprise a generator line 1071, a test line 1072 and a control line 1073. The generator line may comprise the immobilised GOx. When the hydrogen peroxide is generated, the hydrogen peroxide flows to the test line and the control line. The control line 1073 may comprise a control antibody in the control line 1073. The bound analyte of the sample, where present, binds to the test line. The electrode assembly may comprise a reference electrode 1091, a first working electrode 1092 and a second working electrode 1093. These are illustrated in Figure 11 as being directly below the substrate 1060, but the electrode assembly may instead be located above the substrate 1060.

Claims

CLAIMS:

1. A method of detecting the presence or absence of an analyte in a sample using an electronic assay device, wherein the electronic assay device is configured to output an electronic signal indicative of the presence of an analyte in a sample and wherein the electronic assay device comprises: a sample receiving region; a detection reagent comprising a catalytic agent; capture molecules; a test region comprising an electrode assembly and a test line configured to bind to the capture molecules; and a battery configured to be activated by contact with a liquid; the method comprising: depositing a sample and a solution on the sample receiving region, such that: the analyte, where present, binds to the detection reagent via a first binding site of the analyte and binds to the capture molecule via a second binding site of the analyte; the solution is incident on the battery, activating the battery and triggering a sequence of potentials applied to the electrode assembly; and the bound analyte, where present, and the solution are incident on the test region, wherein the solution carries indicator molecules to the test region; after a first period of time from activation of the battery, applying a first potential to the electrode assembly to produce hydrogen peroxide in the test region; generating oxidised indicator molecules at the test region, wherein the oxidised indicator molecules are produced via hydrogen peroxide oxidation of the indicator molecules in the presence of the catalytic agent; after a second period of time from activation of the battery, applying a second potential to the electrode assembly such that the oxidised indicator molecules are reduced, generating the indicator molecules and a charge transfer; measuring the charge transfer, wherein the charge transfer is indicative of the presence or absence of the analyte; and outputting data indicative of the charge transfer.

2. The method of claim 1, wherein the solution comprises the indicator molecules.

3. The method of claim 1 or 2 wherein the first potential is applied to the electrode assembly in the presence of an electron donor.

4. The method of claim 3 wherein either: the solution comprises the electron donor; or the electrode assembly comprises the electron donor.

5. The method of any of claims 1 to 4, wherein the detection reagent is in a dried state on the electronic assay device and wherein depositing the solution releases the detection reagent.

6. The method of any preceding claim, wherein the capture molecule is in a dried state on the electronic assay device and wherein depositing the solution releases the capture molecule.

7. The method of any preceding claim wherein the capture molecule binds to the test line via an intermediate molecule.

8. The method of any preceding claim, wherein the sample receiving region comprises: a first region configured to receive the sample and a buffer solution; and a second region, configured to receive a reagent solution comprising the electron donor and the indicator molecule.

9. The method of claim 8, wherein the method comprises depositing the sample and a buffer solution on the first region and depositing the reagent solution on the second region.

10. The method of claim 8 or 9, wherein the first region comprises the capture molecule and wherein depositing the buffer onto the first region releases the capture molecule.

11. The method of any of claims 8 to 10, wherein the first region comprises the detection reagent and wherein depositing the buffer onto the first region releases the detection reagent.

12. The method of claim 11 wherein the first region comprises a first pad comprising the capture molecule and a second pad comprising the detection reagent, wherein the method comprises depositing the sample and solution on the first pad and wherein the sample and solution subsequently flow through the second pad.

13. The method of any preceding claim, wherein the electrode assembly comprises a first working electrode and a counter electrode, wherein the second potential is applied between the first working electrode and the counter electrode.

14. The method of claim 13 wherein the first potential is applied between the first working electrode and the counter electrode.

15. The method of claim 13 wherein the electrode assembly further comprises a second working electrode, wherein the first potential is applied between the second working electrode and the counter electrode.

16. The method of claim 13 wherein the electrode assembly further comprises a second working electrode and a third working electrode, wherein the first potential is applied between the second working electrode and the third working electrode.

17. The method of claim 16 wherein the first working electrode is adjacent to the counter electrode, and the second working electrode is adjacent to the third working electrode.

18. The method of claim 13, wherein the electrode assembly further comprises a first reference electrode.

19. The method of claim 18 wherein the first potential is applied between the first working electrode and the counter electrode, and wherein the second potential is referenced to the reference electrode.

20. The method of claim 18 wherein the first working potential is applied between the first working electrode and the second working electrode and wherein the second potential is applied: between the first working electrode and the counter electrode; and subsequently between the reference electrode and the counter electrode.

21. The method of claim 18 wherein the electrode assembly further comprises a second reference electrode, wherein the first potential is applied between the first working electrode and the counter electrode and wherein the second potential is applied: between the first working electrode and the counter electrode; subsequently between the first reference electrode and the counter electrode; and subsequently between the second reference electrode and the counter electrode.

22. The method of claim 21 wherein the first working electrode is between the first reference electrode and the second reference electrode.

23. The method of any of claims 13 to 22 wherein the test region is aligned with the first working electrode.

24. The method of any of claims 13 to 23 wherein the test region intersects with the counter electrode.

25. The method of any of claims 13 to 24 wherein the counter electrode is shaped such that the counter electrode is adjacent to each other electrode of the electrode assembly.

26. The method of any preceding claim wherein in an event that an analyte is present, the data indicative of charge transfer is further indicative of a concentration of the analyte such that the method further comprises determining the concentration of the analyte.

27. The method of any preceding claim wherein the detection reagent comprises a molecular binding reagent labelled with the catalytic agent.

28. The method of any preceding claim wherein the catalytic agent comprises catalytic platinum nanoparticles.

29. The method of any of claims 1 to 27 wherein the catalytic agent comprises horseradish peroxidase.

30. The method of any preceding claim wherein the capture molecule comprises Biotin- Ab1.

31. The method of any preceding claim wherein the electron donor comprises a quinone.

32. The method of any of claims 1 to 30 wherein the electron donor comprises flavin mononucleotide.

33. The method of any preceding claim wherein the indicator molecule comprises 3,3',5,5'-Tetramethylbenzidine, TMB.

34. The method of any of claims 7 to 33 wherein the intermediate molecule comprises Polystreptavidin, PSA.

35. An electronic assay device configured to output an electronic signal indicative of the presence of an analyte in a sample, wherein the electronic assay device comprises: a detection reagent comprising a catalytic agent configured to bind to a first binding site of the analyte, where present; capture molecules configured to bind to a second binding site of the analyte, where present; a test region comprising an electrode assembly and a test line configured to bind to the capture molecule; a battery configured to be activated by contact with a liquid; a sample receiving region configured to receive a sample and a solution such that the solution is incident on the test region and on the battery; and a microprocessor configured to:initiate a sequence of potentials applied to the electrode assembly upon activation of the battery; after a first period of time from activation of the battery, apply a first potential to the electrode assembly to produce hydrogen peroxide in the test region, wherein the hydrogen peroxide oxidises indicator molecules in the presence of the catalytic agent to produce oxidised indicator molecules, wherein the indicator molecules are carried to the test region by the solution; after a second period of time from activation of the battery, apply a second potential to the electrode assembly such that the oxidised indicator molecules are reduced, generating the indicator molecules and a charge transfer; measure the charge transfer, wherein the charge transfer is indicative of the presence or absence of the analyte; and output data indicative of the charge transfer.

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