Methods and fuel cell devices for detecting analytes

Abstract

The present invention concerns methods, test strips, systems and kits of parts suitable for detecting the presence of analytes within samples through the use of fuel cells. Use of the test strips, systems and kits in detecting the presence or absence of a target in a sample is also included. When target analytes are present in the sample, sandwich complexes are formed comprising the analyte, a magnetically moveable particle and a detectable species, such as a catalyst or redox mediator. Complexes are separated from other components of the sample by magnetic separation and detected as a response or change in response from a fuel cell.

Description

[0001] METHODS AND FUEL CELL DEVICES FOR DETECTING ANALYTES

[0002] FIELD

[0003] The present invention concerns methods, test strips, systems and kits of parts suitable for detecting the presence of analytes within samples through the use of fuel cells. Use of the test strips, systems and kits in detecting the presence or absence of a target in a sample is also included. When target analytes are present in the sample, sandwich complexes are formed comprising the analyte, a magnetically moveable particle and a detectable species, such as a catalyst or redox mediator. Complexes are separated from other components of the sample by magnetic separation and detected as a response or change in response from a fuel cell.

[0004] BACKGROUND

[0005] High sensitivity immunoassays are a crucial tool in the point of care diagnostics industry. The need for high sensitivity immunoassays is increasing as new and clinically important biomarkers are identified, many of which are present in very low concentrations. Lab based high sensitivity immunoassays exist but typically require numerous assay steps which need to be carried out by trained laboratory technicians. Many immunoassays incorporate labelled reagents conjugated on to antibodies which bind specifically to target antigens.

[0006] Enzyme-linked immunosorbent assays (ELISA) are used to detect the presence of an analyte, such as an antigen, in a sample through the use of probes (such as antibodies) that bind to the analyte. The analyte is immobilised on a solid surface to which the probe is applied such that the probe binds to the analyte. The probe is typically linked to a redox catalyst such as an enzyme. Thus, when analyte is present, a complex comprising analyte, probe and redox catalyst is formed. Any unbound probe is removed from the assay by washing. Then, a substrate of the redox catalyst is added to the assay such that, when analyte is present, the redox catalyst of the complex formed is able to act on the substrate and catalyse its conversion to another product. This conversion reaction may be detectable, for example, by a change in colour, light emission or a change in or production of an electrical signal.

[0007] Sandwich ELISA is a type of ELISA in which the analyte is not itself directly immobilised on a surface. Rather, an additional probe (further to that bonded to a redox catalyst), such as another antigen, is used to bind to a different position on the analyte and immobilise the analyte on a surface. The result is the formation of a sandwich complex comprising the immobilisable probe, the target analyte and the probe bonded to a redox catalyst. Again, any unbound probe is removed from the assay by washing before addition of the substrate of the redox catalyst and, when analyte is present, the redox catalyst of the sandwich complex formed is able to act on the substrate and catalyse its conversion to another product. See D. Neupane and K. J. Stine, Appl. Sci., 2021, 11, 7087 for a review of electrochemical sandwich assays for biomarkers incorporating aptamers, antibodies and nanomaterials for detection of specific protein biomarkers.

[0008] These assays require wash steps to remove any unbound labelled antibody so that after the wash step the only redox catalyst that remains is that bonded to a probe which is bonded to the captured analyte (i.e. that forming part of a sandwich complex with the target analyte). In this way, a signal can be generated which is proportional to the amount of analyte present in the sample. Many products are currently under development which attempt to automate the assay approach, often by carrying out an automated wash step. Such automated wash steps require complex and expensive equipment and often require special wash solutions. These existing automated products are expensive, complex and have multiple potential failure modes. Expensive instrumentation in turn means that such point of care solutions cannot be provided to users at low cost and this limits product adoption.

[0009] In WO 2010 / 004241 (The Secretary of State for Innovation Universities & Skills of her Majesty’s Britannic Government), an assay is described in which an antigen becomes attached to a carrier device, a magnetic particle and to a silver sol particle. It is described that the antigens can be made to move, for example by application of a magnetic field, and can then be detected using the silver of the label. In WO 2020 / 032294 A1 (BBB Inc.), a further example of a biosensor using magnetic nanoparticles is described.

[0010] In WO 2007 / 010368 (Inverness Medical Switzerland GmbH), an assay device is described including a first reagent including a magnetic particle and a second reagent including a detectable component. The first and second reagent can each independently bind to an analyte in a sample and it is described that applying a magnetic field can selectively concentrate the detectable component in a detection zone.

[0011] In US 10,509,032 B (Alere Switzerland GmbH), an assay method and device are described. The device is configured to create a sample liquid-air interface with the sample liquid and magnetically susceptible particles can be located (via an applied magnetic field) at the liquid-air interface when a second liquid contacts the interface to form a liquid-liquid interface. The magnetically susceptible particles are configured to transport an analyte across the interface into the second liquid.

[0012] In US 2008 / 0160634 A 1 (Intel Corp), a device for detecting an analyte in a sample is described. The device comprises a fluidic network and an integrated circuitry component. The fluidic network comprises multiple zones such as a sample zone, a cleaning zone and a detection zone. The fluidic network contains a magnetic particle and / or a signal particle. A sample containing an analyte is introduced, and the analyte interacts with the magnetic particle and / or the signal particle through affinity agents. A microcoil array or a mechanically movable permanent magnet is functionally coupled to the fluidic network, which are activatable to generate a magnetic field within a portion of the fluidic network, and move the magnetic particle from the sample zone to the detection zone. A detection element is present which detects optical or electrical signals from the signal particle, thus indicating the presence of the analyte.

[0013] In PCT / GB2024 / 051473 (Aureum Diagnostics Limited), methods, systems and kits of parts suitable for detecting the presence of analytes within samples are described. The analytes are described as forming sandwich complexes comprising the analyte, a magnetically moveable particle and a catalyst. Complexes are separated from other components of the sample by magnetic separation and detected. The focus of the application is on detection of the analyte using an electrode set up.

[0014] Fuel cells convert chemical energy into electrical energy. An enzymatic biofuel cell (EBFC) is a fuel cell that utilises enzymes as electrocatalysts and molecules from living systems such as glucose or lactate as fuels. The most common EBFC configuration comprises an anode and cathode connected via a salt bridge or separated by a membrane selectively permeable to specific ions. A glucose-consuming enzyme such as glucose oxidase or glucose dehydrogenase is commonly deployed at the anode and an oxygen-consuming enzyme such as Laccase is commonly employed at the cathode. For a review of EBFCs, see N. Karim, “Mini-Review: Recent Technologies of Electrode and System in the Enzymatic Biofuel Cell (EBFC)”, Appl. Sci., 2021, 11, 5197; or R. A. S. Luz et al., “Enzyme Biofuel Cells: Thermodynamics, Kinetics and Challenges in Applicability”, ChemElectroChem, 2014, 1, 11, 1751-1777).

[0015] The efficiency of charge generation in EBFCs can be improved by direct electron transfer (DET) from the enzyme to the electrode. The use of polymeric and conductive films to facilitate long range electron transfer through the surface of devices is described in C. Bunte et al., “Enzyme Containing Redox Polymer Networks for Biosensors or Biofuel Cells: A Photochemical Approach”, Langmuir, 2010, 26, 8, 6019-6027, and the use of nanomaterials (in particular carbon nanomaterials) to directly ‘wire’ the electrode to the active sites of redox enzymes is described in L. Cao et al., “Research Progress in Enzyme Biofuel Cells Modified Using Nanomaterials and Their Implementation as Self-Powered Sensors”, Molecules, 2024, 29, 1 , 257.

[0016] EBFCs are known for use in biosensing. For example, Y. Song et al., in “Multiple signal amplification strategy induced by biomarkers of lung cancer: A self-powered biosensing platform adapted for smartphones”, International Journal of Biological Macromolecules, 2024, 264, 2, 130661, describe a DNA-mediated dual strand displacement assay is used to obtain a signal from an EBFC in order to detect micro RNA. Magnetic beads comprising two types of double-stranded DNA are used for chain displacement reactions, which lead to signal amplification. R. Cai et al., in “Creation of a point-of-care therapeutics sensor using protein engineering, electrochemical sensing and electronic integration”, Nature Communications, 2024, 15, 1689, describe an engineered GDH-PQQ, which is capable of detecting tamoxifen in an EBFC configuration. Finally, D. Galyamin et al., in “Immuno-battery: A single use self-powered immunosensor for REASSURED diagnostics”, Biosensors and Bioelectronics, 2023, 220, 114868, describe a self-powered paper-based immunosensor that generates energy in the presence of the biomarker in the sample. The anode and cathode of the immune-battery are pinned together and magnets are used to help keep the resultant device sealed.

[0017] Many of the assays and devices described above suffer from one or more drawbacks including low sensitivity (e.g. due to high background signal), multiple fluidic chambers, wash steps, complex and / or expensive test strip designs and / or the need for relatively large volumes of sample. There remains a need for low cost high sensitivity immunoassays that deliver a high level of target signal amplification made possible, for example, by enzyme amplification, but that do not require a wash step and do not require an external power source. The immunoassays are preferably easy to use and reliably produced at low cost and high volume. The present invention provides alternative methods, systems and kits to address one or more of these needs and the above-noted problems and / or limitations.

[0018] SUMMARY

[0019] The present invention is based on the finding that a test strip comprising a fuel cell may be used to detect a target, such as an analyte, in a sample. In particular, by using the devices and methods described herein, the target may be detected in a sandwich ELISA-type process, wherein detection is by the production of a charge or an increase in the production of a charge from a fuel cell. Accordingly, an external power source is not required for detection of the target. The result is an effective immunoassay on a low-cost test strip, with a high sensitivity that enables the assay to operate on, for example, a blood sample. In some cases, the method may not require any washing steps.

[0020] When present, the target is detected as part of a sandwich complex comprising the target, a target binding moiety tethered to a magnetically moveable particle, and a target capture moiety conjugated to a detectable species, such as a catalyst or a redox mediator. The resulting sandwich complex is movable to and immobilisable at a second position on or within a test strip by activating or generating (e.g. applying) a magnetic field. When magnetically moved to the second position, the complex is separated from unbound target capture moiety conjugated to a detectable species such that the only detectable species present at the second position is that which is conjugated to a target capture moiety that is bonded to a target.

[0021] The ability to selectively move the complex to the second position may avoid or minimise the need for wash steps. By positioning any additional reagents needed for spontaneous amplification of a reaction modulated by the detectable species at only the second position, any signal generated by the detectable species is restricted to those species which are bound as part of a complex. For example, where the detectable species is a catalyst, an excess of catalyst substrate and optionally also a redox mediator may be positioned at the second position. Alternatively, where the detectable species is a redox mediator, an excess of catalyst and catalyst substrate may be positioned at the second position. The signal detected is a response (e.g. an electrochemical or electrical response) from a fuel cell and is generated in some way (e.g. directly or indirectly) by the detectable species.

[0022] The fuel cell is typically set up such that one of the anode and cathode is supplied with the fuel needed to generate or consume electrons. This typically comprises a catalyst, the catalyst’s substrate and optionally a redox mediator. The other of the anode and cathode is typically absent at least one of the components required for effective charge generation, such as the catalyst or a redox mediator, and this is made available on movement of the complex to the second position. This means that, prior to movement of the complex to the second position, the fuel cell produces either no response, or only a small background response. Movement of the complex to the second position allows the other of the anode and cathode to either generate charge or generate charge more effectively, leading to a response or an increased response from the fuel cell.

[0023] After the complex has been moved to the second position, it may be immediately detected with amplified signal. This is because once the detectable species has moved to the second position, it is able to immediately turn the fuel cell on, or increase the response from the fuel cell, by undergoing multiple response-producing chemical reactions. For example, when the detectable species is a first catalyst, and an excess of its substrate is positioned at or in proximity to the second position, the first catalyst is able to immediately act on its substrate once the first catalyst is moved (as part of a complex) to the second position. In other words, and without being bound by theory, the inventors believe that by positioning suitable amplification reagents at or in proximity to the second position (and, for example, not elsewhere on the test strip), conditions can be created which can allow spontaneous catalytic (e.g. enzymatic) signal amplification to occur and to be detected as a response in a fuel cell. In other words, once the detectable species reaches the electrode area, it can start to convert substrate and generate signal.

[0024] Movement of the complex to the second position may allow the detectable species to chemically react with one or more other species to produce one or more products, and this process may produce or consume electrons, leading to movement of electrons into the anode of the fuel cell or movement of electrons out of the cathode of the fuel cell. In some cases, a signal may be produced by chemical reaction of the one or more products (or products derived from the one or more products) with one or more other species, leading to the production or consumption of electrons and a response from the fuel cell.

[0025] Accordingly, viewed from a first aspect, there is provided a method of detecting a target in a sample, the method comprising:

[0026] (i) contacting a mixture on or within a test strip, the mixture comprising:

[0027] (a) a target binding moiety tethered to a magnetically moveable particle;

[0028] (b) a target capture moiety conjugated to a detectable species; and

[0029] (c) a sample,

[0030] wherein component (b) is positioned on or within the test strip at a first position, and the test strip further comprises:

[0031] (d) a fuel cell comprising an anode and a cathode, wherein one of the anode or cathode is positioned at a second position; (ii) incubating (a), (b) and (c) for a period of time such that, when the target is present, a complex is formed, the complex comprising components (a) and (b) each independently bound to the target;

[0032] (iii) activating or generating a magnetic field to cause the complex to move to the second position such that the complex is separated from one or more other components of the mixture and such that the movement of the detectable species to the second position turns the fuel cell on or increases a response from the fuel cell; and

[0033] (iv) detecting a response from the fuel cell.

[0034] Viewed from a second aspect, there is provided a test strip suitable for use in the method of the first aspect. Specifically, a test strip comprising:

[0035] (i) a sample chamber positioned on or within the test strip;

[0036] (ii) a target capture moiety conjugated to a detectable species positioned on or within the sample chamber at a first position;

[0037] (iii) a fuel cell comprising an anode and a cathode, wherein one of the anode or cathode is positioned at a second position and the other is positioned at a third position;

[0038] optionally wherein the one of the anode and cathode positioned at the second position is configured for fluid communication with the sample chamber and the other of the anode and cathode positioned at the third position is not in fluid communication with the sample chamber.

[0039] Viewed from a third aspect, there is provided a system suitable for use in the method of the first aspect. Specifically, there is provided a system comprising:

[0040] (a) a test strip as defined in the second aspect; and

[0041] (b) a target binding moiety tethered to a magnetically moveable particle.

[0042] Viewed from a fourth aspect, there is provided a kit of parts, comprising, as separate components:

[0043] (a) a system as defined in the third aspect; and

[0044] (b) an electromagnet positionable such that activation of the electromagnet generates a magnetic field capable of moving the magnetically moveable particle to the second position or a permanent magnet positionable such that a magnetic field capable of moving the magnetically moveable particle to the second position is generated. Viewed from a fifth aspect, is provided the use of the test strip of the second aspect, the system of the third aspect, or the kit of the fourth aspect to detect the presence or absence of a target in a sample. In some embodiments of any of the above aspects, the detectable species may comprise:

[0045] a first catalyst and the test strip further comprises:

[0046] (e) a first catalyst substrate positioned at or in proximity to the second position; and

[0047] (f) a second catalyst and a second catalyst substrate , each positioned at or in proximity to a third position, wherein the third position is at the other of the anode and cathode; and

[0048] wherein movement of the first catalyst to the second position turns the fuel cell on.

[0049] In alternative embodiments of any of the above aspects, the detectable species may comprise:

[0050] a redox mediator and the test strip further comprises:

[0051] (e) a first catalyst and a first catalyst substrate positioned at or in proximity to the second position; and

[0052] (f) a second catalyst and a second catalyst substrate, each positioned at or in proximity to a third position, wherein the third position is at the other of the anode and cathode; and

[0053] wherein movement of the first catalyst to the second position increases an electrical response from the fuel cell.

[0054] In some embodiments of any of the above, the fuel cell may be a biofuel cell, in particular an enzymatic biofuel cell. Where the fuel cell is an enzymatic biofuel cell: the first catalyst may be a first enzyme; the first catalyst substrate may be a first enzyme substrate; the second catalyst may be a second enzyme; and the second catalyst substrate may be a second enzyme substrate.

[0055] BRIEF DESCRIPTION OF THE FIGURES

[0056] Fig. 1 is a scatter graph of charge density as a function of the pH of the C-P buffer, including error bars and a polynomial fit line. Each measurement used 2.5 mg / mLABTS with 2.5 mg / mL LacAB in C-P buffers which range from pH 3-7. There was a 5 minute incubation at room temperature before each CA measurement.

[0057] Fig. 2 is a graph of average potential against time showing VOC over a 40-second measurement duration. Each measurement is of 2.5 mg / mL ABTS with 2.5 mg / mL LacAB in the C-P buffer pH range 3-7 (plots from highest to lowest average potential: pH 4, 5, 3, 6 and 7). Fig. 3 is a graph of charge density against the concentration of ABTS, including error bars and a polynomial fit line. Each measurement used 5 mg / mL LacAB in 5 mM PBS. The concentrations of ABTS were: 0.625 mg / mL, 1.25 mg / mL, 6.25 mg / mL, 12.5 mg / mL, 25 mg / mL, and 50 mg / mL. There was a 5 minute incubation at room temperature before each CA measurement.

[0058] Fig. 4 is a bar chart of charge density as a function of the conditions of glycine wash and presence of redox mediator for MB-GDH-PQQ suspensions, including error bars. Each measurement used 5 mM glucose in 5 mM PBS. Samples with redox mediator had 500 pM ferricyanide. There was a 5 minute incubation at room temperature before each CA measurement.

[0059] Fig. 5 is a first test strip design. The features are as follows: 1. sample chamber rear vent, 2. sample chamber channel, 3. sample chamber inlet for sample deposition, 4. working electrode (bioanode), 5. sample chamber front vent, 6. counter chamber inlet for biocathode reconstitution, 7. salt bridge area, 8. counter electrode (biocathode), 9. counter chamber vent, 10. counter electrode connector pad, 11. working electrode connector pad.

[0060] Fig. 6 is an iteration of the test strip design altered to include space for a salt bridge to be cured after layer lamination. The new features are as follows: 1. sample chamber front vent (deviated), 2. salt bridge gel inlet, 3. narrow salt bridge channel, 4. counter chamber inlet (deviated).

[0061] Fig. 7 is a cyclic voltammogram showing the classic duck shape for ferricyanide oxidation and reduction on LIG electrodes prepared using the settings reported by Thana et al (Thaweeskulchai, T.; Schulte, A. Diode Laser and Polyimide Tape Enables Cheap and Fast Fabrication of Flexible Microfluidic Sensing Devices. Micromachines 2022, 13, 2214).

[0062] Fig. 8 is a cyclic voltammogram showing the classic duck shape for ferricyanide oxidation and reduction on LIG electrodes prepared using the settings reported herein.

[0063] Fig. 9 shows an exemplary test strip configuration for carrying out the described methods. The features are as follows: 1. sample chamber front vent (deviated), 2. salt bridge gel inlet, 3. narrow salt bridge channel, 4. counter chamber inlet (deviated).

[0064] Fig. 10 is an exemplary test strip configuration comprising (A) a first layer (an electrode layer) (B) a second layer (a microfluidic layer) and (C) a lid layer. (D) shows a composite of the layers depicting the relative arrangement in the test strip.

[0065] Fig. 11 is a bar chart showing the current density measured for the further test strip design of Fig. 10, using different amounts of peroxidase from horseradish type II (HRP) as the detectable species at the working electrode (the cathode in this example) and TMB and hydrogen peroxide as its substrate. Glucose oxidase type X-S (GOx) was used as a second catalyst positioned at or in proximity to a third position, which was at the counter electrode, in this case the anode. D-glucose was used as the substrate of GOx. A redox mediator (potassium ferricyanide) was used at the anode and a UV-curable polyelectrolyte (PE) gel was used as a medium for selective transport of ions between the anode and cathode.

[0066] Fig. 12 is a bar chart showing the current density measured for the experimental set up described in Test A at different HRP concentrations.

[0067] Fig. 13 is a bar chart showing the current density measured for the experimental set up described in Test A for the detection of Glial Fibrillary Acidic Protein (GFAP). The supernatant from a plate assay prepared using a Human GFAP DuoSet™ ELISA kit was applied to the experimental set up described in Test A, in place of the HRP:TMB / H2O2 mixture.

[0068] Fig. 14 is a bar chart showing the current density measured for the further test strip design of Fig. 10, using different amounts of Glucose oxidase type X-S (GOx) as the detectable species at the working electrode (the anode in this example) and D-glucose as its substrate. The same set-up and procedures were used as in Test A, Fig.

[0069] 11, except that the HRP concentration was fixed at 8 ng / mL and the GOx concentration was varied.

[0070] Fig. 15 is an exemplary set up for recording an electrical signal from the further test strip design of Fig. 10, altered such that the working chamber inlet (or sample chamber inlet) is positioned 2 mm away from the working electrode. In this exemplified set-up, a permanent magnet is positioned underneath the working electrode to move magnetic particles tethered to a detectable species to the working electrode. The strips are in a palmsens potentiostat connector, were wired to a palmsens single potentiostat and 8-channel multiplexer.

[0071] Fig. 16 is a bar chart showing the current density for the experimental set up described in Test A, using HRP-coated magnetic beads and magnetically concentrating the beads at the cathode. The strip design was altered slightly: in this example, the working chamber inlet was positioned just 2 mm away from the working electrode. This allowed for better concentration of the HRP-MBs by the magnet beneath the working electrode, which is fixed in place.

[0072] DETAILED DESCRIPTION In the discussion that follows, reference is made to a number of terms, which have the meanings provided below, unless a context indicates to the contrary. The nomenclature used herein for defining compounds, in particular the compounds according to the invention, is in general based on the rules of the IIIPAC organisation for chemical compounds, specifically the “IIIPAC Compendium of Chemical Terminology (Gold Book)”.

[0073] Throughout this specification, the terms “comprise”, “comprising” and / or “comprises” is / are used to denote aspects, embodiments and examples of the disclosure that “comprise” a particular feature or features. It should be understood that this / these terms may also encompass aspects, embodiments and examples which “consist essentially of” or “consist of’ the relevant feature or features.

[0074] The term “comprising” or variants thereof will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0075] The term “consisting” or variants thereof will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, and the exclusion of any other element, integer or step or group of elements, integers or steps.

[0076] The term “about” herein, when qualifying a number or value, is used to refer to values that lie within ± 5% of the value specified. For example, where the second position is separated from the first position by at least about 1.0 cm, a range of 0.95 cm to 1.05 cm is included.

[0077] The term “in proximity”, as used herein, means an entity is close enough to relevant position to perform a desired function. By way of example, where a second catalyst and second catalyst substrate are positioned in proximity to cathode, these entities are positioned sufficiently close to the cathode such they can act to complete the fuel cell circuit when a first catalyst has been moved to the second position. In some examples, “in proximity”, may mean, for example within about 1 cm, about 0.5 cm, about 0.25 cm, or about 0.10 mm of the relevant position and includes overlap with the relevant position.

[0078] As described above, the first aspect is to a method of detecting a target in a sample, the method comprising:

[0079] (i) contacting a mixture on or within a test strip, the mixture comprising:

[0080] (a) a target binding moiety tethered to a magnetically moveable particle;

[0081] (b) a target capture moiety conjugated to a detectable species; and

[0082] (c) a sample, wherein component (b) is positioned on or within the test strip at a first position, and the test strip further comprises:

[0083] (d) a fuel cell comprising an anode and a cathode, wherein one of the anode or cathode is positioned at a second position;

[0084] (ii) incubating (a), (b) and (c) for a period of time such that, when the target is present, a complex is formed, the complex comprising components (a) and (b) each independently bound to the target;

[0085] (iii) activating or generating a magnetic field to cause the complex to move to the second position such that the complex is separated from one or more other components of the mixture and such that the movement of the detectable species to the second position turns the fuel cell on or increases a response from the fuel cell; and

[0086] (iv) detecting a response from the fuel cell.

[0087] The fuel cell may be any one of the various types of fuel cell known in the art, such as an enzymatic biofuel cell, where enzymes take part in at least some of the chemical reactions taking place at each of the anode and cathode, by which reactions electrons are produced or consumed. For a review of enzymatic biofuel cells, see N. Karim or R. A. S. Luz et al. (supra). Alternatively, the fuel cell may be a microbial fuel cell, in which microbes take part in at least some of the chemical reactions taking place at each of the anode and cathode, by which reactions electrons are produced or consumed. For a review of microbial fuel cells, see P. Jalili et al., “A comprehensive review of microbial fuel cells considering materials, methods, structures, and microorganisms”, Heliyon, 2024, 10, 3, e25439. Another alternative fuel cell is a solid oxide fuel cell, in which electrons are produced by oxidising a fuel, such as hydrogen or carbon monoxide, at the anode, and reducing a species, such as oxygen, at the cathode. Oxide ions or protons (in proton-conducting-solid oxide fuel cells) are transported between the anode and the cathode through a medium for selective transportation of ions. Where oxide ions are transported, this medium is typically a solid oxide or ceramic electrolyte. For a review of solid oxide fuel cells, see Amit Talukdar et al., “A Review on Solid Oxide Fuel Cell Technology: An Efficient Energy Conversion System”, International Journal of Energy Research, 2024, 6443247.

[0088] In some embodiments, the detectable species comprises:

[0089] (i) a first catalyst and the test strip further comprises:

[0090] (e) a first catalyst substrate positioned at or in proximity to the second position, wherein movement of the first catalyst to the second position turns the fuel cell on; or (ii) a redox mediator and the test strip further comprises: (e) a first catalyst and a first catalyst substrate positioned at or in proximity to the second position,

[0091] wherein movement of the first catalyst to the second position increases a response from the fuel cell.

[0092] Where the detectable species is a first catalyst, the substrate of the first catalyst is positioned at or in proximity to the second position, i.e. at or in proximity to the electrode, for example by adsorption or deposition at the surface of the electrode. This way, when the target is present in the sample, and the complex formed with the target moves to the second position, the first catalyst is able to act on its substrate, and either directly or indirectly generate or consume electrons. By directly is meant that the action of the first catalyst on its substrate generates or consumes the electrons that are detected. By indirectly is meant that the action of the first catalyst on its substrate generates products that themselves react further with other species present and it is these further reactions that generate or consume the electrons that are detected. Once the complex has been magnetically moved and immobilised at the second position, detectable response may be immediately produced. The response detected may be directly proportional to the concentration of the complex at the second position, which, in turn, may be directly proportional to the concentration of target. Since the first catalyst is required in order to produce any detectable response, prior to movement of the complex to the second position, the fuel cell produces no response.

[0093] By positioning the first catalyst substrate at the second position, it is unavailable to any unbound target capture moiety, thus any unbound target capture moiety conjugated to a first catalyst cannot convert the first catalyst substrate to another product. In contrast, the first catalyst substrate is available to the complex that, on activating or generating a magnetic field, moves to the second position. Consequently, the first catalyst comprised within the complex at the second position is able to catalytically convert the first catalyst substrate to one or more other products, thereby generating or consuming electrons and generating a response in the fuel cell.

[0094] Provided the first catalyst substrate is available to any complex that, on activating or generating a magnetic field, moves to the second position, the first catalyst comprised within the complex at the second position will be able to catalytically convert the first catalyst substrate to one or more other products, and thus amplify the signal per target.

[0095] In particular embodiments, both the target capture moiety conjugated to a first catalyst and the target binding moiety tethered to a magnetically moveable particle are each positioned at the same one end of the test strip and the first catalyst substrate and the electrode are positioned on the other end. For example, where the test base is dumbbell-like in shape, the target capture moiety conjugated to a first catalyst and the target binding moiety tethered to a magnetically moveable particle may each be positioned at the same one end of the dumbbell-like shape and the first catalyst substrate and electrode may be positioned at the other end of the dumbbell-like shape.

[0096] Where the detectable species is a redox mediator, a first catalyst and its substrate is positioned at or in proximity to the second position i.e. at or in proximity to the electrode, for example by adsorption or deposition at the surface of the electrode. In this set-up, the enzyme is able to act on its substrate at the second position in the absence of the complex, and either directly or indirectly generate or consume electrons, thus a background signal is generated and detectable. When the target is present in the sample, and the complex formed with the target moves to the second position, the redox mediator is able to act on the first catalyst. The redox mediator acts as an electron shuttle and allows for more efficient electron transfer between the first catalyst (or products derived from the action of the first catalyst on its substrate) and the electrode, thereby allowing the first catalyst to more efficiently convert back to its active state and to act on further substrate. This means that movement of the complex, comprising the redox mediator, to the second position causes an increase in the charge generated in the fuel cell and thus an increase in the response from the fuel cell. The increase in the response detected may be directly proportional to the concentration of the complex at the second position, which, in turn, may be directly proportional to the concentration of target.

[0097] The reaction that takes place at the other of the anode and the cathode does not involve the complex and simply completes the fuel cell circuit. Typically, the test strip further comprises a second catalyst and a second catalyst substrate, each of which are positioned at or in proximity to a third position, wherein the third position is at the other of the anode and cathode. The second catalyst is able to act on its substrate, and either directly or indirectly generate or consume electrons. This takes place regardless of the presence or absence of target in the sample. However, movement of the complex to the second position allows a reaction at the second position to complete the fuel cell or to increase a response from the fuel cell. A redox mediator may also be present at the third position, allowing for more efficient electron transfer between the second catalyst (or products derived from the action of the second catalyst on its substrate) and the electrode, thereby allowing the second catalyst to more efficiently convert back to its active state and to act on further substrate.

[0098] The first catalyst substrate is positioned on orwithin the test strip (e.g. on orwithin the sample chamber) at or in proximity to the second position. In some embodiments, the first catalyst substrate is positioned on or within the test strip e.g. on or within the sample chamber) at the second position, for example by any suitable means of contacting. In some embodiments, the first catalyst substrate is deposited onto orwithin the strip as a solution or suspension, for example by spraying or dripping, and the strip is allowed to dry. In some embodiments, the first catalyst substrate is printed onto the test strip, e.g. onto the base.

[0099] A catalyst substrate is a chemical species which may be acted upon by a catalyst and catalytically converted by the catalyst to one or more different products. For example, a catalyst substrate may be cleaved at one or more positions by a catalyst to produce one or more different products. Any one type of catalyst may have more than one suitable catalyst substrate, which it is capable of catalytically converting to one or more different products. Any suitable substrate may be used in combination with its corresponding catalyst.

[0100] In some embodiments, one of the first catalyst and the second catalyst catalyses chemical oxidation (resulting in electrons moving from the catalyst or products derived from the catalyst acting on its substrate to the anode) and the other of the first and the second catalyst catalyses chemical oxidation (resulting in electrons moving from the cathode to the catalyst or to products derived from the catalyst acting on its substrate).

[0101] In some embodiments, the first catalyst comprises an enzyme. The mixture contacted on orwithin the test strip may comprise a buffer for stabilisation of the enzyme. The skilled person is able to select a suitable buffer for the specific enzyme and substrate employed without burden. In some embodiments, the buffer is selected from phosphate buffered saline, e.g. for moderate pH, about 7 to 8, Tris buffer (tris(hydroxymethyl)aminomethane ), e.g. for high pH, greater than or equal to about 8, and citrate-phosphate (e.g. for low pH, about 2 to about 8 or about 2 to about 6). For example, where the enzyme is laccase, the buffer may be a citrate phosphate buffer. Where the enzyme is horseradish peroxidase, glucose oxidase, glucose dehydrogenase, bilirubin oxidase, or tyrosinase, the buffer may be phosphate buffered saline. Where the enzyme is alkaline phosphatase, the buffer may be Tris buffer (tris(hydroxymethyl)aminomethane). The first catalyst may be any enzyme, provided it is stable enough for contact with the sample. For the avoidance of doubt, in such embodiments the first catalyst substrate is an enzyme substrate.

[0102] In some embodiments, the second catalyst comprises an enzyme. A buffer may be used to stabilise the enzyme. Again, the skilled person is able to select a suitable buffer for the specific enzyme and substrate employed without burden. In some embodiments, the buffer is selected from phosphate buffered saline, e.g. for moderate pH, about 7 to 8, Tris buffer (tris(hydroxymethyl)aminomethane ), e.g. for high pH, greater than or equal to about 8, and citrate-phosphate (e.g. for low pH, about 2 to about 8 or about 2 to about 6). For example, where the enzyme is laccase, the buffer may be a citrate phosphate buffer. Where the enzyme is horseradish peroxidase, glucose oxidase, glucose dehydrogenase, bilirubin oxidase, or tyrosinase, the buffer may be phosphate buffered saline. Where the enzyme is alkaline phosphatase, the buffer may be Tris buffer (tris(hydroxymethyl)aminomethane). In some embodiments, the second catalyst is not stabilised by a buffer. Any enzyme may be used provided the reaction that proceeds at the other of the anode and cathode completes the circuit of the fuel cell. The second catalyst and its substrate are each positioned at or in proximity to a third position, which is at the other of the anode and cathode (typically at the cathode).

[0103] Suitable enzymes for use at the cathode or at the anode of enzyme-based biofuel cells are well known in the art (see, for example, Jia Lei Zhang et al., “Enzyme-based biofuel cells for biosensors and in vivo power supply”, Nano Energy, 2021, 84, 105853; and Jingsheng Cai et al., “Enzymatic biofuel cell: A potential power source for selfsustained smart textiles”, iScience, 2024, 27, 2, 108998). Accordingly, the skilled person is able to select a suitable enzyme for use at the anode and a suitable enzyme for use at the cathode (e.g. a suitable enzyme pairing) without burden. Thus, it will be appreciated that the selection of a suitable enzyme for each of the first and second catalyst is dependent upon which of the anode and cathode is at the second position, and which is at the third position.

[0104] Suitable enzymes may include, but are not limited to, oxidases, reductases, dehydrogenases, peroxidases, phosphatases, oxidoreductases etc. In particular, suitable enzymes for use as the first and second catalysts may be selected from the group consisting of glucose oxidase, horseradish peroxidase, bilirubin oxidase, tyrosinase, alkaline phosphatase, glucose dehydrogenase, fructose dehydrogenase, formate dehydrogenase, lactate dehydrogenase, lactose oxidase, glutathione reductase, xanthine oxidase, laccase, glutaredoxin, cytochrome c oxidase, alcohol dehydrogenase, pyruvate dehydrogenase, and sorbitol dehydrogenase. Alternatively, suitable enzymes for use as the first and second catalysts may be selected from the group consisting of glucose oxidase, glucose dehydrogenase, fructose dehydrogenase, formate dehydrogenase, lactate dehydrogenase, lactose oxidase, horseradish peroxidase, alkaline phosphatase, glutathione reductase, xanthine oxidase, laccase, glutaredoxin, cytochrome c oxidase, alcohol dehydrogenase, pyruvate dehydrogenase, sorbitol dehydrogenase and bilirubin oxidase.

[0105] By way of further example, suitable enzymes for use at the anode (which may typically be at the second position or, more typically, at the third position) may be oxidoreductase enzymes. In some embodiments, suitable enzymes for use at the anode may be any one selected from the group consisting of: glucose oxidase, glucose dehydrogenase, lactose oxidase, alcohol dehydrogenase, fructose dehydrogenase, formate dehydrogenase and lactate dehydrogenase. Alkaline phosphatase may also be suitable for use at the anode. Where the anode is at the second position, the first catalyst may be any of those listed above as being suitable for use at the anode. In other embodiments, where the cathode is at the second position, the second catalyst (e.g. the catalyst positioned at or in proximity to the third position) may be any of those listed above as being suitable for use at the anode.

[0106] By way of further example, suitable enzymes for use at the cathode (which may typically be at the third position, or, more typically, at the second position) may be oxidase enzymes, peroxidase enzymes, and / or oxidoreductase enzymes, such as oxidase enzymes, and / or peroxidase enzymes. In some embodiments, suitable enzymes for use at the cathode may be any one selected from the group consisting of laccase, bilirubin oxidase, and horseradish peroxidase. Tyrosinase may also be a suitable enzyme for use at the cathode. Where the anode is at the second position, the second catalyst (e.g. the catalyst positioned at or in proximity to the third position) may be any of those listed above as being suitable for use at the cathode. In other embodiments, where the cathode is at the second position, the first catalyst may be any of those listed above as being suitable for use at the cathode.

[0107] For the avoidance of any doubt, and as stated above, in the methods as described herein, the first catalyst may be moved (as part of a complex) to the second position. Additionally, the second catalyst may be positioned at (or in proximity to) a third position.

[0108] In some embodiments, where the anode is positioned at the second position and the cathode is positioned at the third position, the first catalyst is glucose oxidase, glucose dehydrogenase, or alkaline phosphatase, such as glucose oxidase or glucose dehydrogenase (e.g. glucose oxidase), and the second catalyst is horseradish peroxidase, laccase, bilirubin oxidase, or tyrosinase, such as horseradish peroxidase. Alternatively, the second catalyst is laccase or bilirubin oxidase. In some particular examples, the first catalyst is glucose dehydrogenase and the second catalyst is laccase. In further particular examples, the first catalyst is glucose oxidase and the second catalyst is horseradish peroxidase.

[0109] In other embodiments, where the cathode is positioned at the second position and the anode is positioned at the third position, the first catalyst is horseradish peroxidase, bilirubin oxidase, or tyrosinase, such as horseradish peroxidase, and the second catalyst is glucose oxidase, glucose dehydrogenase, or alkaline phosphatase, such as glucose oxidase. In some particular examples, the first catalyst is horseradish peroxidase and the second catalyst is glucose oxidase.

[0110] Suitable catalyst (e.g. enzyme) substrates will be selected in accordance with the type of catalyst or enzyme selected. Thus, the enzyme substrate may be any substrate of the enzymes described herein. In some examples, the enzyme substrate may be glucose, 3,3',5,5'-tetramethylbenzidine (TMB), bilirubin, oxygen, tyrosine (such as L-tyrosine), a hydroxyphenol (such as catechol, 3,4-dihydroxyphenylalanine (DOPA), or 3,4-dihydroxybenzaldehyde (DOBA)), p-nitrophenylphosphate (PNPP), or hydrogen peroxide (e.g. urea hydrogen peroxide). The enzyme substrate may be selected from one or more of those listed above, for example TMB and hydrogen peroxide. In some examples, the enzyme substrate may be glucose or hydrogen peroxide (e.g. urea hydrogen peroxide). For the avoidance of doubt, the “substrate” may be any molecule acted on by the catalyst. For example, where the catalyst comprises a laccase enzyme, it is able to act on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), phenols, diphenols, methoxy phenols, aromatic amines and phenolic acids, amongst others. These species are considered herein to function as laccase substrates. Laccase is considered to be an oxidase enzyme, a sub-group of oxidoreductase enzymes, but is able to reduce oxygen to water and to then to oxidise substrate molecules, such as ABTS. Thus, laccase is able to catalyse chemical oxidation and chemical reduction. This is true of all oxidoreductase enzymes. For the avoidance of doubt, the first catalyst substrate positioned at or in proximity to the second position may be any one of the substrates that can be acted on by the first catalyst. For the avoidance of doubt, the second catalyst substrate positioned at or in proximity to the third position may be any one of the substrates that can be acted on by the second catalyst. Typically, by acting on its substrate, a catalyst is converted from its active form to its inactive form. The inactive form is then converted back to the active form by reaction with a further species. Where further species are required in order for the catalyst to re-generate, these are available to the catalyst at the second and / or third position. For example, where the catalyst comprises laccase, the substrate may be ABTS, and oxygen may be available to the catalyst at the second and / or third position e.g. by the test strip being open to the air and / or by the catalyst being provided in a solution or mixture comprising oxygen.

[0111] In particular embodiments, where the first or second catalyst is horseradish peroxidase, the enzyme substrate used is TMB and / or hydrogen peroxide. Where the first or second catalyst is glucose oxidase, the enzyme substrate used may be glucose. Where the first or second catalyst is bilirubin oxidase, the enzyme substrate may be oxygen. Where the first or second catalyst is tyrosinase, the enzyme substrate may be tyrosine (such as L-tyrosine) or a hydroxyphenol (such as catechol, 3,4-dihydroxyphenylalanine (DOPA), or 3,4-dihydroxybenzaldehyde (DOBA)). Where the first or second catalyst is alkaline phosphatase, the enzyme substrate may be p-nitrophenylphosphate (PNPP).

[0112] In more particular embodiments, the first catalyst is horseradish peroxidase and the enzyme substrate is TMB and / or hydrogen peroxide, and the second catalyst is glucose oxidase, and the second catalyst substrate is glucose. In even more particular embodiments, the concentration of the glucose oxidase is about 10 to 1000 pg / mL, such as about 50 to about 500 pg / mL, about 100 to about 400 pg / mL, about 200 to about 300 pg / mL, or about 250 pg / mL. In such embodiments, the concentration of glucose may be about 5 to about 30 mM, such as about 5 to about 25 mM or about 10 to about 20 mM.

[0113] In some embodiments, the first and / or second catalyst is part of (e.g. an enzyme of) an enzyme cascade, i.e. a biocatalytic process that uses a series of sequential reactions to synthesize a product. In some preferred embodiments, the first catalyst is part of (e.g. an enzyme of) an enzyme cascade.

[0114] Where the catalyst is part of an enzyme cascade, any one or more of the products generated by the cascade may generate or consume the electrons that give rise to the detectable response from the fuel cell. In other words, where the catalyst is part of an enzyme cascade, the catalyst indirectly generates or consumes electrons that transfer to or from the anode or cathode. An example of such an enzyme cascade is that of the enzyme Recombinant Factor C acting on endotoxin to produce activated Recombinant Factor C, which is the first step of the horseshoe crab clotting cascade (see, for example, T. Osaki and S. Kawabata, “Structure and function of coagulogen, a clottable protein in horseshoe crabs”, CMLS, Cell, Mol. Life Sci., 2004, 61, 1257-1265). Any one of the enzymes making up part of this cascade could be employed as the catalyst.

[0115] In some embodiments, electrons travel directly, by direct electron transfer, between the first catalyst and the anode or cathode. Alternatively or additionally, in some embodiments, electrons travel directly, by direct electron transfer, between the second catalyst and the anode or cathode.

[0116] Having electrons travel by direct electron transfer, avoids the need for a redox mediator to shuttle electrons from the enzyme to the electrode. Using redox mediators can lead to signal loss and a reduction in sensitivity of the method. Thus, having electrons travel by direct electron transfer can be advantageous as it can avoid the need for redox mediators, and thus avoid potential signal loss. In some embodiments, redox mediators are not used in the method.

[0117] Direct electron transfer is promoted by the use of a redox cofactor. Accordingly, in some embodiments, the first and / or second catalyst comprises a redox cofactor. Redox cofactors are molecules that change their oxidation state during catalytic redox reactions of a substrate. Accordingly, redox cofactors participate in oxidation-reduction reactions and can promote direct electron transfer from a catalyst to an electrode. For a review of recent advances in enzymatic fuel cells capable of direct electron transfer, see Sooyoun Yu and Nosang V. Myung, “Recent Advances in the Direct Electron Transfer-Enabled Enzymatic Fuel Cells”, Front. Chem., 2020, 8, 620153.

[0118] Redox cofactors are well known in the art. Those often used in fuel cells include pyrroloquinoline quinone (PQQ), nicotine adenine dinucleotide (NAD), flavin adenine dinucleotide (FAD), and haem. In some embodiments, the first and / or second catalyst comprises one or more redox cofactors selected from PQQ, NAD, FAD, haem, and analogues thereof. By analogues is meant compounds that mimic any of the cofactors listed, by which is meant that they change their oxidation state by the same mechanism. Typically, so that electrons can more easily directly transfer from the catalyst to the electrode, the redox cofactor is positioned on the outside of the first and / or second catalyst. In some embodiments, the first and / or second catalyst comprises PQQ or an analogue thereof. In particular embodiments, the first and / or second catalyst comprises PQQ.

[0119] In some preferred embodiments, the anode is positioned at the second position and the cathode is positioned at the third position. In such configurations, and by way of example only, the first catalyst may comprise glucose dehydrogenase and a redox cofactor (e.g. PQQ), and the first catalyst substrate may be glucose. In such configurations, and by way of example only, the second catalyst may be laccase, the second catalyst substrate may be ABTS, and, typically, oxygen is available to the second catalyst.

[0120] In other preferred embodiments, the cathode is positioned at the second position and the anode is positioned at the third position. In such configurations, and by way of example only, the first catalyst may comprise horseradish peroxidase, and the first catalyst substrate may be TMB and / or hydrogen peroxide. In such configurations, and by way of example only, the second catalyst may be glucose oxidase, and the second catalyst substrate may be glucose, and, typically, oxygen is available to the second catalyst.

[0121] The method of the first aspect comprises activating or generating a magnetic field to cause the complex to move to the second position such that the complex is separated from one or more other components of the mixture and such that the movement of the detectable species to the second position turns the fuel cell on or increases a response from the fuel cell. The second position is at one of the anode or cathode (typically the cathode). Typically, the first catalyst substrate is also positioned at or in proximity to the one of the anode and cathode (typically the cathode), i.e. the first catalyst substrate is typically positioned at or in proximity to the second position. In some embodiments, the first catalyst substrate is positioned at the second position, i.e. at the anode or cathode (typically the cathode).

[0122] Where the detectable species is a redox mediator, a first catalyst and its substrate is positioned at or in proximity to the second position. In such embodiments, the first catalyst and the first catalyst substrate are each typically positioned at the second position, i.e. at the anode or cathode (typically the anode).

[0123] In some embodiments, the test strip comprises a counter chamber configured to allow for fluid communication with the other of the anode and the cathode. In some embodiments, the counter chamber is separated from the sample chamber; optionally they are separated by the separator or medium for the selective transportation of ions. In some embodiments, the separator or medium for the selective transportation of ions is housed and / or contained in a separation chamber. Thus, the separation chamber may comprise the separator or medium. In some embodiments, the counter chamber houses a single liquid or a single body of fluid or liquid that comprises the second catalyst and the second catalyst substrate. The counter chamber may be positioned on or within the test strip (e.g. or on or within the base of the test strip). The counter chamber may define a volume on or within the test strip and / or base into which the second catalyst and the second catalyst substrate may be introduced and / or retained during the methods as described herein. The counter chamber may comprise a single fluid chamber. The third position may be at an end of the counter chamber.

[0124] In some embodiments, the first catalyst and the second catalyst are each enzymes. In such embodiments, the fuel cell is an enzymatic biofuel cell. Such fuel cells are advantageous owing to their ability to simultaneously and continuously produce a response (i.e. an external stimulus is not required: a response is simultaneously produced on movement of the complex to the second position), their good biocompatibility with targets and complexes formed from targets, their low toxicity, simplicity and biodegradability.

[0125] As described above, the detectable species may be a redox mediator. Where the detectable species is a first catalyst, the test strip may further comprise a redox mediator. This may be positioned, for example, at or in proximity to the second position to assist electron transfer between the first catalyst and the electrode. Typically, the redox mediator is positioned at the second position. In some embodiments, the redox mediator is positioned at the anode or cathode (positioned at the second position), for example, the anode or cathode may be coated with the redox mediator.

[0126] Where the detectable species is a first catalyst, a redox mediator may be positioned on or within the test strip (e.g. on or within the sample chamber). In some embodiments, the redox mediator is positioned on or within the test strip e.g. on or within the sample chamber) at the second position, for example by any suitable means of contacting. In some embodiments, the redox mediator is deposited onto or within the strip as a solution or suspension, for example by spraying or dripping, and the strip is allowed to dry. In some embodiments, the redox mediator is printed onto the test strip, e.g. onto the base. In some embodiments, the first catalyst and redox mediator are premixed and simultaneously deposited or printed onto the test strip, e.g. onto the base.

[0127] Both the redox mediator and the first catalyst substrate may each be positioned on or within the test strip (e.g. on or within the sample chamber) at the second position. The redox mediator may also be positioned elsewhere on the test strip, for example it may overlap with the second position or it may be positioned across or within a partial or an entire surface of a base including at the second position. Alternatively, the redox mediator may be positioned only at the second position, and the first catalyst substrate may be positioned at the second position and elsewhere on the test strip. In this case, the first catalyst substrate may overlap with the second position or it may be positioned across or within a partial or an entire surface of a base including at the second position.

[0128] A redox mediator is a chemical species which acts as an electron shuttle between a catalyst’s oxidising and reducing forms. The redox mediator may be any one selected from the group consisting of ferri / ferrocyanide; ruthenium (II) and (III) complexes (e.g. ruthenium (III) hexamine chloride; ferrocene and ferrocenium derivatives; cobaltocene, rhodocene and other metallocenes; p-nitrophenyl phosphate (pNPP); quinones; NAD; NADP; FAD; methylene blue and derivatives; 2,6-dichlorophenolindopheno; and phenylenediamine and derivatives.

[0129] The skilled person is able to assess which redox mediators are compatible with which catalysts. By way of example only, horseradish peroxidase, bilirubin oxidase and laccase are each compatible with at least 3,3',5,5'-tetramethylbenzidine, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid, diaminobenzidine and phenylenediamine. Glucose dehydrogenase and glucose oxidase are each compatible with at least ferricyanide, ruthenium (III) hexamine (and salts, such as chloride salts thereof), tris(bipyridine)ruthenium(ll I) chloride, ferrocene and ferrocene derivatives.

[0130] In some cases, e.g. for horseradish peroxidase, tyrosinase and alkaline phosphatase, the catalyst substrate, once acted on by the catalyst, may act as a redox mediator.

[0131] In some embodiments, the test strip further comprises a gel or polymer at or in proximity to the second position, wherein the gel or polymer is redox active or conductive. The gel or polymer may comprise metal redox-active complexes, or organic redox-active moieties, such as those described by Raquel Garcia and David Mecerreyes in “Polymers with redox properties: materials for batteries, biosensors and more”, Polym. Chem., 2013, 4, 2206. In some embodiments, the gel or polymer comprises metal redox-active complexes such as osmium redox-active complexes. The first catalyst substrate may be embedded within the gel or polymer.

[0132] Typically, the first catalyst substrate and / or redox mediator are present in the system in molar excess relative to each of the target binding moiety tethered to a magnetically moveable particle and the target capture moiety conjugated to a detectable species. Often, the target binding moiety tethered to a magnetically moveable particle and the target capture moiety conjugated to a detectable species are present in substantially equal amounts (i.e. the mean molar concentration of each lie within ± 10% of each other). Typically, the redox mediator and / or the first catalyst substrate are present in molar amounts of 101to 1O20times greater than each of the target binding moiety tethered to a magnetically moveable particle and the target capture moiety conjugated to a detectable species, such as 103to 1018, 105to 1015. In some embodiments, the redox mediator and / or the first catalyst substrate are present in molar amounts of at least 1O10times greater than each of the target binding moiety tethered to a magnetically moveable particle and the target capture moiety conjugated to a detectable species.

[0133] Typically, the anode and cathode are isolated (e.g. fluidly isolated) by a separator. The separator is typically a medium for the selective transportation of ions. As described above, the medium for the selective transportation of ions may be housed and / or contained in a separation chamber. Thus, the separation chamber may comprise the medium. As used herein, the term “separation chamber” may refer to a third chamber that is configured to separate the sample chamber and the counter chamber from one another and / or to separate the anode and cathode from one another. In particular, the separation chamber may be a third chamber that is configured to prevent direct fluid communication between the sample chamber and counter chamber and / or to prevent direct fluid communication between the anode and the cathode.

[0134] The use of a separator allows the anode and cathode to be fluidly isolated from one another, allowing the reactions taking place at each electrode to be separated, but also allows specific ions to transport between the electrodes, thereby completing the circuit. The selectivity of the medium may arise from its properties, e.g., as a result of the chemical structure (such as charged groups) or physical properties (such as porosity). Any medium for the selective transportation of ions may be used in the test strip, including an ion-permeable membrane or a salt bridge, such as an agar salt bridge. The term “salt bridge” is well known in the art and refers to a physical separation between an anode and a cathode of a fuel cell that allows the passage of ions, typically a select species of ions. The particular species and polarity (i.e. , anionic or cationic) of the ions will depend on the type of cell, the electrolytes, and the type of salt bridge. For example, the salt bridge in a fuel cell may allow the selective transportation of H+ions. Thus, in some embodiments, the salt bridge allows the passage of H+ions.

[0135] The medium for the selective transportation of ions may be a fluid or gel. The medium for the selective transportation of ions may be solidifiable material, by which is meant a fluid material such as a liquid, gel, paste, resin, serum, emulsion, suspension, slurry, or solution etc. that can form a solid, for example by drying or curing. In some embodiments, the medium for the selective transportation of ions is a solidifiable material that is configured to solidify to form a medium for selective transportation of ions, such as a salt bridge, between the anode and the cathode. The drying or curing process may comprise any one or more of heating, cooling, evaporating, drying, irradiating, or chemically activating. Therefore, in some embodiments, the medium for the selective transportation of ions is a solidifiable material that is configured to solidify under irradiation (e.g., under electromagnetic irradiation such as UV irradiation) and / or heat, or in the presence of a chemical activator. In some embodiments, the solidifiable material solidifies by a polymerization and / or cross-linking reaction. The skilled person is familiar with polymerization reactions and cross-linking reactions. Nonetheless, for the avoidance of doubt, a polymerization reaction is a chemical reaction in which one or more monomeric species undergoes a process to form a polymer, a large chain composed of repeating backbone of monomeric units, sometimes comprising sidechains; a cross-linking reaction therefore is where the backbones or side-chains of polymers react / interact with each other such that they link together, either intra- or inter-molecularly.

[0136] The skilled person is able to select appropriate monomers and reagents to effect a curing process that comprises polymerization and / or cross-linking reactions. Nonetheless, in some embodiments the solidifiable material, such as a polymerizable liquid, comprises one or more, or all, of:

[0137] (i) monomers (e.g., acrylate-, methacrylate-, vinyl, saccharide or amino acidcomprising monomers, such as diallyldimethylammonium chloride, 2-acrylamido-2-methylpropane sulfonic acid, glucosamine, galactose, 3,6-anhydro-galactopyranose, / V-acetylglucosamine, glucuronic acid, uronic acid, galacturonic acid, mannuronic acid, guluronic acid, glucopyranose, acrylic acid, styrene sulfonate, allylamine, acrylamide, sulfated / V-acetyl-galactosamine, 3,4-ethylenedioxythiophene)sulfonate, 3,4-ethylenedioxythiophene, aniline, pyrrole or ethylene glycol);

[0138] (ii) cross-linking agents (e.g., acrylate-, or methacrylate-, or vinyl-containing cross-linking agents, such as / V, / V'-methylenebis(acrylamide)); and

[0139] (iii) photoinitiators (such as 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone).

[0140] A cross-linking agent is a species (typically a monomeric species) that comprises reactive groups capable of forming linkages between polymer chains. The reactive groups may react to form covalent bonds between polymer chains, or they may comprise ionic groups that are capable of forming ionic interactions between polymer chains. The reactive groups are often vinyl or alkene containing groups, such as acrylate groups, methacrylate groups, vinyl ether groups, vinyl ester groups, styrene groups, and maleimide groups. In some embodiments, the cross-linking agent is selected from, ethylene glycol diacrylate, di(ethylene glycol) diacrylate, tetra(ethylene glycol) diacrylate, ethylene glycol dimethacrylate, di(ethylene glycol) dimethacrylate, tri(ethylene glycol) dimethacrylate, / V, / V’-methylenebis(acrylamide), / V, / V-(1 ,2-dihydroxyethylene)bis(acrylamide), A / -(1 -hydroxy-2, 2-dimethoxyethyl)acrylamide, and divinylbenzene, typically / V, / V'-methylenebis(acrylamide).

[0141] A photoinitiator is a chemical species that absorbs light, typically UV irradiation, to form a radical or ionic species that can initiate polymerization and / or cross-linking reactions by starting a chain reaction. Photoinitiators typically comprise relatively weak covalent bonds that are labile under certain conditions, such as under UV irradiation. In some embodiments, the photoinitiator is selected from benzoin methyl ether, benzoin ethyl ether, 2,2-dimethoxy-2-phenylacetone, camphorquinone, triarylsulfonium hexafluorophosphate, and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, typically 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone. The skilled person understands that the required wavelength of light to initiate polymerisation is dependent on the photoinitiator. For example, in some embodiments, the photoinitiation wavelength is about 365 nm, which is suitable for at least 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone.

[0142] In some embodiments, the solidifiable material solidifies to form a polyelectrolyte gel. The term “polyelectrolyte gel” is herein understood to refer to a gel that comprises polymer chains, wherein the polymer chains comprise charged groups, either on the backbone, the side chains, or the cross-linkers, such that the gel is capable of acting as an electrolyte. In some cases, a polyelectrolyte gel may be described as a three-dimensional matrix of cross-linked polymer chains. The term “gel” is understood to refer broadly to a solid material that, despite comprising a substantial amount of liquid, such as water, forms a solid structure due to the molecular interactions of the gel’s constituent compounds.

[0143] A polyelectrolyte gel may be formed from a polymerizable liquid that, as described above, comprises one or more, or all, of (i) monomers, (ii) cross-linking agents, and (iii) photoinitiators. The skilled person will recognise that the polyelectrolyte gel that forms from a polymerizable liquid is dependent on the constituents of the polymerizable liquid. For example, the polymerizable liquid may comprise the following constituents: diallyldimethylammonium chloride, / V, / V'-methylenebis(acrylamide), and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone. Such a polymerizable liquid may form a polyelectrolyte gel, which may be referred to as poly(diallyldimethylammonium chloride) (pDADMAC).

[0144] In some embodiments, the polyelectrolyte gel may be, or may comprise components, selected from poly(2-acrylamido-2-methyl-1 -propanesulfonic acid) (PAMPS), chitosan, hyaluronic acid, heparin, pectin (polygalacturonic acid), alginate (alginic acid), carboxymethyl cellulose, polypeptides, polyacrylic acid, polystyrene sulfonate, polyallylamine, carboxymethyl cellulose, polyacrylamide, gelatin / agar, chondroitin sulfate, poly(3,4-ethylenedioxythiophene)sulfonate (PEDOTS), poly(3,4-ethylenedioxythiophene) (PEDOT), polyaniline, polypyrrole, polyethyleneglycols (PEG), and poly(diallyldimethylammonium chloride) (pDADMAC).

[0145] The constituents of the polymerizable liquid may be construed by the skilled person with respect to the polyelectrolyte gel - in some cases, a polymerizable liquid may not be required, and instead required a (pre-cursor) solidifiable material to be cured to form a solid as described above. In some embodiments, the polyelectrolyte gel is formed from a polymerizable liquid comprising diallyldimethylammonium chloride, / V, / V'-methylenebis(acrylamide), and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, sometimes referred to as poly(diallyldimethylammonium chloride) (pDADMAC).

[0146] The anode and cathode of the test strip are typically electronically connected (for example, by electrical wires) to a sensor, such as an ammeter or voltmeter, in order to measure the response from the fuel cell. In some embodiments, the test strip further comprises an anode connector pad that is electronically connected to the anode, to which pad a wire may be attached, and a cathode connector pad that is electronically connected to the cathode, to which pad a wire may be attached. The anode and cathode connector pads allow for easier electrical connection of the anode and the cathode to a sensor. In some embodiments, the one or more electrode connector pads are configured to be compatible with a commercially available test strip connector, such as a Metrohm, PalmSens, orTaiDoc connector.

[0147] The response is detected after the activation or generation of a magnetic field. The time in between activating or generating the magnetic field and detecting the response need not be limited, provided (where the target is present) at least some of the complex is able to move to the second position on or within the strip and generate a signal. The inventors have found that magnetic field-induced movement of the complex to the second position is surprisingly quick. Thus, in some embodiments, the response is detected about 0.1 s to 20 s after the activation or generation of a magnetic field. However, it may be beneficial to allow more time in between activation or generation of a magnetic field and detection of the response, for example to allow for more complex to move to the second position and thus greater amounts of detectable signal to build up, thereby allowing for a greater response to be detected. In some embodiments, the method comprises a first incubation step in which the complex is formed and a second incubation step, following movement of the complex to the second position. In embodiments comprising a second incubation step, the method further comprises incubating the complex at the second position and / or in the detection zone. This second incubation step may be for a period of time to allow an increased amount of detectable signal to build up. In some embodiments, the response is detected several minutes after the activation of a magnetic field. The response may be detected from about 0.5 seconds to about 15 minutes, about 1 seconds to about 10 minutes, about 2 seconds to about 5 minutes, about 5 seconds to about 60 seconds, or about 10 to about 30 seconds after the activation of a magnetic field.

[0148] As described above and herein, activating or generating a magnetic field causes the complex to move to the second position such that the complex is separated from one or more other components of the mixture and such that the movement of the detectable species to the second position turns the fuel cell on or increases a response from the fuel cell. In some embodiments, the complex is immobilised or immobilisable at the second position. For the avoidance of doubt, activating or generating a magnetic field causes the complex to move to the second position, thereby magnetically immobilising the complex at the second position. The complex may be further immobilised at the second position by interactions (for example, by bonding, adsorption, entrapment, or encapsulation) between the complex and the electrode surface or of the complex at the electrode surface. For example, the complex may be cross linked to the surface of the electrode.

[0149] The second catalyst may be immobilised at the third position. Again, the immobilisation may be by any one or more selected from adsorption, covalent bonding (such as cross-linking), entrapment or encapsulation.

[0150] The test strip comprises a target capture moiety conjugated to a detectable species. The target capture moiety may be conjugated to the detectable species via a linker, i.e. a molecule bonded to both the target capture moiety and the detectable species. The linker may be a particle, such as a latex particle. Alternatively, the linker may form by conjugation techniques known in the art, such as biotin-streptavidin or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-N-hydroxysuccinimide (NHS). For example, the detectable species may be conjugated to biotin and the target capture moiety may be conjugated to streptavidin. The biotin and streptavidin may bind to one another, thereby linking the target capture moiety and the detectable species.

[0151] Similarly, the target binding moiety may be conjugated to the magnetically moveable particle via a linker, i.e. a molecule bonded to both the target binding moiety and the magnetically moveable particle. The linker may be a particle, such as a latex particle. Alternatively, the linker may form by conjugation techniques known in the art, such as biotin-streptavidin or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-N-hydroxysuccinimide (NHS). For example, the magnetically moveable particle may be conjugated to streptavidin and the target binding moiety may be conjugated to biotin. The biotin and streptavidin may bind to one another, thereby linking the magnetically moveable particle and the target binding moiety.

[0152] In use, following introduction of a sample to the test strip, a mixture is formed comprising the target binding moiety tethered to a magnetically moveable particle, the target capture moiety conjugated to a detectable species and the sample. The mixture on the test strip, comprising the target binding moiety tethered to a magnetically moveable particle, the target capture moiety conjugated to a detectable species and the sample, may be a liquid. For example, the sample comprising the target may be a liquid which, when added to or within the strip, forms the mixture, i.e. forms a liquid comprising the target binding moiety tethered to a magnetically moveable particle, the target capture moiety conjugated to a catalyst and the sample.

[0153] In some embodiments, the method comprises incubating the target binding moiety tethered to a magnetically moveable particle; the target capture moiety conjugated to a detectable species; and the sample for a period of time such that, when the target is present, a complex is formed, the complex comprising the target binding moiety tethered to a magnetically moveable particle and the target capture moiety conjugated to a detectable species, each being independently bound to the target. When the target is present, the complex often forms within minutes or seconds. The incubation time typically ranges from about 0.5 seconds to about 10 minutes, about 2 seconds to about 5 minutes, about 5 seconds to about 60 seconds, or about 10 to about 30 seconds. In some embodiments, the incubation time ranges from about 2 minutes to about 5 minutes.

[0154] The method of the first aspect further comprises activating or generating a magnetic field to cause the complex to move to the second position such that the complex is separated from one or more other components of the mixture and moved to the anode or cathode for detection. The magnetic field may be activated or generated by activating an electromagnet or mechanically activating, e.g. moving, a permanent magnet into proximity with the second position.

[0155] The magnetic field strength is at least strong enough to attract and cause the magnetically moveable particles of the complex to move towards the second position on or within the strip, thereby separating the complex from one or more other components of the mixture. In some embodiments, the complex is separated from at least any unbound target capture moiety conjugated to a redox catalyst within the mixture. The magnetic field typically has a strength of about 0.05 mT to about 5 T, such as about 0.1 mT to about 2 T, about 1 mT to about 500 mT, or about 10 mT to about 50 mT. In some embodiments, the magnetic field has a strength of approximately 1.8 Tesla.

[0156] The magnetically moveable particle may be a paramagnetic particle capable of movement by the magnetic field. The paramagnetic particle may be a paramagnetic bead, such as a “magnetic bead”. Magnetic beads are well known in the art and may comprise any suitable paramagnetic material, such as iron oxide, e.g. magnetite (FesC ).

[0157] In some embodiments, the surface of the magnetically moveable particle is modified with a plurality of molecules. By a plurality is meant two, five, ten or fifty or more molecules. The inventors have found that modifying the surfaces of the magnetically moveable particles with a plurality of molecules may prevent aggregation of the magnetically moveable particles and thus increases the efficiency of target binding. Furthermore, the inventors have found that certain molecules, such as certain amino acids, are able to reduce aggregation of the magnetically moveable particles and also immobilise the magnetically moveable particle at the electrode, thereby increasing the response detected from the fuel cell. Accordingly, in some embodiments, each molecule of the plurality of molecules is selected from an amino acid or a polymer, optionally wherein each molecule of the plurality of molecules is glycine.

[0158] The shape and the precise position of the magnetic field at the second position need not be limited, provided that the second position is physically separate from (e.g. not overlapping with) the first position. To reduce the probability of diffusion of any unbound target capture moiety conjugated to a redox catalyst to the second position, it may be beneficial to position the first position as far away as practically possible from the second position on or within the test strip.

[0159] The second position is positioned away from the first position, i.e. the first and second positions do not overlap. In some embodiments, the first and second positions are separated by at least about 0.1, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5 or 3 cm. For example, the first and second positions may be separated by at least about 1 cm. In some embodiments, the first and second positions are separated in the range of from about 0.5 cm to about 3 cm.

[0160] The test strip may comprise a base and a lid. For the avoidance of doubt, the first position may be positioned anywhere on the base or the lid. In some embodiments the first position is within the test strip, for example the first position may be enclosed by the positioning of the base with respect to the lid. In some embodiments, the first position is on the base and within the test strip, e.g. on the base and enclosed by the lid of the strip. As used herein, the base (e.g. the base of the test strip) may be considered as a test substrate and the two terms may be used interchangeably within the context of the present disclosure.

[0161] In some embodiments, the test strip comprises a sample chamber configured to allow for fluid communication with one of the anode and the cathode. In some embodiments, the sample chamber houses a single liquid mixture or a single body of fluid or liquid that comprises the mixture. In some embodiments, the method does not comprise contacting the sample with one or more fluidic interfaces (e.g. on the test strip). In some embodiments, the method does not require the use of one or more wash steps (e.g. with a buffer or the like).

[0162] The sample chamber may be positioned on or within the test strip (e.g. or on or within the base of the test strip). The sample chamber may define a volume on or within the test strip and / or base into which a sample may be introduced and / or retained during the assays and methods as described herein. The sample chamber may comprise a single fluid chamber. The sample chamber may comprise a plurality of zones in fluid communication with one another (for example, at least two zones). The sample chamber may comprise a reagent zone at the first position. The sample chamber may comprise a detection zone at the second position. The first position and / or reagent zone may be at a first end of the sample chamber. The second position and / or detection zone may be at a second end of the sample chamber. The first end of the sample chamber and the second end of the sample chamber may be opposing ends of the sample chamber.

[0163] The sample chamber may be enclosed, optionally by a lid of the strip. The assay chamber may be partially enclosed (e.g. one portion of the sample chamber, such as the reagent zone and / or first position, may be enclosed, and another portion of the sample chamber, such as the detection zone and / or second position, may not be enclosed). In some embodiments, the lid may be transparent and / or at least a portion of the lid may be transparent. For example, the portion of the lid enclosing the detection zone and / or second position may be transparent.

[0164] In some embodiments, the sample chamber may comprise a first enlarged end (comprising the first position and / or reagent zone) and / or a second enlarged end (comprising the second position and / or detection zone). The first and second enlarged ends may be connected by a connecting channel. The connecting channel may be narrower than the first and second enlarged ends. The first enlarged end of the sample chamber may taper towards the connecting channel. The second enlarged end of the sample chamber may taper towards the connecting channel. In some embodiments, the first and / or second ends (e.g. enlarged ends) taper towards a sample inlet. Such configurations may facilitate and / or promote the desired flow of sample following introduction of the sample into the sample chamber.

[0165] The test base and / or the sample chamber may be dumbbell-like in shape. The first position may be at one end of the dumbbell-like shape. The second position may be at a second end of the dumbbell-like shape. The base and / or sample chamber may comprise an hourglass-like shape. For example, an hourglass-like shape with an elongated mid-portion. The first position may be at one end of the hourglass-like shape. The second position may be at a second end of the hourglass-like shape.

[0166] Alternatively, the test base and / or the sample chamber may comprise a C-shaped channel (by which is meant that the cross-section of the channel is C-shaped), which may be enclosed by the lid.

[0167] The sample chamber is typically a channel, and the first and second position are typically at opposite ends of the channel. For example, where the sample chamber is dumbbell-like in shape or is a C-shaped channel, the first position may be at one end of the dumbbell-like shape and the second position may be at the other end. In particular embodiments, both the target capture moiety conjugated to the detectable species and the target binding moiety tethered to the magnetically moveable particle are positioned at the first position. This is typically at the same one end of the sample chamber. In particular embodiments, where the detectable species comprises a first catalyst and the test strip further comprises a first catalyst substrate positioned at or in proximity to the second position, the target capture moiety conjugated to the first catalyst and the target binding moiety tethered to the magnetically moveable particle are positioned at the first position at the same one end of the sample chamber and the first catalyst substrate is positioned at the other end of the sample chamber. For example, where the sample chamber is dumbbell-like in shape or is a C-shaped channel, the target capture moiety conjugated to a catalyst and the target binding moiety tethered to a magnetically moveable particle may each be positioned at the same one end of the dumbbell-like shape or C-shaped channel and the catalyst substrate (and optionally the redox mediator) may be positioned at the other end of the dumbbell-like shape or C-shaped channel.

[0168] The test strip may comprise a sample inlet that is in fluid communication with the sample chamber. The sample inlet may be positioned at, or in proximity to, the first position or may be positioned between the first and second positions. The positioning of the sample inlet between the first and second positions is sometimes referred to herein as a “centre-fill” design or configuration. In the “centre-fill” design, the sample inlet does not need to be positioned exactly halfway between the first and second positions. In some embodiments, the sample inlet may be positioned away from the first and / or second positions. In some embodiments, the sample inlet may be positioned approximately midway between the first and second positions. In some embodiments, the sample inlet is positioned or located such that the sample is introduced into the connecting channel of the sample chamber.

[0169] The test strip may be configured such that the sample is inserted into the sample chamber by a sample inlet positioned between the first position and second position and the sample travels or flows across the first position in a direction away from the second position. In other words, the end of the sample chamber comprising the first position is filled by the sample at the same time as the end comprising the second position, thereby preventing sample flow from the first position to the second position. Without being bound by theory, this configuration of test strip is particularly advantageous in minimising the risk of cross-contamination and / or unwanted signal from unbound / uncomplexed detectable species moving (e.g. diffusing) towards the second position when the test strip is used in the various methods described herein. By positioning the sample inlet between the first and second positions, following the introduction of the sample, the flow of sample across the first position means that any target capture moiety conjugated to a detectable species that is re-suspended in the sample fluid is less likely to move and / or diffuse towards the second position (as the flow of fluid is away from the second position). Thus, the risk of unwanted signal associated with unbound / uncomplexed detectable species may be reduced.

[0170] The sample inlet may be positioned adjacent to the sample chamber. In some embodiments, the sample inlet is located such that the inlet substantially lies in the same plane as the assay chamber. In such configurations, the sample is introduced into the sample chamber such that the sample is delivered substantially parallel to the surface of the base and / or travels or flows across the surface of the base in a direction that is substantially parallel to the surface of the base.

[0171] The sample inlet may be located on, positioned on, or defined by the lid. In some embodiments, the sample inlet may be located such that the inlet lies substantially above the sample chamber. In such configurations the sample is introduced into the sample chamber such that the sample is delivered from a first direction that is substantially perpendicular to the surface of the base (e.g. from above when the test strip is held in a horizontal configuration). In some embodiments, the sample may strike and / or impinge the surface of the base prior to travelling or flowing across the first position in a direction that is away from the second position (and optionally also to facilitate the sample to travel or flow across the second position in a direction that is away from the first position).

[0172] The sample may be deposited in the sample inlet. The inventors have found that it is advantageous to position the sample in the sample inlet in the described configurations to minimise the risk of potential diffusion of the target binding moiety tethered to a magnetically moveable particle and / or the target capture moiety conjugated to a detectable species to the second position on sample deposition.

[0173] In use, the sample may be deposited in a sample inlet, the sample inlet being positioned at the first position or in between the first and second positions. For example, the sample may be deposited in a sample inlet positioned at one end of the sample chamber (or base) or in between the two ends of the sample chamber (or base).

[0174] The issue of unwanted signal is a particular issue when using sandwich complexes formed from a target analyte, a magnetically moveable particle and a detectable species such as those described herein - as the signal associated with any unbound / uncomplexed detectable species is also subject to amplification, for example by the first catalyst acting on its substrate. Thus, in these types of assays, it is critical to keep unbound / uncomplexed detectable species away from the second position. In order to minimise unwanted signal resulting from such unbound / uncomplexed catalysts, prior art methods typically employ multiple fluidic chambers, wash steps and / or complex strip designs. However, surprisingly, the present inventors have identified that positioning a sample inlet between a first and second position and causing the fluid flow of a sample to move across the first position and away from the second position can minimise unwanted signal associated with unbound / uncomplexed catalyst and avoid the need for such complex and expensive test strips. Furthermore, the present inventors have additionally identified that these configurations can allow the distance between the first and second positions to be reduced, minimising the overall size of the test strip and / or the volume of sample required. Typically strip designs with unidirectional fluid flow paths (e.g. a unidirectional flow of fluid following introduction of a sample, from a sample inlet through a reagent zone to a detection zone) require longer flow paths, multiple fluid chambers and / or wash steps, meaning that relatively larger volumes of sample are required.

[0175] In some embodiments, the sample inlet is positioned between the first and second positions such that introduction of a sample causes a substantially bi-directional fluid flow, one portion of sample flowing away from the sample inlet and across the first position in a direction that is away from the second position, and a second portion of the sample flowing away from the sample inlet and across the second position in a direction that is away from the first position.

[0176] In some embodiments, the sample inlet is positioned such that it is substantially equidistant or substantially midway between the first position and the second position. In some embodiments, the sample inlet may be positioned asymmetrically (e.g. asymmetrically on or within the test strip with respect to the first and second positions). In some embodiments, the sample inlet may be positioned closer to the first position than the second position. In some embodiments, the sample inlet may be positioned closer to the second position than the first position.

[0177] In some embodiments, the flow of liquid from the sample inlet furcates into two or more different directions (e.g. bifurcates). The result of this substantially bi-directional fluid flow (or a multi-directional fluid flow) may be to push the respective components at the first and second positions apart (the introduction of the sample fluid may be considered to wash the resuspended components in opposite directions and / or towards opposite ends of the sample). In some embodiments, the flow of liquid from the sample inlet bifurcates into 2 or more different directions such that diffusion between the first position and the second position is substantially reduced or eliminated. The test strip configurations as described herein assist in minimising the amount of unbound / uncomplexed detectable species that contacts the second position.

[0178] In alternative embodiments, the sample inlet is positioned at, or in proximity, to one end of the sample chamber. Typically, the sample inlet may be positioned at the end comprising the first position, or in proximity to the first position. This positioning of the sample inlet allows for a simpler strip design, and also allows for easier access to the sample inlet, for example when the strip is inserted into a device. In some embodiments, activating or generating a magnetic field causes the complex to move in a lateral direction, e.g. along the test strip to the second position such that the complex is separated from one or more other components of the mixture (e.g. any unbound target capture moiety conjugated to a catalyst within the mixture). By a lateral direction is meant that the complex moves across the horizontal rather than the vertical plane of the test strip. For example, where the strip comprises a substrate that is a thin sheet of material, the complex may move horizontally across the surface with the largest surface area.

[0179] As stated above, the target capture moiety conjugated to the detectable species is positioned on or within the test strip (e.g. on or within the sample chamber) at a first position. The positioning of the target capture moiety may be by depositing the target capture moiety at the first position, for example by any suitable means of contacting. In some cases, the target capture moiety conjugated to a detectable species may be deposited onto the strip as a solution or suspension, for example by spraying ordripping, and the strip is allowed to dry. In some embodiments, the target capture moiety conjugated to a detectable species is printed onto the test strip at the first position. As stated above, the first position may be at one end of the test strip, for example at one end of a base, particularly at one end of the sample chamber.

[0180] In some embodiments, the target binding moiety tethered to a magnetically moveable particle is positioned on or within the test strip. As will be appreciated, this component may be positioned at any suitable position at the start of the method as described herein provided that it is present at the first position at some point for the incubation stage. In some embodiments the target binding moiety tethered to a magnetically moveable particle is positioned on or within the test strip by any suitable means of contacting. In some embodiments, the target binding moiety tethered to a magnetically moveable particle is deposited onto or within the strip as a solution or suspension, for example by spraying or dripping, and the strip is allowed to dry. In some embodiments, the target binding moiety tethered to a magnetically moveable particle is printed onto the test strip, e.g. onto the base.

[0181] In some embodiments, the target binding moiety tethered to a magnetically moveable particle is positioned on or within the test strip at the first position. In such embodiments, the target binding moiety tethered to a magnetically moveable particle is already present at the first position prior to the incubation stage. In some embodiments, the target binding moiety tethered to a magnetically moveable particle is positioned on or within the test strip at a different position to the first position. For example, the target binding moiety tethered to a magnetically moveable particle may be positioned between the first and second positions, or at the second position. In such embodiments, the target binding moiety tethered to a magnetically moveable particle may be moved to the first position prior to the incubation stage. For example, the target binding moiety tethered to a magnetically moveable particle may be magnetically moved to the first position. Additionally, or alternatively, the target binding moiety tethered to a magnetically moveable particle may be moved to the first position following introduction of the sample (e.g. may be moved with the sample as the sample travels or flows across the sample chamber).

[0182] In some embodiments, the target binding moiety tethered to a magnetically moveable particle and the sample may be contacted on or within the test strip by any suitable means. For example, each may be deposited separately on or within the test strip. Alternatively, both components may be added to the strip simultaneously. In some cases, the target binding moiety tethered to a magnetically moveable particle and the sample may be independently deposited onto or within the strip as a solution or suspension, for example by spraying or dripping.

[0183] The target binding moiety tethered to a magnetically moveable particle and the target capture moiety conjugated to a detectable species may each be separately or simultaneously deposited onto or within the strip as a solution or suspension, for example by spraying or dripping, and the strip is allowed to dry. The target binding moiety tethered to a magnetically moveable particle and the target capture moiety conjugated to a detectable species may each be separately or simultaneously printed onto or within the test strip, e.g. at the first position.

[0184] The target binding moiety tethered to a magnetically moveable particle may be positioned at one end of the test strip, such as one end of a base, for example at the same end as the target capture moiety conjugated to a detectable species. Where the test strip comprises a sample chamber and / or base that is dumbbell-like in shape or is a C-shaped channel, both the target capture moiety conjugated to a detectable species and the target binding moiety tethered to a magnetically moveable particle may be positioned at the same one end of the dumbbell-like shape or C-shaped channel.

[0185] The inventors have found that combining the target binding moiety tethered to a magnetically moveable particle and the target capture moiety conjugated to a detectable species before deposition onto the test strip can result in aggregation of the two, increasing the chance of false positive results. Thus, the target binding moiety tethered to a magnetically moveable particle is typically deposited onto or within the strip separately from the target capture moiety conjugated to a detectable species. Additionally or alternatively, the magnetically moveable particles may be surface modified in order to reduce aggregation. This is further described below. In some embodiments, the target binding moiety tethered to a magnetically moveable particle is positioned on or within the test strip at a position, such as at one end of the sample chamber, that does not overlap with the target capture moiety conjugated to a detectable species. In some embodiments, the target binding moiety tethered to a magnetically moveable particle and target capture moiety conjugated to a detectable species are each present (e.g. deposited) in different sections of the reagent zone (e.g. such that they do not overlap or do not substantially overlap). During the test assay these components may become re-suspended as the sample travels or flows across the first position.

[0186] As above, and for the avoidance of doubt, the second position may be positioned anywhere on the base or the lid. In some embodiments the second position is within the test strip, for example the second position may be enclosed by the positioning of the base with respect to the lid. In some embodiments, the second position is on the base and within the test strip, e.g. on the base and enclosed by the lid of the strip.

[0187] The test strip may comprise a base, which may comprise a film. The film may comprise a hydrophilic or hydrophobic polymer, or a hydrophilic or hydrophobic treatment of a polymer to either promote or hinder wetting or fluid movement or diffusion. Hydrophilic polymers, such as some polyesters, may be beneficial owing to improved wetting of the base by the sample, and improved wicking of the sample across the surface of the base.

[0188] The test strip, e.g. the base, may further be treated with a polymer suitable for hindering diffusion of large molecules (such as molecules of a molecular weight greater than about 100 Da, in some embodiments molecules of a molecular weight greater than about 4000 Da, in some embodiments molecules of a molecular weight in the range of 1 MDa) across the surface of the base. This way, the diffusion of any unbound target capture moiety conjugated to a catalyst to the second or third positions on the base may be hindered. For the avoidance of doubt, the polymer suitable for hindering diffusion of large molecules may also hinder, but to a much smaller extent, the movement of the complex to the second position on activation or generation of the magnetic field. In some embodiments, the polymer is positioned at least at the first position. The polymer may comprise or consist of one or more of the group consisting of carboxymethyl cellulose, polyethylene glycol, dextran, dextrin, and polystyrene. The polymer suitable for hindering diffusion of large molecules may also hinder the diffusion of the redox mediator and / or the catalyst substrate so that diffusion of redox mediator and / or catalyst substrate to the first position may be hindered.

[0189] The first and second positions on or within the test strip may be at least partially or entirely covered by the mixture. In particular embodiments, the first and positions on or within the strip are at least partially or entirely covered by the mixture, which comprises a single body of fluid, i.e. the mixture is not broken up.

[0190] The target capture moiety and target binding moiety need not be limited, provided they are capable of binding to the target at separate binding sites. For example, in certain embodiments the target binding moiety and the target capture moiety may each independently comprise amino acids and / or nucleic acids, or derivatives thereof. In some embodiments, the target binding moiety and the target capture moiety may each independently comprise at least one selected from the group of: DNA (e.g. single stranded DNA), RNA, PNA or LNA molecule, a morpholino, a peptide, a protein, an antibody (including a nanobody), or an antigen binding fragment thereof, an antibody mimetic, a cell surface marker, a cell surface receptor or its ligand, ora biologically active fragment of a cell surface marker, a cell surface receptor or its ligand, an aptamer, a molecular imprinted polymer, an enzyme, a lipid, a glycan, a glycoprotein, a glycolipid, a proteoglycan, or a molecularly imprinted polymer (MIP) protein, DNA and RNA, and combinations thereof. In some embodiments the target capture moiety and target binding moiety include a fusion of one or more of the above moiety types, e.g. a hybrid aptamer-molecularly imprinted polymer (apta-MIP). Moreover, the target capture moieties and target binding moieties may be fused or otherwise bound to the magnetically moveable particle or redox catalyst using techniques well known in the art.

[0191] MIPs are synthetic receptors for a targeted molecule and comprise artificial receptor sorbents of a target molecule on a polymer network. A concise review of MIPs is provided by T. Sajini and Beena Mathew, in “A brief overview of molecularly imprinted polymers: Highlighting computational design, nano and photo-responsive imprinting”, Taianta Open 4, 2021, 100072.

[0192] Tagging with nucleic acid sequences is well-known in the art and may be carried out using standard bio-conjugation chemistries (see, for example O. Koniev and A. Wagner, Chem. Soc. Rev., 2015, 44, 5495; and C. Sornay et al., R. Soc. Open Sci., 2022, 9:211563). For example, N-hydroxysuccinimidyl (NHS) ester coupling with amines, carbodiimide (such as 1-ethyl-3-(3-dimethylaminopropyl)cabodiimide (EDO), dicyclohexylcarbodiimide (DOC) or diisopropylcarbodiimide (DIG)) coupling with amines, biotin coupling with Streptavidin (see, for example, C. M. Dundas et al., Appl. Microbiol. Biotechnol., 97, 9343-9353 (2013)); or maleimide coupling with thiol moieties (see, for example, S. S. Ghosh et al., Bioconjug. Chem., 1990, 1:71-6) may be used.

[0193] In some embodiments, the target binding moiety and the target capture moiety each independently comprise one or more selected from the group consisting of protein, DNA and RNA. The target binding moiety and the target capture moiety may each independently comprise one or more selected from the group consisting of an antibody, an antigen-binding antibody fragment, an antibody mimetic and a nucleic acid strand (e.g. a DNA or RNA strand). Often, the target binding moiety and the target capture moiety each independently comprise one or more selected from the group consisting of an antibody, an antigen-binding antibody fragment and an antibody mimetic.

[0194] As used herein, the phrase “antigen-binding fragment thereof”, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigenbinding fragment thereof” include (i) a Fab fragment, a monovalent fragment consisting of the VH, VL, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VH and VL domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544 546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VH and VL, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VH and VL regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423 426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879 5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment thereof”. These antigen binding fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0195] The inventors have found that the method of the invention is capable of detecting the presence of target in a sample at concentrations less than 50 pg / mL, e.g. as low as 10 pg / mL or 1 pg / mL. In some cases, the method of the invention is capable of detecting the presence of target in a sample at concentrations less than 1 pg / mL, such as around 500 fg / mL or lower. In some embodiments, the methods described herein may be used to detect the presence of a target at concentrations less than or equal to 100pg / mL. For example, the methods described herein may be used to detect the presence of a target at concentrations from 500 fg / mL to 100pg / mL, or1 pg / mL to 100pg / mL, such as 1 pg / mL to 100pg / mL. In particular embodiments, the method is capable of detecting the presence of a target at a concentration of 500 fg / mL, 1pM, 5 pM or 10 pM in a sample, such as 1pM, 5 pM or 10 pM, e.g. 500 fg / mL, 1 pM or 10 pM or more of a target in a sample, such as 1 pM or 10 pM or more of a target in a sample.

[0196] The electrodes of the fuel cell may be made of any material suitable for conducting electrons. It is preferable that the material is resistant to corrosion, and is able to conduct a suitable current load. Sometimes, the current load required is in the range of ± 1 nA to ± 1 mA; ± 10 nA to ± 0.1 mA; or ± 100 nA to ± 0.01 mA (with an error of ± 1%). Suitable materials include any one or a selection from the group consisting of graphene, graphite, gold, silver, platinum, palladium, titanium, carbon, brass, tungsten, ruthenium, iridium, titanium, nickel, aluminium, tin, chromium, copper, or one or a selection of their oxides. Examples of carbon electrodes include electrodes comprising carbon ink (e.g. conductive carbon ink, such as EDAG carbon, e.g. LOCTITE EDAG supplied by Henkel), carbon paste (such as D3 carbon paste supplied by Sun Chemical under product code C2090225D), or carbon nanotubes (single- or multi-walled). Often, the electrodes of the system are made from any one or a selection from the group consisting of graphene, graphite, gold, silver, platinum, palladium, titanium, graphite and carbon. Typically, the electrodes are made from any one or a selection from the group consisting of graphene, graphite, gold, silver, platinum and palladium, preferably graphene. Alternatively, the electrodes are made from carbon, such as carbon inks (e.g. EDAG carbon as described above), carbon pastes (e.g. D3 carbon as described above), or carbon nanotubes (single- or multi-walled). In particular embodiments, the electrodes are made from EDAG carbon where a TMB substrate is used. In particular embodiments, the electrodes are made from D3 carbon where a ferricyanide redox mediator is used. Commonly, the electrodes are made of one type of material, i.e. they are not made of a selection of materials.

[0197] The electrodes may be produced via additive printing processes, such as 3D-printing or screen-printing. These are suitable approaches for the production of cost-effective electrode systems and sensors (Tan, C., Nasir, M. Z. M., Ambrosi, A., Pumera, M., 2017. Anal. Chem. 89, 8995-9001). The electrode system may be microfabricated and may be produced by depositing the desired material, patterning the material with the desired micro features (e.g. by UV photolithography), and if necessary, removing or etching material. The electrodes may be screen-printed. Screen printed electrodes (SPEs) feature many advantages over more traditional electrodes such as ease of fabrication and cleaning procedures, reliability, low-cost, repeatability and provide rapid time to result. SPEs are amenable to mass production, whereby a large volume of electrodes can be produced at relatively low-cost compared to traditional macro or microelectrodes (Hayat, A., Marty, J. L., 2014. Sensors. 14, 10432-10453). Due to these advantages, SPEs lend themselves nicely to prototyping and for the development of novel sensing technologies, as reported here.

[0198] The electrodes may be porous. The inventors have found that porous electrodes are particularly useful owing to their much larger surface area, which improves the efficiency of electron transfer between the catalyst and the electrode.

[0199] Typically, where the electrodes are graphene electrodes, they comprise laser-induced graphene (LIG). LIG is a porous, conductive graphene material with a high surface area, and is typically formed through the burning of a polymer film with a CO2 infrared laser such that the polymer forms a graphene layer. See Zhengfan Wan et al., “Laser induced graphene for biosensors”, Sustainable Materials and Technologies, 2020, 25, e00205, for a review of LIG. In some embodiments, the electrodes are each formed through laser-induced graphene burning of a layer of polymer, such as polyimide. In some embodiments, the LIG is formed from a layer of polyimide (for example, a layer with a thickness of about 125 pm) using a diode laser (optionally with an output of about 1.6 W), operating at about 240 mW (or about 15% power for a 1.6 W laser), at a speed of about 500 mm / min, and at a line interval of about 0.01 mm.

[0200] The skilled person is aware that a response may be detected from the fuel cell at multiple time points. For example, a response may be detected before activating or generating the magnetic field and after activating or generating the magnetic field and the two responses compared to assess whether or not the target is present in the sample. This is particularly preferred where the detectable species comprises a redox mediator.

[0201] The test strip as described herein may comprise a plurality of layers. The first layer may comprise the anode and the cathode, and may be referred to herein as the electrode layer. The second layer may comprise the chambers such as the sample chamber, the separation chamber, and / or the counter chamber, and may be referred to herein as the microfluidic layer. In such embodiments, the first layer may be laminated to the second layer and the second layer may be laminated to the lid. In such embodiments, the lid may be considered as essentially or substantially a third layer.

[0202] Where there are greater than two layers, some layers may be laminated together prior to the formation of the complete assembled object. That is to say, some layers may be laminated to form a precursor strip before additional layers are added and laminated to form either a further precursor strip or the test strip. In other words, lamination of all layers does not have to occur in a single lamination step. For example, an inlet may be present and configured to deliver fluid or gel to the separation chamber after lamination.

[0203] Each of the layers may comprise and / or be formed of suitable materials, and / or be sized and dimensioned to facilitate the use of the test strip in the electrochemical detection of a target.

[0204] The first layer may comprise and / or be formed of a material that facilitates the formation of electrodes (e.g. by facilitating the deposition of electrode materials or by facilitating the generation of electrode materials underaction of irradiation, such as laser, heat, light or the like). The first layer may comprise and / or be formed of a transparent and / or a hydrophilic material. In some examples, the first layer may comprise and / or be formed of a polyimide material (e.g. Kapton tape). Where a polyimide material is used as the first layer, graphene electrodes may be formed under laser irradiation. In some examples, the first layer may comprise and / or be formed of a polymeric material. Suitable electrode materials (as described herein) may be deposited onto the polymeric material by screen printing or the like. Representative examples of the polymeric material include, but are not limited to, polyester (such as polyethyleneterephthalate) and polycarbonate. In some examples, the first layer may comprise and / or be formed of polyethyleneterephthalate. In some embodiments, the first layer may comprise and / or be formed of a heat stabilised polyester with a print receptive coating. In some examples, where the electrode material comprises a metal (e.g. titanium, chromium, copper, palladium and the like), the metal may facilitate adhesion of the layers. The first layer may comprise a thickness from about 10 pm to about 500 pm, from about 50 pm to about 400 pm, or from about 75 pm to about 300 pm, or from about 100 pm to about 250 pm. In some examples, the first layer may comprise a thickness of about 250 pm, about 175 pm or about 125 pm.

[0205] The second layer may comprise and / or be formed of a material that facilitates the formation of the microfluidic system and / or the adhesion of the second layer to the first and / or third layer. In some examples, the second layer may comprise and / or be formed of an adhesive material, such as a double-sided adhesive material. The second layer may comprise and / or be formed of a polymeric material, such as polyester. The polymeric material (e.g. polyester) may be coated on one or both sides with an adhesive material (e.g. an acrylate based adhesive). By way of example, the second layer may be a polyester material coated on both sides with an acrylate adhesive, such as 3MTM9965 tape, or ARCare® 8939 tape supplied by Adhesives Research®. The second layer may comprise a thickness from about 10 pm to about 500 pm, from about 50 pm to about 250 pm, or from about 75 pm to about 200 pm, or from about 80 pm to about 150 pm. In some examples, the second layer may comprise a thickness of about 90 pm, or about 125 pm.

[0206] The third layer may comprise and / or be formed of a material that is configured to adhere to the second layer. The third layer may comprise and / or be formed of a transparent and / or a hydrophilic material. The third layer may comprise, be treated with, and / or be coated with a surfactant material, for example to increase the hydrophilicity of the surface. The third layer may comprise and / or be formed of a polymeric film, such as a polyester film. Representative examples include, but are not limited to, 3MTM9984 tape and Lumirror® supplied by Toray Films. In other embodiments the third layer may be an injection-moulded piece. An injection-moulded piece may allow for larger volumes in the channels. Representative materials that can be used to form such an injection-moulded piece include, but are not limited to, polycarbonate, polyether ether ketone, and polyaryletherketone. In some examples, the third layer may comprise and / or be formed of polycarbonate. The third layer may comprise a thickness from about 10 pm to about 5 mm. By way of example, where the third layer comprises a polymeric film, the third layer may comprise a thickness from about 10 pm to about 500 pm, from about 25 pm to about 250 pm, or from about 50 pm to about 200 pm, or from about 75 pm to about 150 pm, such as about 100 pm. By way of further example, where the third layer comprises an injection-moulded piece, the third layer may comprise a thickness from about 0.5 mm to about 5 mm, or from about 2 mm to about 3 mm.

[0207] The second aspect of the invention provides a test strip for use in a method of detecting a target in a sample (e.g. for use in the first aspect), the test strip comprising:

[0208] (i) a sample chamber positioned on or within the test strip;

[0209] (ii) a target capture moiety conjugated to a detectable species positioned on or within the sample chamber at a first position;

[0210] (iii) a fuel cell comprising an anode and a cathode, wherein one of the anode or cathode is positioned at a second position and the other is positioned at a third position; optionally wherein the one of the anode and cathode positioned at the second position is configured for fluid communication with the sample chamber and the other of the anode and cathode positioned at the third position is not in fluid communication with the sample chamber.

[0211] Typically, the detectable species of the test strip comprises:

[0212] (i) a first catalyst and the test strip further comprises:

[0213] (e) a first catalyst substrate positioned at or in proximity to the second position; and

[0214] (f) a second catalyst and a second catalyst substrate each positioned at or in proximity to the third position; or

[0215] (ii) a redox mediator and the test strip further comprises:

[0216] (e) a first catalyst and a first catalyst substrate positioned at or in proximity to the second position; and

[0217] (f) a second catalyst and a second catalyst substrate, each positioned at or in proximity to the third position.

[0218] One of the anode and cathode positioned at the second position is configured for fluid communication with the sample chamber and the other of the anode and cathode positioned at the third position is not in fluid communication with the sample chamber. Typically, the anode and cathode are isolated (e.g. fluidly isolated) by a separator or a medium for the selective transportation of ions, such as a salt bridge (optionally an agar salt bridge or a polymeric material).

[0219] The anode and cathode are typically (e.g. electrically or electronically) connected to a sensor, such as an ammeter, such that a response from the fuel cell may be detected.

[0220] In some embodiments, the test strip further comprises a target binding moiety tethered to a magnetically moveable particle, optionally positioned on or within the sample chamber at the first position.

[0221] The test strip may be configured such that the introduction of a sample into the sample chamber via the sample inlet causes the sample to travel or flow across the first position in a direction that is away from the second position. For example, the sample inlet may be configured to deliver the sample (e.g. in the form of a fluid) into the sample chamber at between the first and second positions. The test strip may be configured such that the introduction of a sample into the sample chamber via the sample inlet causes the sample to travel or flow across the first position in a direction that is towards the second position. For example, the sample inlet may be configured to deliver the sample (e.g. in the form of a fluid) into the sample chamber at a first end of the sample chamber, the first end being in proximity to the first position and / or at a reagent zone.

[0222] The third aspect of the invention provides a system for use in a method of detecting a target in a sample, the system comprising:

[0223] (a) a test strip as defined in the second aspect; and

[0224] (b) a target binding moiety tethered to a magnetically moveable particle.

[0225] When a sample comprising a target is contacted with a target capture moiety conjugated to a detectable species and a target binding moiety tethered to a magnetically moveable particle, a complex is formed, the complex comprising the target capture moiety conjugated to a detectable species and the target binding moiety tethered to a magnetically moveable particle, each being independently bound to the target. In such cases, when the magnetic field is activated or generated, the complex moves to the second position such that the complex is separated from one or more other components of the mixture and such that the catalyst catalyses the conversion of the catalyst substrate to another product.

[0226] In some embodiments, the system further comprises one or more of the following: (a) a magnetic field generator configured to activate or generate a magnetic field; and

[0227] (b) a detector (or sensor) configured to detect the electrical response of the biofuel cell.

[0228] In some embodiments, the magnetic field generator may be an electromagnet positionable such that activation of the electromagnet generates a magnetic field capable of moving the magnetically moveable particle to the second position. In some embodiments, the magnetic field generator may be a permanent magnet positionable such that a magnetic field capable of moving the magnetically moveable particle to the second position is generated.

[0229] The detector or sensor may be any detector or sensor suitable to sense the electrical response of the biofuel cell that is characteristic of a reaction at the electrode, such as catalysis of a first catalyst substrate by a first catalyst. For example, the detector may be an ammeter. In some embodiments, the system of the third aspect further comprises a sample that may or may not comprise a target.

[0230] The fourth aspect of the invention provides a kit of parts, comprising, as separate components the system as defined in the third aspect.

[0231] By way of example, the kit of parts may comprise, as separate components: (a) a test strip as defined in the second aspect; and

[0232] (b) a target binding moiety tethered to a magnetically moveable particle.

[0233] The kit may further comprise:

[0234] a magnetic field generator configured to activate or generate a magnetic field; and

[0235] a detector (or sensor) configured to detect the electrical response of the biofuel cell.

[0236] The kit may further comprise one or more electronic connections capable of completing a circuit between the electrodes of the test strip of the system and the detector or sensor.

[0237] The fifth aspect of the invention provides for the use of the test strip of the second aspect, the system of the third aspect, or the kit of the fourth aspect to detect the presence or absence of a target in a sample. For example, the test strip of the second, third and fourth aspects may further comprise a sample that may or may not comprise a target. The sample may be added to the test strip to form a mixture as defined in the first aspect. The first and second positions may be at least partially or entirely covered by the mixture.

[0238] For the avoidance of doubt, each of the embodiments described in relation to the method of the first aspect apply mutatis mutandis to the test strip of the second aspect, the system of the third aspect, the kit of parts of the fourth aspect, and the use of the fifth aspect. For example, the detectable species may be a first catalyst or a redox mediator; the first catalyst substrate may be positioned at or in proximity to the second position; first catalyst may be a horseradish peroxidase or an oxidoreductase enzyme, such as an oxidoreductase enzyme; and / or the anode and cathode may comprise carbon, such as EDAG carbon, D3 carbon or carbon nanotubes (single- or multi-walled) or LIG, for example, LIG.

[0239] The methods, test strips, systems, kits and uses described herein are intended for use in detecting the presence of a target in a sample. The target may be an analyte. In particular, the target may be a component found (or potentially found) in a biological sample. In other examples, the target may be a component (such as a contaminant) found (or potentially found) in a sample taken from a manufacturing process.

[0240] In some examples, the target may be, or comprise, a chemical compound, a biological molecule or a pathogenic organism. In some embodiments, the target may be, or comprise, a protein, a peptide, a nucleic acid (e.g. a specific nucleic acid sequence), a lipid, or a small molecule. In some embodiments, the target may be a pathogenic organism (e.g. a bacteria, virus, fungus, or the like). In some embodiments, the target may be a hormone, a metabolite, a saccharide or polysaccharide, a drug (such as an antibiotic) or drug metabolite, or the like.

[0241] The sample may be a biological fluid. By way of example only, the biological fluid may be blood, plasma, serum, urine, sweat, stool, cerebrospinal fluid, interstitial fluid, saliva, sputum, nasal fluid or any other bodily fluid.

[0242] The sample may be a liquid sample. The sample may be diluted and / or mixed with a liquid (e.g. a solution or buffer) prior to introduction to the sample inlet. Accordingly, in some examples, the sample may comprise a biological fluid diluted and / or mixed in a liquid (e.g. a solution or buffer).

[0243] As stated above, the test strips and systems described herein can allow the distance between the first and second positions to be reduced, minimising the overall size of the test strip and / or the volume of sample required. In particular, the volume of a biological sample required may be minimised. In some embodiments, the methods may be suitable to detect the presence of a target in from about 1 pL to 1 mL of a biological sample (e.g. from about 1 pL to about 100 pL, or from about 1 pL to about 50 pL, e.g. from about 5 pL to about 20 pL of a biological sample). Such biological samples may be diluted and / or mixed with a biologically compatible liquid or buffer (e.g. a citratephosphate buffer) prior to introduction to the sample inlet.

[0244] Each and every patent and non-patent reference referred to herein is hereby incorporated by reference in its entirety, as if the entire contents of each reference were set forth herein in their entirety.

[0245] The invention may be further understood with reference to the following examples.

[0246] EXAMPLES

[0247] Methods

[0248] Four areas were initially investigated, which combine to form a working test strip. These were cathode, anode, strip design (microfluidics, electrodes and EBFC integration) and laser induced graphene (LIG) electrode creation. A further exemplary test strip design was used to test specific enzyme / substrate combinations, the effects of these on detection limits, the ability to detect Glial Fibrillary Acidic Protein (GFAP), and the ability to magnetically concentrate the detectable species.

[0249] Cathode Optimisation

[0250] Laccase from Agaricus Bisporus (LacAB) was chosen as a biocathode enzyme as it was commercially available (Merck) and has a high activity (>4 ll / rng by mass). 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was selected as a substrate as it has a high reduction potential (~0.7 V) and good activity with LacAB. A citratephosphate (C-P) buffer was used as it is reported to reduce the detrimental effects of peroxide generation on laccase enzyme activity and has a wide pH range for investigation of the optimal pH for enzyme activity.

[0251] The electrochemical signal was improved through two areas: buffer pH and substrate concentration. C-P buffer solutions of varying pH (3-7) were prepared by first preparing solutions of 0.1 M citric acid (58 mg of citric acid in 3 mL of dH2O) and 0.2 M sodium phosphate dibasic (96 mg of sodium phosphate dibasic in 3 mL of distilled H2O). The buffers were made by combining the 0.1 M citric acid and 0.2 M sodium phosphate dibasic solutions according to Table 1.

[0252] Table 1 - Volumes of 0.1 M citric acid and 0.2 sodium phosphate dibasic to combine to prepare 1.2 mL C-P buffer solutions of pH ranging 3-7.

[0253] pH Amount of 0.1 M Citric Amount of 0.2 M Total Volume

[0254] Acid (mL) Na2HPO4(mL) (mL)

[0255] 3 0.954 0.247 1.2

[0256] 4 0.738 0.463 1.2

[0257] 5 0.582 0.618 1.2

[0258] 6 0.443 0.758 1.2

[0259] 7 0.212 0.989 1.2

[0260]

[0261] 2.5 mg of LacAB was added to 0.5 mL of each C-P buffer. Separately, 2.5 mg of ABTS was added to 0.5 mL of each C-P buffer. The final concentrations of the LacAB stock solutions and ABTS stock solutions were 5 mg / mL (reported solubility limit for LacAB in distilled H2O). 30 pL of the tested LacAB solution and 30 pL of the tested ABTS solution were pipetted onto Flex Medical circular gold with Ag / AgCI pseudo-reference test strips (12.57 mm2working electrode surface area) and incubated at room temperature for 5 minutes. Electrochemical measurements were conducted using a PalmSens4 instrument. Each pH was measured in triplicate. Cyclic voltammetry (CV) was used to determine the ABTS reduction potential (Vc), chronoamperometry (CA) to obtain the charge density (pC / mm2) and open circuit potentiometry (OCP) to find the open circuit potential (Voc). The settings used for each measurement are set out in Tables 2, 3 and 4.

[0262] Table 2 - CV setings used for the confirmation of the reduction potential of ABTS.

[0263] Cyclic Voltammetry Value

[0264] E Begin 0 V

[0265] E vertexl 1.2 V

[0266] E vertex2 0 V

[0267] E Step 0.01 V

[0268] Scan Rate 0.1 V / s

[0269] t Interval 0.1 s

[0270]

[0271] Table 3 - CA setings used for the reduction of ABTS.

[0272] Chronoamperometry Value

[0273] Applied Potential 0.25 V

[0274] t Interval 0.1 s

[0275] t Run 30 s

[0276]

[0277] Table 4 - OCP setings used for the measurement of ABTS VOc.

[0278] Open Circuit Value

[0279] Potentiometry

[0280] t Run 30 s

[0281]

[0282] For optimisation of the substrate concentration, stock solutions of ABTS in 5 mM phosphate buffered saline (PBS) at pH 7.4 were prepared by serial dilution: 50 mg / mL, 25 mg / mL, 12.5 mg / mL, 5 mg / mL, 2.5 mg / mL and 1.25 mg / mL. 10 mg / mL LacAB was also prepared in 5 mM PBS at pH 7.4. The high solubility of LacAB here was unexpected, but the solution clarified. 30 pL of LacAB solution and 30 pL of ABTS solution were pipetted onto Flex Medical circular gold with Ag / AgCI pseudo-reference test strips (12.57 mm2working electrode surface area) and incubated at room temperature for 5 minutes. Electrochemical measurements were conducted using a PalmSens4 instrument - each concentration was measured in quadruplicate. CA was measured to obtain the charge density (pC / mm2). Table 3 notes the settings used for the electrochemical measurement.

[0283] Anode Optimisation

[0284] The sample of interest in this test was whole blood. Where glucose is used as the substrate of the catalyst at the anode, the concentration of substrate will be dictated by the concentration of glucose in a blood sample (around 5 mM average) and the pH will be about physiological pH (about pH 7.4).

[0285] Magnetic beads were conjugated with Pyrroloquinoline quinone-dependent glucose dehydrogenase (GDH-PQQ) to form MB-GDH-PQQ. This suspension was split into two equal aliquots and one was washed with glycine to perturb aggregation (MB-GDH-PQQ-Gly), the other MB-GDH-PQQ remained unwashed.

[0286] Solutions of 10:1 mM ferricyanide:glucose in 5 mM PBS and 1 mM glucose in 5 mM PBS were prepared as redox mediator or redox mediator-less substrate stock solutions. The suspensions of MB-GDH-PQQ or MB-GDH-PQQ-Gly were prepared from 1% solids stock suspensions of both. The beads were left to separate from the storage buffer on permanent magnet for 1 minute. 90% of the storage buffer solution was removed by pipette and replaced with 5 mM PBS.

[0287] 30 pL of MB-GDH-PQQ or MB-GDH-PQQ-Gly suspensions and 30 pL of ferricyanide:glucose or glucose solutions were pipetted onto Integrate Graphene Gii Sens ‘3D Graphene’ with Ag / AgCI pseudo-reference test strips (12.57 mm2working electrode surface area). The MBs were concentrated on the working electrode using a magnet and incubated at room temperature for 5 minutes. Electrochemical measurements were conducted using a PalmSens4 instrument - each condition was measured in triplicate. CA was measured to obtain the charge density (pC / mm2). The setting used for each measurement are set out in Table 5.

[0288] Table 5 - CA setings used for the oxidation of ferricyanide.

[0289] Chronoamperometry Value

[0290] Applied Potential -0.2 V

[0291]

[0292] t Interval 0.1 s t Run 30 s

[0293] Strip Design

[0294] FreeCAD was used to develop all the CAD files for test strip designs. An Epilog Helix Mini 50 W laser cutting instrument was then used to cut strip design elements. The microfluidic layer used 125 pm thick double-faced polyester tape with medical pressuresensitive adhesive, and the top lid was a 100 pm thick layer of transparent film treated with surfactant for hydrophilicity. The laser cutter was operated on vector mode with the settings outlined in Tables 6 and 7.

[0295] Table 6 - Vector settings for laser cutting the micro fluidic layer

[0296] Microfluidic Layer

[0297] Laser Power 10% (5 W)

[0298] Laser Speed 25%

[0299] Frequency 750 Hz

[0300]

[0301] Table 7 - Vector settings for laser cutting the top lid layer

[0302] Top Lid Layer

[0303] Laser Power 10% (5 W)

[0304] Laser Speed 30%

[0305] Frequency 750 Hz

[0306]

[0307] The salt bridge was formed from a polyelectrolyte gel using a solution of 65:2:2% w / v Diallyldimethylammonium chloride:2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone:N,N'-methylenebis(acrylamide) in distilled H2O. The monomer, photoinitiator and crosslinker undergo a rapid photopolymerisation process under a 365 nm UV lamp. Three experiments were conducted for the formation of the salt bridge by initiating the polymerisation: before placement of the top lid (1 minute), partially (10 seconds) and then full curing once the top lid has been secured (50 seconds), and after the top lid has been applied (1 minute). The initial testing was to determine the effectiveness of the salt bridge in creating a physical barrier between chambers. This was shown visually with blue food dye pipetted into sample chamber inlet and the counter chamber inlet in the top lid.

[0308] Laser-induced Graphene Electrodes Laser-induced graphene (LIG) electrodes were burnt into 125 pm polyimide film using the Snapmaker 3-in- 1 instrument with 1.6 W diode laser. These electrodes would allow for the replacement of Flex Medical and Integrated Graphene test strips when testing the EBFC immunoassay. Thana et al. reported using the Snapmaker for LIG preparation using the settings outlined in Table 8 (Thaweeskulchai, T.; Schulte, A. Diode Laser and Polyimide Tape Enables Cheap and Fast Fabrication of Flexible Microfluidic Sensing Devices. Micromachines 2022, 13, 2214).

[0309] Table 8 - Setings reported by Thana et al. for the production of LIG electrodes.

[0310] Thana et al.

[0311] Line Interval 0.05 mm

[0312] Laser Power 12.5% (200 mW) Laser Speed 500

[0313]

[0314] mm / min

[0315] Laser power and line interval were briefly investigated. Electrodes were subsequently prepared using updated settings that produced an entirely different type of electrochemical response than Thana etal.. The settings used are outlined in Table 9.

[0316] Table 9 - Setings reported by this work for the production of LIG electrodes.

[0317] This Work

[0318] Line Interval 0.01 mm

[0319] Laser Power 15% (240 mW)

[0320] Laser Speed 500 mm / min

[0321]

[0322] The LIG electrodes were tested by CV of 1 mM ferricyanide. The redox peak separation and general shape of the peaks were considered.

[0323] Table 10 - CV setings used for the evaluation of Thana et al. LIG electrodes.

[0324] Cyclic Voltammetry Value

[0325] E Begin 0 V

[0326] E vertexl 0.2 V

[0327] E vertex2 -0.7 V

[0328] E Step 0.01 V

[0329] Scan Rate 0.1 V / s

[0330] t Interval 0.1 s

[0331]

[0332] Table 11 - CV setings used for the evaluation of LIG electrodes from this work.

[0333] Cyclic Voltammetry Value

[0334] E Begin 0 V

[0335] E vertexl 0.5 V

[0336] E vertex2 -0.5 V

[0337] E Step 0.01 V

[0338] Scan Rate 0.1 V / s

[0339] t Interval 0.1 s

[0340]

[0341] Results

[0342] Anode

[0343] The physical separation of the sample chamber and counter chamber (where LacAB was housed) allowed for the maximisation of signal through the optimisation of factors affecting LacAB activity and electron transport between LacAB and the electrode.

[0344] Fig. 1 shows the charge density calculated from the CA data collected for the reaction of 2.5 mg / mL ABTS and 2.5 mg / mL LacAB in the C-P buffers over the pH range 3-7. All concentrations mentioned were the on-strip concentrations during electrochemical measurement.

[0345] The choice of substrate determines the optimal pH for LacAB activity. For comparison, pH 6 is best for LacAB acting on catechol. From Fig. 1, the maximum signal achieved for LacAB and ABTS is around pH 4. Compared with neutral, pH 7, which is closer to physiological pH, this is at least a 5-times increase in signal.

[0346] The Voc will also be affected by LacAB activity as it measures the concentration of charged species at the electrode surface - the greater the activity of LacAB, the higher the ratio of oxidised ABTS at the electrode surface compared with reduced ABTS and hence a different Voc. This principle is demonstrated in Fig. 2.

[0347] The greatest positive Voc was observed at pH 4 and was around 0.28 V. Voc dependence on pH is such that more positive values correspond to higher LacAB activity with ABTS. The lowest activity shown in Fig. 1 was found at pH 7, the lowest Voc in Fig.

[0348] 2 is also found at pH 7.

[0349] Cathode In this example, the substrate also acts as a redox mediator. The concentration of the substrate / redox mediator can be high to allow for efficient transfer of electrons to the electrode from the LacAB. Since, in this set up, the cathode is in a counter chamber and will not contact the sample (whole blood), there is no concern for signal loss on shuttling electrons using a redox mediator. In Fig. 3 a plateau is seen to form at on-strip concentrations above 10 mg / mL ABTS with 5 mg / mL LacAB in 5 mM PBS at pH 7.4.

[0350] There seems to be a decrease in signal when approaching 25 mg / mL ABTS which means the solution is oversaturated with substrate / redox mediator and the electrochemistry and / or the activity of the enzyme is impeded. The optimal concentration in 5 mM PBS at pH 7.4 is around 12.5 mg / mL ABTS to 5 mg / mL LacAB.

[0351] Direct Electron Transfer to the anode

[0352] In the sample chamber, the absence of a redox mediator is preferable to prevent signal loss during the shuttling of electrons. Redox mediator replacements include redox cofactors used for DET. Fig. 4 shows different scenarios for testing the DET response of GDH-PQQ conjugated to magnetic beads: ones washed with glycine and ones not washed with glycine, and those with or without the redox mediator (500 pM ferricyanide).

[0353] The charge density is greatest when the redox mediator is present and the MB-GDH-PQQ is not washed with glycine. When washed with glycine and in the presence of redox mediator, there is a small decrease in the charge density compared with the unwashed sample. DET behaviour can be seen with the glycine-washed MB-GDH-PQQ without redox mediator, where there is a clear ~3 pC / mm2signal. The signal is around half that achieved by the redox-mediated and glycine-washed sample. In contrast to the glycine-washed sample, the unwashed MB-GDH-PQQ sample does not give any signal without a mediator. Without being bound by theory, the inventors think that the magnetic beads of the glycine-washed sample might be charged, thereby attracting the enzyme to and potentially immobilising the enzyme at the electrode surface.

[0354] Test Strip Microfluidics

[0355] The first design of an EBFC immunoassay test strip is shown in Fig. 5. The features are as follows: 1. sample chamber rear vent, 2. sample chamber channel, 3. sample chamber inlet for sample deposition, 4. working electrode (bioanode), 5. sample chamber front vent, 6. counter chamber inlet for biocathode reconstitution, 7. salt bridge area, 8. counter electrode (biocathode), 9. counter chamber vent, 10. counter electrode connector pad, 11. working electrode connector pad. The sample chamber channel was made 1 mm wide to reduce the sample volume to around 5 pL. Each vent has a cross-like design between double-faced layer and top lid layer to prevent small deviations in lamination of these layers altering the size and shape of the vent, thus leading to changes in airflow and fluidics.

[0356] The anode was 1 mm long, and the cathode was 2 mm long to account for the lower activity of LacAB with ABTS compared to GDH-PQQ with glucose. The design of the double-faced layer can be altered to change the shape or size of the electrode surface area.

[0357] The counter chamber is separated from the sample chamber by the salt bridge between anode and cathode. The counter chamber can hold around 3 pL for reconstitution of the cathode using the counter chamber inlet on the side.

[0358] The working and counter electrodes connector pads are designed to fit into commercially available test strip connectors - Metrohm, PalmSens and TaiDoc connectors have been tested.

[0359] Test Strip Salt Bridge

[0360] The salt bridge of the design in Fig. 5 was positioned such that the polyelectrolyte gel would either be fully cured before test strip lamination or it would be partially cured beforehand and completely cured after the test strip was fully constructed.

[0361] An attempt to fill the sample chamber with blue dye when the salt bridge gel had been cured fully or partially before the test strip layers were fully laminated failed. Upon pipetting blue dye into the sample chamber inlet, the dye fully filled the sample chamber. The dye also began filling the counter chamber. This occurred on strips prepared from both partially and fully cured gels. Without being bound by theory, the inventors concluded that poor contact between layers created spaces between layers where the dye could penetrate: the firmness of the gel prevented the layers from pressing together completely.

[0362] The design was revised to allow for the curing of the gel once the full strip was laminated. In Fig. 6, the new design features are labelled. The new features are as follows: 1. sample chamber front vent (deviated), 2. salt bridge gel inlet, 3. narrow salt bridge channel, 4. counter chamber inlet (deviated).

[0363] The new design allowed for the strip to be fully constructed - electrode layer, microfluidic layer and top lid layer - then firmly sealed by pressing and deposition of ~0.2 pL of gel monomer solution before curing under a UV lamp for 1 minute. The new design did not exhibit any leakage of the dye from the sample chamber into the counter chamber, nor was there any leakage of dye from the counter chamber into the sample chamber. It even prevented dye from seeping into the salt bridge when both the sample and counter chambers were filled with dye.

[0364] LIG Electrode Manufacture

[0365] Thana et al. reported using the Snapmaker instrument to manufacture LIG electrodes. Fig. 7 is a cyclic voltammogram of 1 mM ferricyanide using LIG electrodes prepared with settings from Thana et al.

[0366] The classic duck shape for the reduction and oxidation of ferricyanide is prevalent. Peak separation is around 190 mV. Oxidation peak current is around 60 pA and the reduction peak current is just over 60 pA.

[0367] In contrast, the parameters for laser power and line fill can be tweaked to provide a more capacitive material, shown in Fig. 8. The more rectangular shape of this cyclic voltammogram suggests that the material is capacitive. The peak separation in this case is around 160 mV. The peak currents are far smaller than for Thana’s electrode. Oxidation peak current is around 9 pA and the reduction peak current is around 10 pA. Capacitive behaviour can be indicative of a porous electrode surface, which may be beneficial to DET as it allows for greater surface area for contact with the catalyst.

[0368] Test Strip

[0369] An annotated embodiment of the invention is shown in Fig. 9, depicting a test strip 100. The test strip 100 comprises a first layer 116 that comprises a first electrode (e.g., an anode) 104, a second electrode (e.g., a cathode) 108, a first connector pad 110 (e.g., for a cathode (or counter electrode)), and a second connector pad 111 (e.g., for an anode (or working electrode)). The test strip 100 additionally comprises a second layer 115 that comprises a sample chamber 102 and a separation chamber 107. Additionally, the second layer 115 is laminated to a lid 114, wherein there is an inlet 113 configured to deliver the fluid or gel to the separation chamber 107 after the lamination. In some embodiments, the first electrode 104 is an anode, the second electrode 108 is a cathode, the first connector pad 110 is a cathode connector pad or a counter electrode connector pad, and the second connector pad 111 is an anode connector pad or a working electrode connector pad. Also depicted is a sample chamber deviation channel 112, with a sample chamber front vent 105 positioned at said deviation. Additionally, the sample chamber 102 has a sample chamber rear vent 101 positioned at the other end of the channel. Further, the sample chamber 102 comprises a sample chamber inlet 103. Also shown in this embodiment of a test strip 100 is a counter chamber 117, comprising a counter chamber deviation 118, where the counter chamber inlet 106 is positioned, such that the counter chamber vent 109 is positioned at the other end of the channel.

[0370] Further test strip design

[0371] A further annotated embodiment of the invention is shown in Fig. 10, depicting a test strip 200 comprising three layers. Fig. 10A, 10B, and 10C depict the separated layers for clarity; a composite of the layers is depicted in Fig. 10D. The layers are: (A) a first layer 216, referred to as an electrode layer; (B) a second layer 215, referred to as a microfluidic layer (or an adhesive layer); and (C) a third layer 214, referred to as a lid or a lid layer.

[0372] The first layer 216 comprises a first electrode such as a working electrode (e.g., a cathode) 204, a second electrode such as a counter electrode (e.g., an anode) 208, a first connector pad 210 (such as for a counter electrode (e.g. for an anode)), and a second connector pad 211 (such as for working electrode (e.g. for a cathode)).

[0373] The second layer 215 comprises a sample chamber 202 and a separation chamber 207. Additionally, the second layer 215 is laminated to a lid 214, wherein there is an inlet 213 configured to deliver the fluid or gel to the separation chamber 207 after the lamination. In this particular embodiment, the separation chamber 207 comprises a further deviated channel 219, configured to receive a fluid or gel, wherein the inlet 213 is positioned above one end of the channel 219. In some embodiments, the first electrode 204 is a cathode, the second electrode 208 is an anode, the first connector pad 210 is an anode connector pad or a counter electrode connector pad, and the second connector pad 211 is a cathode connector pad or a working electrode connector pad. Further, the sample chamber 202 comprises a sample chamber inlet 203. In this particular embodiment, the sample chamber inlet is positioned at one end of the sample chamber 202. Also shown in this embodiment of a test strip 200 is a counter chamber 217, comprising a counter chamber inlet 206. In this particular embodiment, the counter chamber inlet is positioned at one end of the counter chamber 217. Additionally, in this particular embodiment, the lid comprises a sample chamber vent 205, positioned at one end of the sample chamber 202 (e.g., the opposite end to where the sample chamber inlet 203 is positioned), and a counter chamber vent 209, positioned at one end of the counter chamber 217 (e.g., the opposite end to where the counter chamber inlet 206 is positioned).

[0374] The altered strip design was used to test specific enzyme / substrate combinations, the effects of these on detection limits, the ability to detect Glial Fibrillary Acidic Protein (GFAP), the ability to magnetically concentrate the detectable species, and the ability to detect recombinant HIV-1 reverse transcriptase and magnetically concentrate the detectable species (horseradish peroxidase in this example).

[0375] Tests A to E: Materials

[0376] All assay solutions were acquired from Roche Reverse Transcriptase Assay, colorimetric. The 500 nm streptavidin-coated magnetic beads were acquired by Ademtech. The test strips were printed by Caiman using EDAG ink acquired from Loctite and Caiman’s own dielectric ink, on Caiman’s own polyester sheets. The TMB and peroxide stabilised substrate solutions A and B were acquired from R&D Systems. Glucose oxidase, potassium ferricyanide, D-glucose, PBS, DADMAC, MA and HEMP were acquired from Fisher Scientific or Merck / Sigma Aldrich.

[0377] Test A: Sensitivity and operation of further test strip design using peroxidase from horseradish type II (HRP) as the detectable species at the working electrode In this example, HRP was exemplified as the detectable species. 3, 3', 5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide were used as example substrates of HRP. TMB is a chromogenic substrate, which is colourless in its neutral, relaxed state, and is blue / yellow in its oxidised state (for example on oxidation with HRP). HRP, TMB and hydrogen peroxide were positioned at the working electrode, i.e. at the second position, which in this example was the cathode. In this example, Glucose oxidase type X-S (GOx) was exemplified as a second catalyst positioned at or in proximity to a third position, which was at the counter electrode, in this case the anode. D-glucose was used as an example substrate of GOx. In this example, a redox mediator (potassium ferricyanide) was used at the anode. In addition, a UV-curable polyelectrolyte (PE) gel was used as a medium for selective transport of ions between the anode and cathode.

[0378] Preparation of GOx:ferricyanide:qlucose solution (for the counter chamber)

[0379] 36 mg of D-glucose was added to 5 mL of phosphate-buffered saline (PBS) to prepare a 40 mM D-glucose solution. 1 mg of GOx was added to 1 mL of PBS to prepare a 1 mg / mL GOx solution. 34 mg of potassium ferricyanide was added to 10.4 mL of PBS to prepare a 10 mM ferricyanide solution. 200 pL of 10 mM ferricyanide solution was added to a 1.5 mL tube. 200 pL of glucose oxidase solution was added to the tube. Then, 400 pL of glucose solution was also added to the tube. The tube was vortexed for 5 s and left on the bench for 20 mins. Preparation of HRP standard dilutions (for the sample chamber)

[0380] 1 mg of HRP was added to 1 mL of PBS (1 mg / mL). This was covered with foil and put on a windmill mixer for 30 mins at room temp. Dilutions were prepared. For a 20 pg / mL HRP solution, 8 pL of 1 mg / mL HRP in PBS was added to 392 pL of PBS. For serial dilutions of 2 pg / mL to 20 pg / mL, 50 pL of each previous dilution was added to 450 pL of PBS. All dilutions were aspirated to mix. The final on-strip concentrations of HRP used were 1 pg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL, blank (0 g / mL).

[0381] Preparation of UV-curable PE Solution (used to form a medium for selective transport of ions between the anode and cathode)

[0382] Diallyldimethylammonium chloride (DADMAC) solution, 65% in H2O, was used as the base of the PE solution. 20 mg of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (HEMP) and 20 mg of N,N'-methylenebis(acrylamide) (MBA) were added to 1 mL of DADMAC in a 1.5 mL tube. The solution was held in a sonic bath for 1 minute and vortexed to dissolve the components.

[0383] Formation of a PE gel from the UV-curable PE solution

[0384] Using the fully assembled strips of the further test strip design, 1.35 pL of PE solution was pipetted into the separation chamber via the separation chamber inlet. A UV lamp was left on for 5 mins to warm up the bulb. The strips were placed on the middle shelf of the UV lamp stand (365 nm). The UV-curable PE solution was exposed to UV for 30s. After this time, the PE solution formed a gel.

[0385] Chronoamperometry measurements of the current density detected at different HRP concentrations

[0386] Strips prepared with the gel (described in the preceding section) were inserted into palmsens potentiostat connectors. These connectors were wired to a palmsens single potentiostat and 8-channel multiplexer. 5 pL of the prepared GOx solution was pipetted into the counter chamber. The chosen HRP dilution was mixed 1:1 with the corresponding TMB / H2O2 substrate solution (comprising 1:1 stabilised TMB:stabilised peroxide, color reagents A and B supplied by R&D systems), 20 pL and 20 pL, using aspiration to mix. 5 pL of the HRP:TMB / H2C>2 mixture was pipetted into the sample chamber through the sample chamber inlet. 5 mins was allowed for incubation of HRP with TMB. Chronoamperometry measurements were taken with the settings set out in the table below:

[0387] Parameter Setting

[0388] Applied Potential 0 V

[0389]

[0390] Interval 0.1 s

[0391] Duration 5 s

[0392] Data Analysis

[0393] The end current value of each chronoamperometry scan was used in calculations of current density. Current Density (nA / mm2) = (Current at 5 s (nA)) I (Area of Working Electrode(mm2)). The area of the working electrode was 1.5 mm2on the strips used for this experiment. The mean and standard deviation of the current densities were calculated for each concentration across 3 replicate measurements. These were plotted in a histogram with error bars at ±1 x standard deviation (see Fig. 11).

[0394] Conclusions

[0395] No bubbles formed over the electrodes, which is beneficial in preventing potential issues in signal measured. A clear signal was achieved for every concentration of HRP tested. All concentrations of HRP, except 10 pg / mL, gave a negative current density. This is expected as the TMB is reduced by the working electrode (the cathode in this example), giving a negative reduction current.

[0396] The blank produced a positive signal, suggesting some oxidation current passed into the working electrode from residual electrochemical effects in the sample chamber. Without being bound by theory, the inventors believe that a species in the sample chamber was present in its reduced form and was oxidised during the measurement of the blank solution. The cell was run using an applied voltage of 0 V. This allowed for any spontaneous electrochemical reactions between the electrodes to occur. It is not possible to determine the cause of unintended spontaneous reactions, only that they are not detrimental provided they do not interfere with the sensitivity of the measurements.

[0397] There was a clear difference in current density (nA / mm2) between the lowest concentration of HRP tested (10 pg / mL) and the blank ( 0 pg / mL). This difference was statistically significant, p=0.004, where a two-tailed t-test was used on these two concentrations. The very high sensitivity of this unoptimized assay was unexpected. Test B: Sensitivity at lower glucose concentrations and lower HRP concentrations of experimental set-up described in Test A

[0398] To assess the sensitivity of the experimental set up described in Test A to the concentration of glucose used at the counter electrode (in this example, the anode), the experimental details and set-up were the same as for Test A, except for the glucose concentration used in the counter chamber and, in some cases, the HRP and TMB / H2O2 concentration used in the sample chamber. 10 mM of D-glucose was investigated, and the HRP concentration was also decreased to a lowest standard solution of 1 pg / mL, rather than 10 pg / mL.

[0399] Preparation of GOx:ferricyanide:glucose solution (for the counter chamber)

[0400] This was the same as that disclosed for Test A, except that 36 mg of D-glucose was added to 1.25 mL of PBS to prepare a 160 mM glucose solution. A 10 mM concentration of D-glucose was made by serial dilution using 1:1 volumes of glucose solution and PBS.

[0401] Preparation of HRP standard dilutions (for the sample chamber)

[0402] The same method was used as disclosed for Test A. The final on-strip concentrations of HRP used were 10 pg / mL, 1 pg / mL and Blank (0 g / mL).

[0403] All other methods were the same as for Test A. The results are shown in Fig. 12.

[0404] Conclusions

[0405] A clear signal was achieved for each concentration of HRP. The extremely high sensitivity was greater than anticipated, particularly in view of the use of an unoptimized method, e.g. without optimisation of enzyme conditions.

[0406] The current densities were positive, suggesting the contribution of spontaneous reactions (excluding the HRP-TMB / H2O2 reaction) were greater than the contribution of the HRP-TMB / H2O2 reaction. However, the balance of the negative reductive current (from the HRP reaction) and positive oxidative current (from the unknown spontaneous reactions) provides dose-response behaviour and a clear difference from the positive current density of the blank.

[0407] There was a clear difference in current density (nA / mm2) between the lowest concentration of HRP tested (1 pg / mL) and the blank ( 0 pg / mL). This difference was statistically significant, p=0.00022, where a two-tailed t-test was used on these two concentrations. The results indicate that even lower concentrations of HRP (such as 500 fg / mL HRP) might be detectable in the 10-20 mM glucose concentration range.

[0408] Test C: Sensitivity and operation of further test strip design to detect Glial Fibrillary Acidic Protein (GFAP)

[0409] To assess the sensitivity of the experimental set up described in Test A to GFAP detection, the supernatant from a plate assay prepared using a Human GFAP DuoSet™ ELISA kit was applied to the experimental set up described in Test A, in place of the HRP:TMB / H2O2 mixture.

[0410] GFAP plate preparation

[0411] The contents of the Human GFAP DuoSet™ ELISA kit are as follows:

[0412] • PBS: 137 mM NaCI, 2.7 mM KCI, 8.1 mM Na2HPO4, 1.5mM KH2PO4, pH 7.2-7.4, 0.2 pm filtered (R&D Systems, Catalog # DY006)

[0413] • Wash Buffer: 0.05% Tween® 20 in PBS, pH 7.2-7.4 (R&D Systems, Catalog # WA126)

[0414] • Block Buffer: 1% BSA in PBS, pH 7.2-7.4, 0.2 pm filtered (R&D Systems, Catalog # DY995).

[0415] • Reagent Diluent: 1% BSA in PBS, pH 7.2-7.4, 0.2 pm filtered (R&D Systems, Catalog # DY995).

[0416] • Substrate Solution: ELISA TMB Substrate (R&D Systems, Catalog # DY999B or DY999B-250). The substrate solution comprises H2O2.

[0417] • Human GFAP Capture Antibody (Part #844350)

[0418] • Human GFAP Detection Antibody (Part #844351)

[0419] • Human GFAP Standard (Part #844352)

[0420] • Streptavidin-HRP B (Part #893975)

[0421] • Microplates: R&D Systems (Catalog # DY990)

[0422] • Plate Sealers: ELISA Plate Sealers (Catalog # DY992)

[0423] Day 1

[0424] The reconstituted capture antibody was diluted from 480 pg / mL to a working concentration of 4 pg / mL by adding 25 pL of the 480 pg / mL solution provided to 2975 pL of plate coating buffer (PBS) and aspirating and inverting to mix. The plate was coated in the diluted capture antibody with 100 pL in each well. The plate was sealed with an adhesive strip and incubated overnight at room temperature. Day 2

[0425] STANDARD WASH PROCEDURE (used for every wash step). All liquid was pipetted from the wells. 400 pL of 1x wash buffer was added to each well. The solution was aspirated 4 times. The wash buffer was removed from the well. This was repeated twice. After the final wash, all of the liquid was removed, the well was inverted and blotted against clean paper towels until no liquid spotted on dry towels.

[0426] The reagent diluent was made up fresh for each assay, using a 1:10 dilution, which was prepared by adding 2 mL of 10x reagent diluent into 18 mL of dH2O.

[0427] The plates were blocked by adding 300 pL of the reagent diluent to each well (no adhesive was needed). The plates were incubated at room temperature for 1 hour.

[0428] The standard wash procedure was then carried out again.

[0429] GFAP Assay Procedure

[0430] Standard GFAP solutions were prepared as follows:

[0431] Std 1 - A reconstituted GFAP stock standard solution (740 ng / mL) was diluted to a working concentration of 20 ng / mL by adding 20 pL of the stock solution to 720 pL of x1 reagent diluent to obtain 20 ng / mL GFAP.

[0432] Std 2 - 350 pL of Std 1 was added to 350 pL of x1 reagent diluent to obtain 10 ng / mL GFAP.

[0433] Std 3 - 350 pL of Std 2 was added to 350 pL of x1 reagent diluent to obtain 5 ng / mL GFAP.

[0434] Std 4 - 350 pL of Std 3 was added to 350 pL of x1 reagent diluent to obtain 2.5 ng / mL GFAP.

[0435] Std 5 - 350 pL of Std 4 was added to 350 pL of x1 reagent diluent to obtain 1.25 ng / mL GFAP.

[0436] Std 6 - 350 pL of Std 5 was added to 350 pL of x1 reagent diluent to obtain 0.625 ng / mL GFAP.

[0437] Std 7 - 350 pL of Std 6 was added to 350 pL of x1 reagent diluent to obtain 0.313 ng / mL GFAP BLANK - this was 400 pL of the reagent diluent

[0438] 100 pL of each standard solution was added to a separate well. The wells were covered with an adhesive strip and incubated for 2 hours at room temperature. The standard wash procedure was carried out. 24 pg / mL of the detection antibody was diluted to a working concentration of 200 ng / mL by adding 27.5 pL of the 24 pg / mL solution provided to 3272.5 pL of 1x reagent diluent. The resultant solution was aspirated and inverted to mix. 100 pL of the diluted detection antibody was added to each well. The wells were covered with a new adhesive strip and incubated for 2 hours at room temperature. The standard wash procedure was carried out.

[0439] A 40-fold dilution of streptavidin-HRP was carried out using 1x reagent diluent by adding 75 pL of the stock streptavidin-HRP solution to 2925 pL of 1x reagent diluent.

[0440] 100 pL of the diluted streptavidin-HRP solution was added to each well. The plate was covered, placed out of direct sunlight, and incubated for 20 minutes at room temperature. The standard wash procedure was carried out.

[0441] 100 pL of the substrate solution was added to each well. The plate was placed out of direct sunlight and incubated for 20 minutes at room temperature.

[0442] 5 pL was removed from each well on the plate and applied to the working chamber inlet (or sample chamber inlet) of the further strip design.

[0443] Measurement

[0444] Two palmsens 8-channel multiplexers were connected to two palmsens single potentiostats. The potentiostats were set up in the software to run 8 channels, with a combined counter reference, in consecutive measurements. The strips were inserted into all 16 channels. 5 pL of the prepared GOx solution was pipetted into the counter chamber inlet. The blank (0 ng / mL) was measured first at 20 minutes of TMB incubation. The standard concentrations were measured after with around 1 minute difference between them. The final standard (20 ng / mL) was measured at around 5 minutes after the blank. The chronoamperometry measurements were taken with the settings set out in Test A, above.

[0445] Data analysis was carried out according to the method described for Test A, above. The limit of blank, detection, and quantification (LoB, LoD and LoQ) were calculated as follows:

[0446] • LoB = Mean Blank + (1 ,645*SD Lowest Standard) = 0.023 O.D.

[0447] • LoD = Mean Blank + (3.3*SD Lowest Standard) = 0.033 O.D.

[0448] • LoQ = Mean Blank + (10*SD Lowest Standard) = 0.069 O.D. The above is the limit in units of absorbance. To calculate the limit in units of concentration, a plot of log Absorbance as a function of log [GFAP] was plotted and the line of best fit of the linear region was solved for x.

[0449] • LoB = 15 pg / mL

[0450] • LoD = 59 pg / mL

[0451] • LoQ = 239 pg / mL

[0452] The current densities measured for each sample are shown in Fig. 13.

[0453] Conclusions

[0454] The LoD is much lower than the lowest standards.

[0455] The plate assay fixes the HRP (which is part of a sandwich complex) to the bottom of the microplate using the detection antibody, and thus the solution pipetted onto the strip does not contain HRP. However, TMB was oxidised by the HRP on the microplate and oxidised TMB was added to the strip for detection. The oxidised TMB acted as a redox mediator and generated a signal at the working electrode. The concentration of oxidised TMB was proportional to the concentration of HRP, which was in turn proportional to the concentration of the target (GFAP).

[0456] The measurements provided a trend in the concentration of GFAP owing to TMB oxidase being a redox mediator, and a detectable species at the working electrode.

[0457] Test D: Sensitivity and operation of further test strip design using glucose oxidase type X-S (GOx) as the detectable species at the working electrode

[0458] In this example, GOx was exemplified as the detectable species. D-glucose was used as an example substrate of GOx. The same set-up and procedures were used as in Test A, except that the HRP concentration was fixed at 8 ng / mL and the GOx concentration was varied. The HRP:TMB / H2O2 solution was pipetted into the counter chamber inlet, while the glucose oxidase:ferricyanide:glucose solution was pipetted into the sample chamber inlet.

[0459] Preparation of GOx:ferricyanide:glucose solution (for the sample chamber)

[0460] 36 mg of D-glucose was added to 5 mL of phosphate-buffered saline (PBS) to prepare a 40 mM D-glucose solution. 1 mg of GOx was added to 1 mL of PBS to prepare a 1 mg / mL GOx solution. Dilutions were prepared from 1 ng / mL by first adding 100 pL of the 1 mg / mL of GOx solution to 150 pL of PBS to prepare a 400 pg / mL solution. To prepare further dilutions, 20 pL of the previous concentration was added to 180 pL of PBS. The final on-strip concentrations of GOx were 1 pg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, and Blank.

[0461] 34 mg of potassium ferricyanide was added to 10.4 mL of PBS to prepare a 10 mM ferricyanide solution. 200 pL of 10 mM ferricyanide solution was added to a 1.5 mL tube. 200 pL of glucose oxidase solution was added to the tube. Then, 400 pL of glucose solution was also added to the tube. The tube was vortexed for 5 s and left on the bench for 20 mins.

[0462] Preparation of HRP standard dilutions (for the counter chamber)

[0463] 1 mg of HRP was added to 1 mL of PBS (1 mg / mL). This was covered with foil and put on a windmill mixer for 30 mins at room temp. HRP solution was diluted in PBS to 16 ng / mL for a final on-strip cone, of 8 ng / mL when combined 1:1 with TMB solution. The dilution was aspirated to mix.

[0464] The PE gel was formed using the same procedures as disclosed above for Test A.

[0465] Chronoamperometry measurements of the current density detected at different HRP concentrations

[0466] Strips prepared with the gel were inserted into palmsens potentiostat connectors. These connectors were wired to a palmsens single potentiostat and 8-channel multiplexer. The GOx samples were made by adding 40 pL of 10 mM ferricyanide solution to a 1.5 mL tube. 40 pL of glucose oxidase solution was then added to the tube, followed by 80 pL of glucose solution. 5 pL of the GOx sample solution was pipetted into the sample chamber. The HRP dilution was mixed 1:1 with TMB / H2O2 substrate solution, 20 pL and 20 pL, with aspiration to mix. 5 pL of the HRP:TMB / H2O2 mixture was pipetted into the counter chamber inlet. 5 mins was allowed for incubation of GOx with ferricyanide and glucose.

[0467] Chronoamperometry measurements were taken with the same settings set out for Test A, above.

[0468] The same data analysis procedure was used as set out for Test A, above. The results are shown in Fig. 14.

[0469] Conclusions

[0470] In this unoptimized configuration, it was possible to detect 1 pg / mL GOx, i.e. the signals obtained differed from the blank when 1 pg / mL of GOx was used. The sensitivity of this set-up was less than that of the set-up of Test A. However, the system had not been optimized and was still able to detect GOx at a concentration of 1 pg / mL.

[0471] As anticipated, the blank signal gave a negative current density as only HRP was present in the counter chamber, and a positive current density was exhibited by a positive sample. This is in contrast to the configuration used in Test A, in which a more negative current density was exhibited by a positive sample.

[0472] Without being bound by theory, the inventors believe that the lower sensitivity of the configuration used in this test relative to that used in Tests A and B was caused by over-oxidation of the TMB to an electrochemically inactive state, resulting in poorer spontaneity of the fuel cell reaction.

[0473] Test E: Operation of experimental set-up disclosed in Test A, using HRP-coated magnetic beads and magnetically concentrating the beads at the cathode

[0474] To assess if HRP could be magnetically concentrated at the working electrode (the cathode in this example), the experimental set up described in Test A was used, replacing HRP with HRP-coated magnetic beads (HRP-MB). The strip design was altered slightly: in this example, the working chamber inlet (or sample chamber inlet) was positioned just 2 mm away from the working electrode. This allowed for better concentration of the HRP-MBs by the magnet beneath the working electrode, which is fixed in place.

[0475] Preparation of HRP-coated Magnetic Beads (HRP-MBs) (for the sample chamber) 10pL of 1% solids HRP-MB in storage buffer (the storage buffer provided by Ademtech, and diluted by a factor of 10) was added to 90 pL of PBS to prepare 0.1% solids HRP-MB in a tube. These were vortexed for 2 s and centrifuged for 2 s. A buffer exchange was completed by contacting the tube with a magnet. The HRP-MBs were allowed to collect on the tube wall for 1 minute. As much of the liquid was pipetted out of the tube as possible. Then, 100 pL of PBS was added. The buffer exchange was repeated. Two samples of HRP-MBs were prepared: CB-1 and CB-2. A blank was prepared, which contained 0.1% MB without conjugated HRP. The same buffer exchange was completed to prepare the blank.

[0476] All other methods were the same as for Test A, with the exception that 5 pL of HRP-MB:TMB / H2C>2 mixture was pipetted into the sample chamber through the sample chamber inlet positioned 2 mm away from the working electrode, a magnet was fixed beneath the working electrode (in this example, the cathode), as shown in Fig. 15, and there were 8 replicates of each sample. The final concentrations of MBs on the strips were 0.05%. The results are shown in Fig. 16.

[0477] Conclusions

[0478] HRP-MB benefits greatly from magnetic concentration. In the best case of this example, the signal was 64x higher with magnetic concentration versus without it.

[0479] The bare MBs (without conjugated HRP) did not contribute significantly to the background signal. The current density was positive, as would be expected in the absence of MBs. Concentration of the bare MBs lowers the current density, i.e. the current moves towards a negative current density.

[0480] The current density of the blank is more negative than previous experiments, suggesting a small contribution by the bare MBs to the signal. Without being bound by theory, the inventors believe this could be the result of catalytic oxidation, or a reducing current being generated by contact between the MBs and the working electrode

[0481] Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein in their entirety by reference.

Claims

CLAIMS:

1. A method of detecting a target in a sample, the method comprising:(i) contacting a mixture on or within a test strip, the mixture comprising:(a) a target binding moiety tethered to a magnetically moveable particle;(b) a target capture moiety conjugated to a detectable species; and(c) a sample,wherein component (b) is positioned on or within the test strip at a first position, and the test strip further comprises:(d) a fuel cell comprising an anode and a cathode, wherein one of the anode or cathode is positioned at a second position;(ii) incubating (a), (b) and (c) for a period of time such that, when the target is present, a complex is formed, the complex comprising components (a) and (b) each independently bound to the target;(iii) activating or generating a magnetic field to cause the complex to move to the second position such that the complex is separated from one or more other components of the mixture and such that the movement of the detectable species to the second position turns the fuel cell on or increases a response from the fuel cell; and(iv) detecting a response from the fuel cell.

2. The method of claim 1 , wherein the detectable species comprises:(i) a first catalyst and the test strip further comprises:(e) a first catalyst substrate positioned at or in proximity to the second position; and(f) a second catalyst and a second catalyst substrate, each positioned at or in proximity to a third position, wherein the third position is at the other of the anode and cathode; andwherein movement of the first catalyst to the second position turns the fuel cell on; or(ii) a redox mediator and the test strip further comprises:(e) a first catalyst and a first catalyst substrate positioned at or in proximity to the second position; and(f) a second catalyst and a second catalyst substrate, each positioned at or in proximity to a third position, wherein the third position is at the other of the anode and cathode; andwherein movement of the first catalyst to the second position increases an electrical response from the fuel cell.

3. The method of claim 2, wherein the first catalyst is an enzyme and optionally wherein the first catalyst substrate is positioned at one of the anode and cathode.

4. The method of claim 2 or claim 3, wherein the second catalyst is an enzyme.

5. The method of any one of claims 2 to 4, wherein the second catalyst and / or the second catalyst substrate is / are positioned at the other of the anode and cathode.

6. The method of any one of claims 2 to 5, wherein the complex is immobilised or immobilisable at the second position, optionally wherein the immobilisation is by any one or more selected from adsorption, covalent bonding (such as crosslinking), entrapment, or encapsulation.

7. The method of claim 6, wherein the second catalyst is immobilised at the third position.

8. The method of claim 6 or claim 7, wherein the surface of the magnetically moveable particle is modified with a plurality of molecules, optionally wherein each molecule of the plurality of molecules is selected from an amino acid or a polymer, such as glycine.

9. The method of any one of claims 6 to 8, wherein electrons travel directly, by direct electron transfer, between the first catalyst and the anode or cathode.

10. The method of any one of claims 2 to 9, wherein at least one or both of the first and second catalysts comprises a redox cofactor, optionally wherein the redox cofactor is pyrroloquinoline quinone.

11. The method of any one preceding claim, wherein the first catalyst substrate is positioned at or in proximity to the cathode and the second catalyst and second catalyst substrate is positioned at or in proximity to the anode.

12. The method of claim 11, wherein the first catalyst catalyses chemical oxidation of the first catalyst substrate.

13. The method of claim 12, wherein the first catalyst is an oxidoreductase, such as a peroxidase, optionally wherein the first catalyst is horseradish peroxidase and the first catalyst substrate is 3,3',5,5'-Tetramethylbenzidine and / or hydrogen peroxidase.

14. The method of claim 12 or claim 13, wherein the second catalyst is an oxidase enzyme, optionally wherein the second catalyst is glucose oxidase.

15. The method of any one preceding claim, wherein the test strip comprises one or more mediators (e.g. redox mediators) at the anode and / or cathode.

16. The method of any one preceding claim, wherein the anode and cathode are isolated (e.g. fluidly isolated) by a separator and / or wherein the anode and cathode are separated by a medium for the selective transportation of ions such as an ion-permeable membrane, optionally a salt bridge, such as an agar salt bridge.

17. The method of claim 16, wherein the medium for the selective transportation of ions is a polymeric material, optionally wherein the polymeric material is UV curable and / or is a polyelectrolyte gel.

18. The method of any one preceding claim, wherein the anode and cathode are electronically connected to a sensor.

19. The method of any one preceding claim, wherein activating or generating a magnetic field causes the complex to move in a lateral direction to the second position such that the complex is separated from one or more other components of the mixture.

20. The method of any one preceding claim, wherein both of the target binding moiety tethered to a magnetically moveable particle and the target capture moiety conjugated to the first catalyst are present at the first position.

21. The method of any one preceding claim, wherein the magnetic field is generated by:(i) activating an electromagnet; or(ii) mechanically activating a permanent magnet into proximity with the second position.

22. The method of any one preceding claim, wherein the target binding moiety and the target capture moiety each independently comprise:(i) amino acids and / or nucleic acids, or derivatives thereof; and / or(ii) one or more selected from the group consisting of DNA (e.g. single stranded DNA), RNA, PNA or LNA molecule, a morpholino, a peptide, a protein, an antibody (including a nanobody), or an antigen binding fragment thereof, an antibody mimetic, a cell surface marker, a cell surface receptor or its ligand, or a biologically active fragment of a cell surface marker, a cell surface receptor or its ligand, an aptamer, a molecular imprinted polymer, an enzyme, a lipid, a glycan, a glycoprotein, a glycolipid, a proteoglycan, or a molecularly imprinted polymer (MIP) protein, DNA and RNA; and combinations thereof.

23. The method of any one preceding claim, wherein the test strip comprises a sample chamber positioned on or within the test strip and a sample inlet in fluid communication with the sample chamber, wherein:the first and second positions are within the sample chamber and the sample inlet is positioned between the first and second positions, andthe sample is introduced to the sample chamber via the sample inlet such that the sample travels or flows across the first position in a direction that is away from the second position, optionally also such that the sample travels or flows across the second position in a direction that is away from the first position.

24. A test strip for use in a method of detecting a target in a sample, the test strip comprising:(i) a sample chamber positioned on or within the test strip;(ii) a target capture moiety conjugated to a detectable species positioned on or within the sample chamber at a first position; and(iii) a fuel cell comprising an anode and a cathode, wherein one of the anode or cathode is positioned at a second position and the other is positioned at a third position;optionally wherein the one of the anode and cathode positioned at the second position is configured for fluid communication with the sample chamber and the other of the anode and cathode positioned at the third position is not in fluid communication with the sample chamber.

25. The test strip of claim 24, wherein the detectable species comprises:(i) a first catalyst and the test strip further comprises:(e) a first catalyst substrate positioned at or in proximity to the second position; and(f) a second catalyst and a second catalyst substrate each positioned at or in proximity to the third position; or(ii) a redox mediator and the test strip further comprises:(e) a first catalyst and a first catalyst substrate positioned at or in proximity to the second position; and(f) a second catalyst and a second catalyst substrate, each positioned at or in proximity to the third position.

26. A system for use in a method of detecting a target in a sample, the system comprising:(a) a test strip as defined in claim 24 or claim 25; and(b) a target binding moiety tethered to a magnetically moveable particle; and optionally comprising one or both of:(c) a magnetic field generator configured to activate or generate a magnetic field; and(d) a detector configured to detect the electrical response of the fuel cell.

27. A kit of parts, comprising, as separate components:(a) a system as defined in claim 26.

28. Use of the test strip of claim 24 or claim 25, the system of claim 26, or the kit of claim 27 to detect the presence or absence of a target in a sample.

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