Test strip

Abstract

Disclosed is a test strip for electrochemically detecting a target, preferably in the form of a biofuel cell, the test strip comprising a first substrate laminated to a lid; the first substrate comprising: a sample chamber; an anode and a cathode, the sample chamber configured to allow for fluid communication with one of the anode and the cathode; and a separation chamber configured to separate the anode and cathode and to receive a fluid or gel; and wherein the first substrate is laminated to a lid and the test strip further comprises an inlet configured to deliver the fluid or gel to the separation chamber after the lamination. Also disclosed are the use of the test strip to detect the presence or absence of a target in a sample; and a method for manufacturing the test strip.

Description

[0001] TEST STRIP

[0002] FIELD

[0003] The present invention concerns a test strip for electrochemically detecting a target; the use of the test strip to detect the presence or absence of a target in a sample; and a method for manufacturing the test strip.

[0004] BACKGROUND

[0005] Electrochemical sensors are devices that utilise an electrical signal generated by a redox reaction to detect one or more analytes. Such sensors can find use in diagnostic tests and methods. An electrochemical biosensor involves a biological process, for example the formation of an immune complex, an enzymatic reaction, and / or the detection of a biomarker. Electrochemical biosensors have been widely utilised in the monitoring of blood glucose for patients with diabetes. Typically, a patient deposits a small volume of whole blood onto a test strip, where the test strip comprises an electrochemical cell that produces an electrical signal proportional to the concentration of glucose in the whole blood sample. The signal is produced by the oxidation of glucose by an enzyme, such as glucose oxidase, which transfers electrons to a mediator compound, which then shuttles the electrons to an anode. The resultant electrical current that flows to a cathode provides the signal to be measured by an instrument, e.g., a strip connector such as a hand-held monitor.

[0006] There are a number of challenges associated with the development of useful diagnostic kits (e.g. test strips) and methods based on electrochemical detection methods, particularly those incorporating biosensors or the like. Ideally, the diagnostic kits and methods may comprise one or more, or all, of the following advantages: inexpensive; durability; capable of reproducible and / or scalable production; only require a small quantity of sample; the ability to utilise whole samples (e.g., whole blood, or untreated saliva); requires no washing steps; compact size; compatibility with commercially available strip connectors; and capable of providing accurate and / or consistent results.

[0007] The present inventors have designed, developed, and manufactured a test strip for use in electrochemical detection methods that addresses some of these needs. SUMMARY

[0008] The present inventors have identified a test strip configuration that can facilitate and / or improve the reliability of the manufacturing process. In particular, the test strip configurations and manufacturing methods as described herein can reduce the risk of non-viable test strips (e.g. test strips that would be ineffective, or less effective, in an electrochemical detection method).

[0009] Without being bound by theory, for certain test strip designs to be viable, there should be no direct fluid communication between the anode and the cathode but there should be selective transportation of ions between the anode and the cathode. As such, the anode and cathode are typically separated by a separation chamber filled with a medium that facilitates the selective transportation of ions, such as a salt bridge. This is required for electrochemical function and to provide reliable and / or reproducible results. Unintended leakage of fluid (e.g. of a sample fluid) between different compartments on a test strip, can lead to a non-viable strip. In a laminated test strip (comprising a plurality of layers), the separation of compartments (to avoid unintended fluid leakage between compartments) requires that good contact between layers is maintained at certain positions.

[0010] The present inventors have identified a test strip and manufacturing method in which an inlet on the strip is configured to deliver the medium (e.g., the salt bridge) after a lamination step. The ability to fill the separation chamber at a later stage of the manufacturing process (specifically after the lamination step) means that the defined and distinct nature of the various compartments on the test strip (e.g. a sample chamber, separation chamber and, where present, a counter chamber) can be established during the lamination step and are not impacted by the fluid or gel present in the separation chamber.

[0011] Therefore, in a first aspect there is provided a test strip for electrochemically detecting a target, the test strip comprising a first substrate laminated to a lid;

[0012] the first substrate comprising:

[0013] a sample chamber;

[0014] an anode and a cathode, the sample chamber configured to allow for fluid communication with one of the anode and the cathode; and

[0015] a separation chamber configured to separate the anode and cathode and to receive a fluid or gel; and wherein the first substrate is laminated to a lid and the test strip further comprises an inlet configured to deliver the fluid or gel to the separation chamber after lamination (e.g. after lamination of the first substrate to the lid).

[0016] As described above, the inventors have shown that the test strip disclosed herein can be used to detect the presence or absence of a target in a sample. In other words, the test strip may be an electrochemical biosensor.

[0017] Therefore, in a second aspect there is provided the use of a test strip as defined in the first aspect to detect the presence or absence of a target in a sample.

[0018] Furthermore, and as described above, the inventors have found that the test strip disclosed herein may be manufactured by a method, comprising the addition of a fluid or gel to the separation chamber by way of an inlet, subsequent to a lamination step.

[0019] Therefore, in a third aspect there is provided a method for manufacturing a test strip for electrochemically detecting a target, the method comprising:

[0020] (i) laminating a lid to a first substrate, the first substrate comprising:

[0021] a sample chamber;

[0022] an anode and a cathode, the sample chamber configured to allow for fluid communication with one of the anode and the cathode; and

[0023] a separation chamber configured to separate the anode and cathode; and (ii) adding a fluid or gel to the separation chamber by way of an inlet configured to deliver the fluid or gel to the separation chamber after lamination;

[0024] wherein the fluid forms, or is configured to form, a medium for selective transportation of ions, such as a salt bridge.

[0025] In some embodiments of any of the above aspects, the inlet is comprised on the lid.

[0026] BRIEF DESCRIPTION OF THE FIGURES

[0027] 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 / mL ABTS 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.

[0028] 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.

[0029] 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.

[0030] Fig. 5 is a first test strip configuration. 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.

[0031] Fig. 6 is an iteration of an exemplary test strip configuration 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).

[0032] 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).

[0033] 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.

[0034] Fig. 9 is an exemplary test strip configuration similar to that shown in Fig. 6 but with a greater level of annotation with respect to the features of the invention.

[0035] Fig. 10 shows the results of blue dye testing on the exemplary test strip of Fig. 6 and 9. The exemplary test strip does not show any leaking of blue dye from: (a) the counter chamber into the sample chamber; (b) the sample chamber into the counter chamber; and (c) into the salt bridge when both chambers are filled with the blue dye. Fig. 11 shows 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.

[0036] DETAILED DESCRIPTION

[0037] 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 IUPAC organisation for chemical compounds, specifically the “IUPAC Compendium of Chemical Terminology (Gold Book)”.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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, a wavelength of about 365 nm is intended to include wavelengths from 346.75 to 383.25 nm.

[0042] Test strip

[0043] As described above, in a first aspect there is provided a test strip for electrochemically detecting a target, the test strip comprising a first substrate laminated to a lid;

[0044] the first substrate comprising:

[0045] a sample chamber;

[0046] an anode and a cathode, the sample chamber configured to allow for fluid communication with one of the anode and the cathode; and

[0047] a separation chamber configured to separate the anode and cathode and to receive a fluid or gel; and wherein the first substrate is laminated to a lid and the test strip further comprises an inlet configured to deliver the fluid or gel to the separation chamber after the lamination.

[0048] A test strip, in the present context, is understood to refer to a small, typically mass-produced (or capable of mass production) device that comprises an electrochemical system (such as an electrochemical biosensor). A well-known example of a class of test strips is the electrochemical blood glucose test strip.

[0049] In some embodiments, the test strip comprises a first substrate, and a lid. The test strip may therefore comprise at least two layers - at least one provided by the first substrate which is laminated to a lid layer.

[0050] As stated above, the first substrate comprises the sample chamber, the anode and cathode, and the separation chamber. The various chambers and electrodes may be deposited (e.g. printed) and / or formed on the first substrate using any suitable method as known in the art.

[0051] In some embodiments, the first substrate may comprise a plurality of layers. For example, the first substrate may comprise a first and a second layer. 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 sample chamber and the separation chamber (and optionally any other chambers as described herein, e.g. a 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.

[0052] As such, there is further provided a test strip for electrochemically detecting a target, the test strip comprising a first and second layer, wherein the first layer comprises an anode and a cathode and is laminated to a second layer, the second layer comprising:

[0053] a sample chamber configured to allow for fluid communication with one of the anode and the cathode; and

[0054] a separation chamber configured to separate the anode and cathode and to receive a fluid or gel; and

[0055] wherein the second layer is laminated to a lid and the test strip further comprises an inlet configured to deliver the fluid or gel to the separation chamber after the lamination.

[0056] However, the skilled person will recognise that the present disclosure is not limited to two or three layers as described above. By way of example, the test strip may comprise additional layers (for example for further functionality, for structural stability, for ease in manufacturing etc.). Thus, in some embodiments, the test strip comprises a plurality of layers, optionally more than two or more than three. In some embodiments, the lid is the “nth” layer.

[0057] For the avoidance of any doubt, the lid layer as defined herein refers to a layer that is formed over the sample chamber, the separation chamber, and (when present) the counter chamber. In some embodiments, the lid layer fully or partially encloses the defined chambers (e.g. to form a lid over such chambers and other components on the test strip). The lid layer may sometimes form an outer layer of the strip.

[0058] The term “laminated” as used herein refers to layers that have undergone laminating, wherein “laminating” is understood to refer a process of joining adjacent faces or sides of two or more layers in a planar fashion to form an assembled object, sometimes referred to as a “sandwich” of layers. Layers may be laminated together with a variety of means, including any combination of an adhesive, heating, fusion or melting, applying pressure, mechanical fastening, chemical bonding, and vacuum or low-pressure sealing. With respect to the present disclosure, the layers may be laminated together such that they together form a single uniform strip, i.e., the test strip.

[0059] In some embodiments, the first layer is laminated to the second layer with an adhesive, such as an adhesive tape, optionally in combination with applying pressure. In some embodiments, the adhesive tape is double-sided adhesive tape, optionally doublesided polyester tape. Thus, in some embodiments, one or more of the layers comprises an adhesive, or is an adhesive material, such that when pressure is applied to two or more layers positioned side-to-side in a planar fashion, the adhesive bonds to the adjacent layers. Layers that comprise - or are - a double-sided adhesive may be laminated to layers that do not comprise adhesive, or layers that comprise adhesive on only one side.

[0060] As discussed above, the test strip may comprise a plurality of layers, optionally greater than two or three. 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. As described above, the inlet is configured to deliver the fluid or gel to the separation chamber after the lamination. More specifically, in some embodiments, the inlet is configured to deliver the fluid or gel to the separation chamber after the lamination of at least the first substrate, and the lid. That is to say, this feature does not preclude additional layers from the test strip; additional layers may be laminated to the test strip after delivery of the fluid or gel, wherein the “nth” layer may instead be considered the lid.

[0061] 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.

[0062] 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 under action 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.

[0063] 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.

[0064] 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.

[0065] As stated above, the test strip comprises a sample chamber. The sample chamber may be configured to receive a sample fluid (e.g. a sample fluid that is to be tested in the test strip). The sample chamber may house and / or be in contact with one of the anode or the cathode. The sample chamber may be configured to hold fluid for the operation of the redox reaction at the one of the anode or cathode. The sample chamber may house and / or be in contact with one of the anode or the cathode such that fluids contained in the sample chamber can contact the one of the anode or the cathode. In some embodiments, the sample chamber has a volume of about 5 pL.

[0066] As stated above, the test strip comprises a separation chamber configured to separate the anode and cathode (e.g. fluidly separate the anode and cathode). The separation chamber is further configured to receive a fluid or gel, in particular a fluid or gel which forms, or is configured to form, a medium for selective transportation of ions, such as a salt bridge. Without being bound by theory, for certain test strip designs to be viable, there should be no direct fluid communication between the anode and the cathode but there should be selective transportation of ions between the anode and the cathode. As such, the anode and cathode are typically separated by the separation chamber which is configured to be filled with a medium that facilitates the selective transportation of ions, such as a salt bridge. The separation chamber may be configured to prevent direct fluid communication between the sample chamber and counter chamber. 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.

[0067] The test strip may further comprise a counter chamber. That is to say, the test strip may comprise a counter chamber in addition to the sample chamber and the separation chamber. In some embodiments, the counter chamber is separated from the sample chamber by the separation chamber. The counter chamber may house and / or be in contact with one of the anode or cathode. The counter chamber may be configured to hold fluid for the operation of the redox reaction at the one of the anode or cathode. The counter chamber may house and / or be in contact with one of the anode or the cathode such that fluids and / or species contained in the counter chamber can contact the one of the anode or the cathode. In some embodiments, the counter chamber has a volume of about 3 pL.

[0068] In some embodiments, the anode and cathode are positioned close to but not in contact with the separation chamber. The skilled person will recognise that the electrochemical operations of the test strip may be dependent on the relative configuration of the electrodes with respect to the separation chamber. For example, it may be preferable that the anode and cathode are not in direct contact with the separation chamber.

[0069] In some embodiments, and as stated above, one of the anode and the cathode is housed in the sample chamber and the other of the anode and cathode is housed in a counter chamber. In some embodiments, the anode is housed in the sample chamber and the cathode is housed in a counter chamber. In some embodiments, the cathode is housed in the sample chamber and the anode is housed in the counter chamber. In some embodiments, the sample chamber, the separation chamber, and optionally the counter chamber may comprise a microfluidic system on the test strip. In other words, these chambers may each be considered to be comprised and / or formed of one or more channels configured to receive, hold, and / or communicate fluid.

[0070] As stated above, these chambers may be formed using any suitable method as known in the art (e.g. deposited or printed on a substrate). In some embodiments, these chambers may be comprised in a microfluidic layer. The microfluidic layer may be laminated to an electrode layer and / or a lid. In some embodiments, the microfluidic layer is a double-sided adhesive, such as a double-sided tape, for example double-sided polyester tape. The double-sided adhesive may enable the microfluidic layer to be laminated to the first layer (for example, an electrode layer) on one side and the lid on the other side. In some embodiments, the microfluidic layer is formed using a laser cutting instrument.

[0071] In some embodiments, one or more of the chambers comprised on the test strip may deviate from linearity. In other words, the chambers may comprise a channel with one or more bends / turns, e.g. one or more bends or turns substantially 90° to the longitudinal axis of the test strip.

[0072] The separation chamber may extend between the sample chamber and the counter chamber. In some embodiments, the portion of the separation chamber extending between the sample chamber and the counter chamber may be considered as the main body of the separation chamber. Thus, the separation chamber may comprise a main body extending between the sample chamber and the counter chamber. The separation chamber may further comprise a channel configured to deliver the fluid or gel to the main body of the separation chamber. The channel may comprise a first end to deliver the fluid or gel to the main body and an additional end positioned at or close to the inlet (e.g. to receive the fluid or gel). The channel configured to deliver the fluid or gel to the main body of the separation chamber may be configured to deliver the fluid or gel to a mid-portion of the main body of the separation chamber. In some embodiments, this channel may be considered to bifurcate to provide the main body of the separation chamber (as described further below). In some embodiments, the channel and main body of the separation chamber may together form a substantially T-shaped or Y-shaped configuration. In some embodiments, the channel may terminate in a substantially T-shaped or Y-shaped configuration.

[0073] In some embodiments, the separation chamber comprises a channel that furcates, for example, bifurcates. Such configurations (or those in which the channel is configured to deliver the fluid or gel to a mid-portion of the main body of the separation chamber) may be useful in facilitating uniform filling of the fluid or gel into the separation chamber and / or a good connection between the salt bridge and the sample chamber and, when present, the counter chamber. In other words, the separation chamber may comprise a channel that splits into branches, e.g. splits into two branches. The channel may split into branches such that a first end of the channel contacts the sample chamber and a second end of the channel contacts the counter chamber. In such embodiments, the portion of the separation chamber extending between the first end and second end may be considered as the main body of the separation chamber. The third end of the channel may be positioned at or close to the inlet.

[0074] In some embodiments, the separation chamber comprises a substantially T-shaped, or Y-shaped configuration. Typically, the separation chamber comprises a substantially T-shaped configuration, in other words, the separation chamber is bifurcated at right angles at an end of the channel. In some embodiments, the channel may terminate in the substantially T-shaped or substantially Y-shaped configuration. In such embodiments, the channel may further comprise a deviated portion extending between an end (e.g. a third end) and the substantially T- or Y-shaped configuration.

[0075] In some embodiments, the inlet is configured to deliver fluid or gel at or close to an end (e.g. a third end) of the channel. For example, the inlet may be configured to deliver the fluid or gel to the third end of a substantially T- or Y-shaped separation chamber. In other examples, the inlet may be configured to deliver the fluid or gel to the deviated portion of the channel as described above.

[0076] In some embodiments, the channel(s) of the separation chamber are narrower than the channel(s) of the sample chamber. In some embodiments, the main body of the separation chamber is narrower than the channel(s) of the sample chamber and / or the counter chamber. The main body of the separation chamber may be narrower than the deviated portion of the channel. In some embodiments, the sample chamber channel has a width of about 1 mm. In some embodiments, the channel of the separation chamber may be less than about 1 mm, optionally about 0.8 mm or less.

[0077] In some embodiments, the distance between the sample chamber and the counter chamber may be defined as the length of the separation chamber. The length of the separation chamber may be about 2mm.

[0078] The sample chamber and / or the counter chamber may each independently comprise a channel extending or oriented along a longitudinal axis of the test strip. The sample chamber channel may extend between a first end positioned at or near a sample inlet and a second end contacting the separation chamber. The counter chamber channel may extend between a first end and a second end contacting the separation chamber.

[0079] The sample chamber and / or the counter chamber may each independently comprise a deviated channel. In other words, each of these chambers may comprise a first channel oriented along a longitudinal axis of the test strip, which deviates and / or bends away from the longitudinal axis (e.g. at substantially 90°). In particular, the deviation in the channel may have an origin at the end of the channel. The end of the channel may be where the channel contacts the separation chamber. The deviation or bend in the channels are formed in proximity to the separation chamber. The sample chamber and / or counter chamber may each comprise a substantially L-shaped configuration.

[0080] Without being bound by theory, the inventors have found that such deviations may help to avoid the build-up of air. This is because the deviations provide a locality for the collection of air, in that it may be pushed to the end of the deviation as fluid is delivered. The deviations may further provide a good position for a vent to be situated, such that the air that is pushed into the deviation can be released. This, in turn, allows the fluid to fill the deviation such that the point of contact of the channel with the separation chamber (where the deviation has its origin) is sufficiently filled with fluid to enable effective contact, and thus effective selective transportation of ions.

[0081] Thus, in some embodiments, the sample chamber comprises a deviated channel, and optionally a vent positioned at the end of the deviated channel (a front vent), further optionally a vent at the other end of the channel (a rear vent). In some embodiments, the sample chamber comprises a substantially L-shaped configuration. In some embodiments, the deviated channel deviates from the main body of the sample chamber in a substantially perpendicular direction to the longitudinal axis of the test strip. The deviated channel may be narrower than the main body of the sample chamber.

[0082] In some embodiments where the test strip comprises a counter chamber, the counter chamber comprises a deviated channel, and optionally a vent positioned at the end of the deviated channel, or at the other end of the channel. In some embodiments, the counter chamber channel comprises a substantially L-shaped configuration. Similarly, an inlet may be positioned at the end of a deviated channel, such that the desired contact area fills with the fluid and air is displaced - the air may then collect at the other end of the channel, where a vent may be positioned. In some embodiments, the deviated channel deviates from the main body of the counter chamber in a substantially perpendicular direction to the longitudinal axis of the test strip. The deviated channel may be narrower than the main body of the counter chamber.

[0083] In some embodiments, the sample chamber deviated channel and the counter chamber deviated channel are parallel, in-line, adjacent, and / or deviate in the same direction. In some embodiments, the sample chamber vent and the counter chamber vent are in-line and / or adjacent.

[0084] As stated above, the test strip comprises a lid. In some embodiments, the lid may comprise one or more inlet(s) and / or one or more vent(s) as described in further detail herein.

[0085] In some embodiments, the lid comprises the inlet. The lid may define an opening which forms the inlet and / or the inlet may be considered to be positioned through the lid. The inlet is configured to deliver fluid or gel to the separation chamber after the lamination (for example after the lamination of the first substrate to the lid, or after lamination of the second layer to the lid). The inlet may be any suitable shape and / or dimension such that the inlet may be configured to deliver the fluid or gel to the separation chamber. In some embodiments, the inlet may comprise a generally circular or oval opening in the lid.

[0086] In some embodiments, the inlet is configured to deliver fluid to the separation chamber in a direction substantially perpendicular to a first (e.g. upper) surface of the first substrate. In some embodiments, the lid is a layer of polymeric film, e.g. a polyester film, for example configured to cover, seal, top, enclose, and / or contain the test strip, e.g., to prevent the leaking of any fluid, and / or to enclose or partially enclose any of the above described chambers. That is to say, the lid may form a wall or surface of any of the above-described chambers. In some embodiments, a surface of the lid is treated with a hydrophilic agent prior to lamination, such that the hydrophilic agent is internal with respect to the chambers. Without being bound by theory, an internal hydrophilic agent can facilitate the flow of fluid in the various chambers forming the microfluidic system. In some embodiments, the hydrophilic agent is a surfactant.

[0087] In some embodiments, the test strip comprises a plurality of inlets. In some embodiments, the lid may comprise a plurality of inlets. That is to say, the test strip may comprise one or more inlets in addition to the inlet configured to deliver the fluid or gel to the separation chamber after the lamination. In some embodiments, the inlet or plurality of inlets may be formed in the lid through the laser cutting of a layer of film.

[0088] In some embodiments, the test strip comprises a sample chamber inlet, configured to deliver a sample to the sample chamber. The sample inlet may be located such that the inlet lies substantially above the sample chamber. The sample chamber inlet may be configured to deliver a sample such that the sample is delivered from a first direction that is substantially perpendicular to the surface of the first substrate (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 first substrate prior to travelling or flowing through the sample chamber in a direction that is away from the one of the anode or cathode housed in the sample chamber. In some embodiments, the sample inlet may be configured to deliver the sample to the sample chamber in between the two ends of the sample chamber (e.g. at an approximately midway point between the two ends of the sample chamber). In other embodiments, the sample inlet may be configured to deliver the sample fluid to a first end of the sample chamber, optionally at an opposing end of the sample chamber to the end which the one of the anode or cathode is located.

[0089] In some embodiments, the test strip comprises a counter chamber inlet, configured to deliver fluid to a counter chamber. The counter chamber inlet may be located such that the inlet lies substantially above the counter chamber. The counter chamber inlet may be configured to deliver a fluid such that the fluid is delivered from a first direction that is substantially perpendicular to the surface of the first substrate (e.g. from above when the test strip is held in a horizontal configuration). In some embodiments, the counter chamber inlet may be configured to deliver the fluid to a first end of the counter chamber, optionally at an opposing end of the counter chamber to the end which the one of the anode or cathode is located. The counter chamber inlet may enable the reconstitution of an electrode that is in contact with the counter chamber. That is to say, a fluid comprising for example electrolytes, catalysts, and / or mediators etc. may be delivered to the counter chamber to provide the necessary components for electrode operation, either initially (optionally immediately before operation), to ensure the continuous operation of the electrode, or to restart the operation of the electrode.

[0090] In some embodiments, the test strip, optionally the lid, comprises one or more vent(s). A vent may allow for the release of, i.e., the venting of, air and / or excess fluid from the chambers, e.g., from a microfluidic layer. Air and fluid may be vented together in the form of a bubble. In some embodiments, the one or more vents may act as a valve to release excess air and / or may have a substantially cross-like configuration.

[0091] Where the vent comprises a substantially cross-like configuration, the cross-like configuration may allow for some misalignment between a second layer (e.g. microfluidic layer) and a lid layer during assembly without affecting air / fluid flow through the chamber(s). In some embodiments comprising a microfluidic layer, the microfluidic layer may comprise a slit (e.g. a narrow straight cut-out) in the microfluidic layer which is orthogonal to a corresponding slit (e.g. narrow straight cut-out) on the lid. When the lid is placed on the microfluidic layer, the two slits may together form a vent with a crosslike configuration. In some embodiments, each slit (e.g. each narrow cut-out) may comprise an excess length to allow for deviation from perfect alignment without affecting the vent’s function. In such configurations, the area of the vent may be unaffected.

[0092] In some embodiments, the test strip comprises one or more sample chamber vents, and / or one or more counter chamber vents. In other words, in some embodiments, the sample chamber comprises one or more vents, and / or the counter chamber comprises one or more vents. For example, in some embodiments, the sample chamber comprises a front vent and a rear vent, the counter chamber comprises a vent. Without being bound by theory, vents can help to avoid the build-up, collection, or obstruction of air, which might prevent or hinder the flow of fluid through the channels. Such a build-up of air is particularly likely upon delivery of a fluid to an inlet, where there may be air in the channels prior to delivery which build-up without proper venting. This may not only prevent the delivered sample reaching the electrode, but also prevent the separation chamber from acting in its intended functions, for example by providing a medium for selective transportation of ions. For example, if there is insufficient fluid at the end of the sample chamber, then there cannot be effective transportation of ions. This also applies mutatis mutandis to a test strip that comprises a counter chamber. Thus, the vents offer a point of escape (venting) for any air in the channels and help to enable reliability between each test strip manufactured.

[0093] As stated above, the test strip comprises an inlet configured to deliver a fluid or gel to the separation chamber after the lamination. In some embodiments the fluid or gel comprises, or is configured to form, a medium for selective transportation of ions. Selective transportation as used herein refers to the passage of some but not all species of ions through the medium. The selectivity may arise from the properties of the medium, e.g., as a result of the chemical structure (such as charged groups) or physical properties (such as porosity). In some embodiments the fluid or gel comprises, or is configured to form, a salt bridge.

[0094] The term “salt bridge” is well known in the art and is understood to refer to a medium between an anode and a cathode (or, more specifically, between the respective electrolytes of an anode and a cathode) of a cell (such as a fuel cell or galvanic cell) that allows the passage of ions, typically a select species of ions, whilst preventing contact between the electrodes (or the respective electrolytes). 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, otherwise referred to as protons. In particular, the purpose of the salt bridge is to allow for selective transport of ions between the electrodes for electronic connection. In the various embodiments as described herein, the salt bridge may not allow a target analyte (e.g. a target analyte found in a sample) to pass through. If present in a sample, any target analyte would be located or locatable on the working electrode (one of the anode or cathode).

[0095] The medium for the selective transportation of ions may be a fluid or gel. In some embodiments, the fluid or gel may be a gel. A gel may be considered as a semi-solid material and / or as a material that is at least partially solidified before injection into the separation chamber. Such gels may demonstrate sufficiently liquid-like properties (e.g. under pressure) to facilitate injection into the separation chamber. In some embodiments, such gels may be wholly solidified following injection into the separation chamber.

[0096] The medium for the selective transportation of ions may be solidifiable material. The solidifiable material may be a fluid material (such as a liquid, gel, paste, resin, serum, emulsion, suspension, slurry, or solution etc.) that is configured to solidify after introduction into the separation chamber. The solidification process may comprise any one or more of heating, cooling, evaporating, drying, irradiating, or chemically activating. In some embodiments, the solidifiable material may solidify under irradiation (e.g. under electromagnetic irradiation such as UV irradiation) and / or heat, or in the presence of a chemical activator.

[0097] In some embodiments, the fluid or gel 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. In some embodiments, the solidifiable material is configured to solidify by a polymerization and / or cross-linking (e.g. curing) 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.

[0098] The skilled person is able to select appropriate monomers and reagents to effect a solidification 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:

[0099] (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);

[0100] (ii) cross-linking agents (e.g., acrylate-, or methacrylate-, or vinyl-containing cross-linking agents, such as / V, / V'-methylenebis(acrylamide)); and

[0101] (iii) photoinitiators (such as 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone).

[0102] A cross-linking agent is often 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).

[0103] 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 therefore 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.

[0104] In some embodiments, the solidifiable material solidifies to form a polyelectrolyte, optionally a polyelectrolyte gel. The term “polyelectrolyte” is used herein to refer to a material that comprises polymer chains, wherein the polymer chains comprise charged groups (e.g. on the backbone, on the side chains, and / or the cross-linkers), such that the material is capable of acting as an electrolyte. In some cases, a polyelectrolyte 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. 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.

[0105] In some embodiments, the polymerizable liquid is formed prior to delivery to the inlet, or the polymerizable liquid is formed in the separation chamber. That is to say, the abovementioned components of the polymerizable liquid may be contacted and / or mixed either outside the separation chamber (prior to addition) or inside the separation chamber.

[0106] 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).

[0107] 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).

[0108] 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).

[0109] As stated above, the first substrate comprises a plurality of electrodes (at least the anode and cathode). The first substrate may additionally one or more electrode connector pads, such as a cathode (or counter electrode) connector pad and an anode (working electrode) connector pad. In some embodiments, the anode and cathode are configured to allow for electrical connection; in other words, they are configured to allow for current to flow. In some embodiments, the anode is in electrical connection with the anode (working electrode) connector pad, and the cathode is in electrical connection with the cathode (counter electrode) connector pad. 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, or TaiDoc connector.

[0110] 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.

[0111] The electrodes may be formed and / or comprised of any suitable material, e.g. 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. In some embodiments, the electrodes may be porous. 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.

[0112] In some embodiments, the electrodes may comprise graphene, such as laser-induced graphene (LIG). LIG is a porous a 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. In some embodiments, the electrode layer is 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.

[0113] In some embodiments, the electrodes, e.g., the anode and the cathode, have a width that is similar to or the same as the width of the chamber in which they are housed, optionally about 1 mm.

[0114] In some embodiments, the test strip comprises a fuel cell. By way of example, in some embodiments, the separation chamber comprises a medium for selective transportation of ions, such as a salt bridge, e.g., a salt bridge as described above, and the anode and cathode form a fuel cell on the test strip. As used herein, a fuel cell refers to an electrochemical cell that generates electricity or an electrical signal through the oxidation of a fuel at an anode, and a corresponding reduction of an oxidant at a cathode. In general terms, a fuel cell can generate electricity as long as the fuel and the oxidant are supplied.

[0115] In some embodiments, in use of the test strip, when a sample comprising the target is introduced to the sample chamber and is brought into proximity with one of the anode or cathode, the fuel cell may be turned on, or a response from the fuel cell is increased.

[0116] In some embodiments, the anode is a bioanode and the cathode is a biocathode and together they form a biofuel cell, e.g., an enzymatic biofuel cell (EBFC). 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). 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 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.

[0117] 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.

[0118] 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.

[0119] In some embodiments, the biocathode has a greater surface area than the bioanode. In some embodiments, the biocathode has a greater length and / or width than the bioanode.

[0120] Where the separation chamber, the anode, and the cathode form a fuel cell on the test strip, 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. In some embodiments where the anode and cathode form a biofuel cell, the fuel is glucose and the oxidant is oxygen.

[0121] The test strip described may find particular application 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.

[0122] 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.

[0123] 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.

[0124] 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). Typically, the sample comprises the target to be detected, and may be blood (ideally whole-blood but the sample may also be for example plasma or serum), saliva, or any fluid that may comprise the target to be detected.

[0125] Uses

[0126] As described above, in a second aspect there is provided the use of a test strip as defined in the first aspect to detect the presence or absence of a target in a sample.

[0127] For the avoidance of doubt, the embodiments of the first aspect of the invention apply mutatis mutandis to the second aspect of the invention.

[0128] As described above, in some embodiments, the separation chamber comprises a medium for selective transportation of ions, such as a salt bridge, e.g., a salt bridge as described above, and the anode and cathode form a fuel cell on the test strip. Such test strips may be used to detect the presence or absence of a target in a sample. In particular, when a sample comprising the target is introduced to the sample chamber and is brought into proximity with one of the anode or cathode, the fuel cell may be turned on, or a response from the fuel cell is increased.

[0129] The skilled person would be able to select the appropriate species and reactants at each of the anode and cathode such that when the target is brought into proximity with one of the anode or cathode, the fuel cell may be turned on, or a response from the fuel cell is increased. In some embodiments, the method may comprise the use of an immunoassay (e.g. an ELISA-based immunoassay).

[0130] By way of example, in some embodiments, the use may comprise a method of detecting a target in a sample, the method comprising:

[0131] (i) contacting a mixture on or within a test strip (as described herein), the mixture comprising: (a) a target binding moiety tethered to a magnetically moveable particle;

[0132] (b) a target capture moiety conjugated to a detectable species; and

[0133] (c) a sample,

[0134] wherein component (b) is positioned on or within the test strip at a first position, and the test strip further comprises:

[0135] (d) a fuel cell comprising an anode and a cathode, wherein one of the anode or cathode is positioned at a second position;

[0136] (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;

[0137] (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

[0138] (iv) detecting a response from the fuel cell.

[0139] In such embodiments, 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. 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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. In some embodiments, the detectable species comprises:

[0144] (i) a first catalyst and the test strip further comprises:

[0145] (e) a first catalyst substrate positioned at or in proximity to the second position; and

[0146] (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

[0147] wherein movement of the first catalyst to the second position turns the fuel cell on; or

[0148] the detectable species comprises:

[0149] (ii) a redox mediator and the test strip further comprises:

[0150] (e) a first catalyst and a first catalyst substrate positioned at or in proximity to the second position; and

[0151] (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

[0152] wherein movement of the first catalyst to the second position increases an electrical response from the fuel cell.

[0153] In some embodiments, the first catalyst is an enzyme; optionally the first catalyst substrate is positioned at one of the anode and cathode. In some embodiments, the second catalyst is an enzyme. In some embodiments, the second catalyst and / or the second catalyst substrate is / are positioned at the other of the anode and cathode.

[0154] In some embodiments, the complex is immobilised or immobilisable at the second position. The immobilisation may be by any one or more selected from adsorption, covalent bonding (such as crosslinking), entrapment, or encapsulation.

[0155] In some embodiments, the second catalyst is immobilised at the third position. In some embodiments, the surface of the magnetically moveable particle is modified with a plurality of molecules. Each molecule of the plurality of molecules may be selected from an amino acid or a polymer, such as glycine.

[0156] In some embodiments, the electrons travel directly, by direct electron transfer, between the first catalyst and the anode or cathode.

[0157] In some embodiments, at least one or both of the first and second catalysts comprises a redox cofactor. The redox cofactor may be pyrroloquinoline quinone. In some embodiments, the first catalyst substrate is positioned at or in proximity to the anode and the second catalyst and second catalyst substrate is positioned at or in proximity to the cathode.

[0158] Where the first and second catalysts are enzymes, 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 one selected from the group consisting of glucose oxidase, horseradish peroxidase, bilirubin oxidase, glucose dehydrogenase, tyrosinase, alkaline phosphatase, 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, and sorbitol dehydrogenase and bilirubin oxidase. 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.

[0159] 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). 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.

[0160] In some embodiments, the first catalyst catalyses chemical oxidation of the first catalyst substrate. In some embodiments, the first catalyst is an oxidoreductase, such as an oxidase, optionally wherein the first catalyst is glucose oxidase or glucose dehydrogenase and the first catalyst substrate is glucose.

[0161] In some embodiments, the second catalyst is an oxidase enzyme. The second catalyst may be laccase.

[0162] 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, or glucose dehydrogenase, or alkaline phosphatase, such as glucose oxidase or glucose dehydrogenase (e.g. glucose oxidase), and the second catalyst is horseradish peroxidase, laccase, or 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.

[0163] 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.

[0164] 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). 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.

[0165] In some embodiments, the test strip may comprise one or more mediators (e.g. redox mediators) at the anode and / or cathode.

[0166] In some embodiments, the anode and cathode are electronically connected to a sensor.

[0167] In some embodiments, 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.

[0168] In some embodiments, 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.

[0169] In some embodiments, the magnetic field is generated by:

[0170] (i) activating an electromagnet; or

[0171] (ii) mechanically activating a permanent magnet into proximity with the second position.

[0172] In some embodiments the target binding moiety and the target capture moiety each independently comprise:

[0173] (i) amino acids, nucleic acids and / or derivatives thereof; and / or

[0174] (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.

[0175] In some embodiments, 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. The first and second positions may be within the sample chamber. The sample inlet may be positioned between the first and second positions. In use, the sample may be 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.

[0176] For the avoidance of doubt, any embodiments that have been described in detail for the second aspect of the invention apply mutatis mutandis to the first and third aspects of the invention.

[0177] Methods of manufacture

[0178] As described above, in a third aspect there is provided a method for manufacturing a test strip for electrochemically detecting a target, the method comprising:

[0179] (i) laminating a lid to a first substrate, the first substrate comprising:

[0180] a sample chamber;

[0181] an anode and a cathode, the sample chamber configured to allow for fluid communication with one of the anode and the cathode; and

[0182] a separation chamber configured to separate the anode and cathode; and (ii) adding a fluid or gel to the separation chamber by way of an inlet configured to deliver the fluid or gel to the separation chamber after lamination;

[0183] wherein the fluid forms, or is configured to form, medium for selective transportation of ions, such as a salt bridge.

[0184] For the avoidance of doubt, the embodiments of the first and second aspects of the invention apply mutatis mutandis to the third aspect of the invention.

[0185] By way of example, in some embodiments, the first substrate comprises a first layer and a second layer. The first layer may comprise the anode and the cathode. The second layer comprise the sample chamber and the separation chamber. The method may further comprise laminating together the first layer, the second layer and the lid.

[0186] By way of further example, in some embodiments, the first layer is an electrode layer that comprises a plurality of electrodes, for example an anode and a cathode, and optionally additionally one or more electrode connector pads, such as a cathode (or counter electrode) connector pad and an anode (working electrode) connector pad, and optionally the electrode layer comprises graphene, such as laser-induced graphene (LIG); the second layer is a microfluidic layer, optionally a double-sided adhesive, such as a double-sided tape, optionally wherein the second layer comprises a counter chamber; the lid comprises the inlet, optionally comprising a plurality of inlets, further optionally the lid comprises one or more vents; the chambers of the microfluidic layer deviate from linearity, optionally such that they are substantially L-shaped; one of the anode and the cathode is housed in the sample chamber and the other of the anode and cathode is housed in a counter chamber; the separation chamber comprises a channel that furcates; optionally the separation chamber comprises a substantially T-shaped configuration; the anode and cathode are positioned close to but not in contact with the separation chamber; the medium for the selective transportation of ions is a solidifiable material, such as a polymerizable liquid, for example a polyelectrolyte gel; the separation chamber comprises a medium for selective transportation of ions, such as a salt bridge, e.g., a salt bridge as described above, and the anode and cathode form a fuel cell on the test strip, optionally the anode is a bioanode and the cathode is a biocathode and together they form a biofuel cell, such as an EBFC.

[0187] As discussed above, the term “laminating” is understood to refer a process of joining adjacent faces or sides of two or more layers in a planar fashion to form an assembled object, sometimes referred to as a “sandwich” of layers. Layers may be laminated together with a variety of means, including any combination of an adhesive, heating, fusion or melting, applying pressure, mechanical fastening, chemical bonding, and vacuum or low-pressure sealing. With respect to the present invention, the layers may be laminated together such that they together form a single uniform strip, i.e. , the test strip. Hence, the step of laminating of the second aspect of the invention may comprise any one or more of: the use of an adhesive; heating, fusion, or melting; applying pressure; mechanical fastening; chemical bonding; and vacuum or low-pressure sealing. In some embodiments, the laminating comprises the use of an adhesive, and optionally applying pressure. In some embodiments, the second layer comprises an adhesive on both planar sides, or is a double-adhesive, such that when pressure is applied to the first layer, the second layer, and the lid positioned side-to-side in a planar fashion, the adhesive bonds to the adjacent layers.

[0188] In some embodiments, the step of adding the fluid or gel comprises one or more of: dropping, such as pipetting; injection, such as syringe-injection, optionally pump-assisted; capillary action; and vacuum filling (that is, applying a vacuum to the separation chamber or test strip such that fluid or gel is drawn through the inlet). The skilled person will recognise that the particulars of the step of adding may be dependent on the properties of the fluid or gel (such as viscosity or melting point), and the properties of the inlet (such as diameter). In some embodiments, where the fluid or gel is a polymerizable liquid, the step of adding comprises adding the polymerizable liquid perse, or adding the individual components of the polymerizable liquid in turn, such that the polymerizable liquid forms inside the separation chamber.

[0189] In some embodiments, where the fluid or gel is a solidifiable material, the method further comprises: solidifying the solidifiable material to form the medium for selective transportation of ions, such as a salt bridge between the anode and the cathode.

[0190] The solidifiable material may be a fluid material (such as a liquid, gel, paste, resin, serum, emulsion, suspension, slurry, or solution etc.) that is configured to solidify after introduction into the separation chamber. The solidification process may comprise any one or more of heating, cooling, evaporating, drying, irradiating, or chemically activating. In some embodiments, the solidifiable material may solidify under irradiation (e.g. under electromagnetic irradiation such as UV irradiation) and / or heat, or in the presence of a chemical activator.

[0191] Thus, in some embodiments, the method comprises: solidifying the solidifiable material using any one or more of heating, cooling, evaporating, drying, irradiating, or chemically activating. In some embodiments, the method comprises: solidifying the solidifiable material using irradiation and / or heat, or a chemical activating species, optionally wherein the method comprises polymerizing and / or cross-linking the solidifiable material.

[0192] Where the solidifiable material is solidified using irradiation, the irradiation is typically UV irradiation. The wavelength of light to be used is dependent on the solidifiable material. As described above, the skilled person is familiar with polymerization reactions and cross-linking reactions that may be comprised within the solidifying, as is the skilled person able to select appropriate monomers and reagents to effect a curing process that comprises polymerization and / or cross-linking reactions. In some embodiments, the solidifying forms a polyelectrolyte gel which typically, as described above, is formed from a polymerizable liquid comprising a monomer (such as diallyldimethylammonium chloride), a cross-linking agent (such as / V, / V'-methylenebis(acrylamide)), and a photoinitiator (such as 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 solidifying is carried out a photoinitiation wavelength of about 365 nm, which is suitable for (at least) 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone.

[0193] In some embodiments, the step of laminating occurs before the step of adding. In some embodiments where the method further comprises a step of solidifying, the step of solidifying occurs after the step of adding; in other words, the order of steps is (i) laminating; (ii) adding; and (iii) solidifying. However, where the test strip comprises three or more layers, this does not necessarily preclude the addition of further layers and laminating steps after the adding step. Nonetheless, additional layers may still be laminated before the adding step.

[0194] Examples

[0195] Methods

[0196] 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.

[0197] Cathode Optimisation

[0198] 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.

[0199] 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. 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.

[0200] pH Amount of 0.1 M Citric Acid Amount of 0.2 M NaaHPCh Total Volume (mL) (mL) (mL)

[0201] 3 0.954 0.247 1.2

[0202] 4 0.738 0.463 1.2

[0203] 5 0.582 0.618 1.2

[0204] 6 0.443 0.758 1.2

[0205] 7 0.212 0.989 1.2

[0206]

[0207] 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).

[0208] 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.

[0209] Table 2 - CV settings used for the confirmation of the reduction potential of ABTS.

[0210] Cyclic Voltammetry Value

[0211] E Begin 0 V

[0212] E vertexl 1.2 V

[0213] E vertex2 0 V

[0214] E Step 0.01 V

[0215] Scan Rate 0.1 V / s

[0216] t Interval 0.1 s

[0217]

[0218] Table 3 - CA settings used for the reduction of ABTS.

[0219] Chronoamperometry Value

[0220] Applied Potential 0.25 V

[0221] t Interval 0.1 s

[0222] t Run 30 s

[0223]

[0224] Table 4 - OOP settings used for the measurement of ABTS Voc.

[0225] Open Circuit Potentiometry Value

[0226] t Run 30 s

[0227] 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.

[0228] 30 L of LacAB solution and 30 L 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.

[0229] Anode Optimisation

[0230] 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).

[0231] 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.

[0232] 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.

[0233] 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.

[0234] Table 5- CA settings used for the oxidation of ferricyanide.

[0235] Chronoamperometry Value

[0236] Applied Potential -0.2 V

[0237] t Interval 0.1 s

[0238] t Run 30 s

[0239]

[0240] Strip Design

[0241] 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.

[0242] Table 6 - Vector settings for laser cutting the microfluidic layer Microfluidic Layer

[0243] Laser Power 10% (5 W)

[0244] Laser Speed 25%

[0245] Frequency 750 Hz

[0246]

[0247] Table 7 - Vector settings for laser cutting the top lid layer

[0248] Top Lid Layer

[0249] Laser Power 10% (5 W)

[0250] Laser Speed 30%

[0251] Frequency 750 Hz

[0252]

[0253] 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.

[0254] Laser-induced Graphene Electrodes

[0255] 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).

[0256] Table 8 - Settings reported by Thana et al. for the production of LIG electrodes.

[0257] Thana et al.

[0258] Line Interval 0.05 mm

[0259] Laser Power 12.5% (200 mW)

[0260] Laser Speed 500 mm / min

[0261]

[0262] 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.

[0263] Table 9 - Settings reported by this work for the production of LIG electrodes.

[0264] This Work

[0265] Line Interval 0.01 mm

[0266] Laser Power 15% (240 mW)

[0267] Laser Speed 500 mm / min

[0268]

[0269] The LIG electrodes were tested by CV of 1 mM ferricyanide. The redox peak separation and general shape of the peaks were considered.

[0270] Table 10- CV settings used for the evaluation of Thana et al. LIG electrodes.

[0271] Cyclic Voltammetry Value

[0272] E Begin 0 V

[0273] E vertexl 0.2 V

[0274] E vertex2 -0.7 V

[0275] E Step 0.01 V

[0276] Scan Rate 0.1 V / s

[0277] t Interval 0.1 s

[0278]

[0279] Table 11 - CV settings used for the evaluation of LIG electrodes from this work.

[0280] Cyclic Voltammetry Value

[0281] E Begin 0 V

[0282] E vertexl 0.5 V

[0283] E vertex2 -0.5 V

[0284] E Step 0.01 V

[0285] Scan Rate 0.1 V / s

[0286] t Interval 0.1 s

[0287]

[0288] Results

[0289] Anode 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.

[0290] 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.

[0291] 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.

[0292] 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.

[0293] 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.

[0294] 2 is also found at pH 7.

[0295] Cathode

[0296] 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.

[0297] 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.

[0298] Direct Electron Transfer to the anode

[0299] 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).

[0300] 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.

[0301] Test Strip Microfluidics

[0302] 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.

[0303] 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.

[0304] 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.

[0305] 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.

[0306] 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. Test Strip Salt Bridge

[0307] 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.

[0308] 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.

[0309] 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).

[0310] 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. (See Fig. 10).

[0311] LIG Electrode Manufacture

[0312] 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.

[0313] 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.

[0314] 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.

[0315] 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.

[0316] Test Strip

[0317] 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.

[0318] A further annotated embodiment of the invention is shown in Fig. 11 , depicting a test strip 200 comprising three layers. Fig. 11 A, 11 B, and 11C depict the separated layers for clarity; a composite of the layers is depicted in Fig. 11D. 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.

[0319] 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)).

[0320] 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).

[0321] 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

CLAIMS1. A test strip for electrochemically detecting a target, the test strip comprising a first substrate laminated to a lid;the first substrate comprising:a sample chamber;an anode and a cathode, the sample chamber configured to allow for fluid communication with one of the anode and the cathode; anda separation chamber configured to separate the anode and cathode and to receive a fluid or gel; andwherein the first substrate is laminated to a lid and the test strip further comprises an inlet configured to deliver the fluid or gel to the separation chamber after the lamination.

2. The test strip of claim 1, wherein the fluid or gel comprises, or is configured to form a medium for selective transportation of ions, such as a salt bridge.

3. The test strip of claim 1 or 2, wherein the fluid or gel is or comprises a solidifiable material, optionally wherein the fluid or gel 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.

4. The test strip of any one of claims 1 to 3, wherein the fluid or gel 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.

5. The test strip of claim 4, wherein the solidifiable material solidifies by a polymerization and / or cross-linking reaction.

6. The test strip of claim 5, wherein the fluid or gel is a solidifiable material, such as a polymerizable liquid, that comprises one or more, or all, of:(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, N-acetylglucosamine, glucuronic acid, uronic acid, galacturonic acid, mannuronic acid, guluronic acid, glucopyranose, acrylic acid, styrene sulfonate, allylamine, acrylamide,sulfated N-acetyl-galactosamine, 3,4-ethylenedioxythiophene)sulfonate, 3,4-ethylenedioxythiophene, aniline, pyrrole or ethylene glycol);(ii) cross-linking agents (e.g. acrylate-, or methacrylate-, or vinyl-containing cross-linking agents, such as N,N'-methylenebis(acrylamide)); and(iii) photoinitiators (such as 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone).

7. The test strip of any one preceding claim, wherein the fluid is a solidifiable material that solidifies to form a polyelectrolyte gel.

8. The test strip of any one preceding claim, wherein the lid comprises the inlet.

9. The test strip of any one preceding claim, wherein the inlet is configured to deliver fluid to the separation chamber in a direction substantially perpendicular to a first (e.g. upper) surface of the first layer.

10. The test strip of any one preceding claim, wherein one of the anode and the cathode is housed in the sample chamber and the other of the anode and cathode is housed in a counter chamber.

11. The test strip of any one preceding claim, wherein the cathode is housed in the sample chamber and the anode is housed in a counter chamber.

12. The test strip of any one preceding claim, wherein the separation chamber comprises a channel that furcates (e.g. bifurcates) such that a first end of the channel contacts the sample chamber and a second end of the channel contacts the counter chamber.

13. The test strip of claim 12, wherein the inlet is configured to deliver fluid or gel at or close to a third end of the channel.

14. The test strip of any one preceding claim, wherein the separation chamber comprises and / or terminates in a substantially T-shaped configuration.

15. The test strip of any one preceding claim, wherein the anode and cathode are positioned close to but not in contact with the separation chamber.

16. The test strip of any one preceding claim, wherein the first substrate comprises a first and a second layer, wherein:the first layer comprises the anode and the cathode and is laminated to the second layer; andthe second layer comprises the sample chamber and the separation chamber; andfurther wherein the second layer is laminated to the lid.

17. The test strip of any one preceding claim, wherein the separation chamber comprises a medium for selective transportation of ion, such as a salt bridge and the anode and cathode form a fuel cell on the test strip, optionally wherein the medium for selective transportation of ions is as defined in any one of claims 2 to 7.

18. The test strip of claim 17, wherein the anode is a bioanode and the cathode is a biocathode and together they form a biofuel cell (such as an enzymatic biofuel cell).

19. The test strip of claim 17 or claim 18, wherein, in use, when a sample comprising the target is introduced to the sample chamber and is brought into proximity with one of the anode or cathode, the fuel cell is turned on, or an electrical response from the fuel cell is increased.

20. Use of a test strip as defined in any one of claims 17 to 19 to detect the presence or absence of a target in a sample.

21. A method for manufacturing a test strip for electrochemically detecting a target, the method comprising:(i) laminating a lid to a first substrate, the first substrate comprising:a sample chamber;an anode and a cathode, the sample chamber configured to allow for fluid communication with one of the anode and the cathode; anda separation chamber configured to separate the anode and cathode; and(ii) adding a fluid or gel to the separation chamber by way of an inlet configured to deliver the fluid or gel to the separation chamber after lamination;wherein the fluid forms, or is configured to form, medium for selective transportation of ions, such as a salt bridge.

22. The method of claim 21, wherein the inlet is comprised on the lid.

23. The method of claim 21 or 22, wherein the fluid or gel is a solidifiable material, and the method further comprises:solidifying the solidifiable material to form the medium for selective transportation of ions.

24. The method of claim 23, wherein the method comprises:solidifying the solidifiable material using irradiation and / or heat, or a chemical activating species,optionally wherein the method comprises polymerizing and / or cross-linking the solidifiable material.

25. The method of any one of claims 21 to 24, wherein the test strip is as defined in any one of claims 1 to 15.

26. The method of any one of claims 21 to 24, wherein the first substrate comprises a first layer and a second layer, wherein:the first layer comprises the anode and the cathode; andthe second layer comprises the sample chamber and the separation chamber; and wherein the method further comprises:laminating together the first layer, the second layer and the lid.

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