Biosensor

Abstract

The present invention provides a novel biosensor that is simpler, has stable performance, and can operate independently. The biosensor is provided with a working electrode formed on a first support body, a counter electrode formed on a second support body, and a separator or a spacer. The biosensor is provided with a structure in which the working electrode and the counter electrode are disposed so as to face each other while the separator or the spacer is interposed therebetween, and the working electrode, the separator or the spacer, and the counter electrode are layered so as to entirely or partially overlap with one another.

Description

Biorecovery

[0001] This invention relates to a biosensor.

[0002] Glucose sensors, a type of biosensor, have been developed and are now commonly used as blood glucose sensors for diabetes. Their origins lie in the monitoring system using enzyme electrodes reported by Clark et al. (Non-Patent Literature 1) and the glucose oxidase immobilized electrode technology reported by Updike (Non-Patent Literature 2).

[0003] Subsequently, a method was developed to detect glucose by transferring electrons derived from glucose to a biosensor electrode (the electrode here is also called the anode or working electrode) via glucose oxidase and a redox mediator (electron mediator, hereinafter sometimes referred to as "RM") (Non-Patent Literature 3). Currently, this method, which similarly uses enzymes and RMs to detect samples other than glucose, is widely applied for electrochemical biosensor applications.

[0004] Recently, a self-generating biosensor that does not require an external power source has been developed by applying a biofuel cell type structure that uses oxygen and oxygen reductase at the counter electrode (Non-Patent Literature 4). This type of biosensor enables autonomous operation and thus autonomous wireless communication. Furthermore, since no power source for operation is required, it is expected that the measurement device will be less expensive and more compact. However, this method has several challenges, including the fact that sensing is performed by combining two types of enzyme reactions, which makes the reaction complex and the voltage and current that result in the signal tend to be unstable; the need for oxygen at the counter electrode, which requires either an open-to-the-air system for the counter electrode or an oxygen introduction channel; and the complexity of the sensor structure.

[0005] Ann. NY Acad Sci. 102, 1962, p. 29-45.Nature. 214, 1967, p. 986.Anal. Cham. 1984, 56, 667-671Adv. Energy Mater. 2013, 3, 60-64

[0006] This invention has been made in view of the above circumstances, and aims to provide a novel biosensor that is simpler, has more stable performance, and is capable of autonomous operation.

[0007] The inventors, through diligent research to achieve the above objectives, have discovered that in a biosensor comprising a counter electrode and a working electrode, by adopting a structure in which both the counter electrode and the working electrode are sheet-like structures, and their active electrode surfaces are arranged facing each other via a separator, it is possible to receive electrons from the working electrode with high sensitivity, accuracy, and in a low-cost and simple configuration. In particular, the counter electrode was more effective because it became self-driving by applying a substance with an oxidation-reduction potential equivalent to or higher than that of the redox mediator functioning at the working electrode as the redox active substance. This method can also be called a discharge-type biosensor because it is self-driving by utilizing the difference in oxidation-reduction potential between the counter electrode and the working electrode. Furthermore, while general biosensors use a structure in which the working electrode and counter electrode are placed in parallel on the same plane, by adopting the above-mentioned facing arrangement, it is possible to detect the sample substance to be sensed with a small amount of sample liquid, the electrode can be easily made large in area, and detection can be performed with low resistance and high sensitivity. A biosensor with this structure can also be called a facing electrode type biosensor. In other words, we have discovered that the face-to-face electrode type biosensor of the present invention exhibits stable and high sensing performance as a self-driving electrochemical biosensor, thereby completing the present invention.

[0008] In other words, the present invention provides the following biosensors: 1. A biosensor comprising a working electrode formed on a first support, a counter electrode formed on a second support, and a separator or spacer, wherein the working electrode and the counter electrode are arranged facing each other via the separator or spacer, and the working electrode, the separator or spacer, and the counter electrode are laminated such that all or part of them overlap. 2. The biosensor of 1 which is self-driving. 3. The biosensor of 1 or 2 in which the working electrode is a laminate comprising a conductive substrate and a working electrode layer laminated on all or part of the conductive substrate. 4. The biosensor of 3 in which the working electrode layer contains a redox mediator and an enzyme. 5. The biosensor of 3 or 4 in which the working electrode layer contains a carbon material. 6. The biosensor of 4 further contains an enzyme crosslinking agent. 7. The biosensor of any of 3 to 6 in which the conductive substrate is a metal foil. 8. The biosensor of 7 in which the portion of the conductive substrate in which the working electrode layer is not formed is waterproofed. 9. 10. Biosensor 1 to 7, wherein the counter electrode is a laminate comprising a conductive substrate and a counter electrode layer laminated on all or part of the conductive substrate. 11. Biosensor 9, wherein the counter electrode layer contains a redox active substance. 12. Biosensor 9 to 11, wherein the conductive substrate is metal foil. 13. Biosensor 12, wherein the portion of the conductive substrate where the counter electrode layer is not formed is waterproofed. 14. Biosensor 1 to 13, wherein the separator is a cellulose-based separator or cellophane. 15. Biosensor 1 to 14, wherein the biosensor is a glucose sensor. 16. Sensing method using biosensor 1 to 15, wherein the voltage between the counter electrode and the working electrode is 0V and sensing is performed by current measurement.

[0009] The biosensor of the present invention can accept electrons from the working electrode without applying an external voltage during sensing, thus enabling sensitive and quantitative detection of sample substances such as glucose without the need for a power source. Furthermore, the biosensor of the present invention can include metal foil as the conductive substrate constituting the working electrode and counter electrode, and because it has low volume resistance, it can be used not only as an electrode but also as wiring in part. In addition, by imparting water resistance to each electrode, the biosensor of the present invention can operate stably for a long time even in sample liquid, making it suitable for continuous measurement applications. The signal voltage can be defined from the potential difference between the oxidation potential of the redox active substance contained in the counter electrode and the reduction potential of the redox mediator contained in the working electrode. In the biosensor of the present invention, since there is no external voltage application or inflow of external current, noise current peaks such as those originating from the electric double layer capacitance or oxidation currents to the redox mediator reduced by some factor do not occur. Therefore, reaction current values ​​can be measured and sensed with high sensitivity, high accuracy, and a high S / N ratio using a simple signal processing method such as an ammeter. Furthermore, if both the redox active substance in the counter electrode and the redox mediator in the working electrode are fast-reactive substances such as organic redox substances, the reaction can proceed rapidly. As a result, the detection current reaches its peak in a short time from the start of measurement, enabling faster measurement. The concentration of the sample substance can then be easily calculated from the peak current, integrated current, etc.

[0010] Furthermore, as described above, the biosensor of the present invention is preferably used in an enzymatic reaction system via a redox mediator. However, it can also be applied to biosensors using the DET (Direct Electron Transfer) method, which reacts directly with the working electrode without the use of a redox mediator, biosensors that apply a redox mediator without involving an enzymatic reaction, and antigen-antibody reaction system biosensors that bind an antibody probe to the working electrode.

[0011] This is a perspective view showing an example of an embodiment of the biosensor according to the present invention. This is a side view of the biosensor shown in Figure 1, viewed from the direction of arrow X. (A) is a plan view showing another aspect of the working electrode, and (B) is a plan view showing yet another aspect. (A) is a plan view showing another aspect of the counter electrode, and (B) is a plan view showing yet another aspect. This is a plan view showing the working electrode or counter electrode fabricated in the example. (A) shows the front surface, and (B) shows the back surface. This is a schematic perspective view showing the structure of the biosensor fabricated in the example. This is a top view showing the biosensor shown in Figure 6. (A) shows the state during assembly, and (B) shows the state after assembly. This is a cross-sectional view along the Y-Y line in Figure 7(B). This is a graph plotting the peak current measured in the biosensor of Example 1 against glucose concentration. This is a graph plotting the peak current measured in the biosensor of Example 2 against glucose concentration.

[0012] The present invention will be described in more detail below. [1] Biosensor The biosensor according to the present invention comprises a working electrode formed on a first support, a counter electrode formed on a second support, and a separator, wherein the working electrode and the counter electrode are arranged facing each other via the separator. The biosensor according to the present invention will be described below with reference to the drawings, but the present invention is not limited thereto.

[0013] Figures 1 and 2 show a biosensor according to one embodiment of the present invention. The biosensor 1 shown herein comprises a working electrode 2 formed on a first support m, a counter electrode 3 formed on a second support n, and a separator or spacer 4, wherein the working electrode 2 and the counter electrode 3 are arranged facing each other via the separator or spacer 4, and the working electrode 2, the separator or spacer 4, and the counter electrode 3 are stacked so that they overlap in whole or in part.

[0014] The supports m and n described above may be shaped according to the structure of the biosensor being manufactured, but in the examples shown in Figures 1 and 2, rectangular (approximately square) plate-shaped supports m and n are used. The thickness of the supports m and n is also not particularly limited for the same reason, but in the present invention, it can be approximately 0.01 to 1 mm.

[0015] Furthermore, the material itself is not particularly limited, as long as it has adequate strength and does not adversely affect sensing accuracy. Specific examples include glass; acrylic resins such as polyethylene terephthalate, polycarbonate, polyacrylate, and polymethacrylate; polyolefins such as polystyrene, polyester, polyethylene, and polypropylene; epoxy, melamine, triacetylcellulose, ABS, AS, and other plastics; and cellulose.

[0016] The working electrode 2 is a laminate in which a working electrode layer 22 is formed in a predetermined area at one end of a rectangular conductive substrate 21. The working electrode layer 22 is positioned near the center of the first support m and is oriented (upward in the drawing) in contact with the separator or spacer 4. The portion of the conductive substrate 21 in which the working electrode layer 22 is not formed (this portion can also be used as wiring for the biosensor, and is therefore sometimes referred to as the "wiring portion of the conductive substrate 21" below) protrudes outward from the first support m.

[0017] The conductive substrate 21 can be appropriately selected from those conventionally used as electrode substrates, and for example, thin films of copper, aluminum, nickel, gold, silver and their alloys, carbon materials, metal oxides, conductive polymers, etc. can be used. The thickness of the conductive substrate 21 is not particularly limited, but in the present invention, for example, a thickness of about 1 to 100 μm can be suitably used.

[0018] As the conductive substrate 21 of the working electrode 2, it is preferable to use a metal foil, and more preferably to use aluminum foil considering processability and the conductivity of the resulting electrode. When a metal foil with excellent conductivity is used, the metal foil can be used as wiring, so the working electrode 2 with wiring can be easily manufactured, and an improvement in the productivity of the biosensor 1 can be expected. Furthermore, since the shape of the working electrode layer 22 is stable due to the conductive substrate 21, it is easy to cut the working electrode layer 22 together with the conductive substrate 21 into a shape corresponding to the shape of the wiring. The working electrode 2 can be cut by known methods such as punching or slitting.

[0019] The above-mentioned active layer 22 contains a redox mediator (RM), and further contains, if necessary, carbon material, a dispersant / binder, enzymes, and enzyme crosslinking agents.

[0020] The working electrode 2 may be configured as shown in Figures 1 and 2, in which a working electrode layer 22 is laminated on a conductive substrate 21, or as shown in Figures 3(A) and (B). Figure 3(A) comprises a conductive substrate 21, a coating film c formed on the conductive substrate 21, and a working electrode layer 22 formed in a predetermined range at one end of the coating film c. Figure 3(B) comprises a conductive substrate 21 and a coating film c formed on the conductive substrate 21, wherein a predetermined range at one end of the coating film c is a working electrode layer 22 including RM (shown as a shaded area in the figure).

[0021] Here, the coating film c contains components other than RM from the components included in the working electrode layer 22 (carbon material, dispersant / binder, enzyme, enzyme crosslinking agent, etc.). The working electrode layer 22 is formed by laminating (see Figure 3(A)) or impregnating (see Figure 3(B)) RM and, if necessary, the other components onto this coating film c. In Figure 3(A), the coating film c and the working electrode layer 22 are depicted as separate layers, but in this case, the coating film c functions integrally with the working electrode 22 as a part of it. Details of the working electrode layer 22 and the coating film c will be described later.

[0022] The counter electrode 3 is a laminate in which a counter electrode layer 32 is formed in a predetermined area at one end of a rectangular conductive substrate 31. The counter electrode layer 32 is positioned near the center of the second support n and is oriented (downward in the drawing) in contact with the separator or spacer 4. The portion of the conductive substrate 31 in which the counter electrode layer 32 is not formed (this portion can also be used as wiring for the biosensor, and is therefore sometimes referred to as the "wiring portion of the conductive substrate 31") protrudes outward from the second support n in the direction opposite to the protrusion direction of the wiring portion of the conductive substrate 21.

[0023] The conductive substrate 31 can be appropriately selected from those conventionally used as electrode substrates, and specific examples include those similar to those exemplified in the description of the working electrode 21.

[0024] As the conductive substrate 31 of the counter electrode 3, it is preferable to use a metal foil, and more preferably to use aluminum foil considering processability and the conductivity of the resulting electrode. When a metal foil with excellent conductivity is used, the metal foil can be used as wiring, so the counter electrode 3 with wiring can be easily manufactured, and an improvement in the productivity of the biosensor 1 can be expected. Furthermore, since the shape of the counter electrode layer 32 is stable due to the conductive substrate 31, it is easy to cut the counter electrode layer 32 together with the conductive substrate 31 into a shape corresponding to the shape of the wiring. The counter electrode 3 can be cut by known methods such as punching or slitting.

[0025] The counter electrode layer 32 contains a redox active substance and, if necessary, also contains carbon material and a dispersant / binder.

[0026] The counter electrode 3 may be configured as shown in Figures 1 and 2, in which a counter electrode layer 32 is laminated on a conductive substrate 31, or as shown in Figures 4(A) and (B). Figure 4(A) comprises a substrate 31, a coating film c formed on the substrate 31, and a counter electrode layer 32 formed in a predetermined range at one end of the coating film c. Figure 4(B) comprises a substrate 31 and a coating film c formed on the substrate 31, wherein a predetermined range at one end of the coating film c is a counter electrode layer 32 including RM (shown as a shaded area in the figure).

[0027] Here, the coating film c contains components other than the redox active substance among the components contained in the counter electrode layer 32 (such as carbon material and dispersant / binder). The counter electrode layer 32 is formed by laminating (see Figure 4(A)) or impregnating (see Figure 4(B)) RM and, if necessary, the other components onto this coating film c. In Figure 4(A), the coating film c and the counter electrode layer 32 are depicted as separate layers, but in this case, the coating film c functions integrally with the counter electrode 32 as a part of it. Details of the counter electrode layer 32 and the coating film c will be described later.

[0028] Furthermore, if the conductive substrates 21 and 31 are metal foils, it is preferable to apply a waterproofing treatment to the portion of the conductive substrates 21 and 31 where the working electrode layer 22 or counter electrode layer 32 is not formed, and to the surface opposite to the surface where the working electrode layer 22 or counter electrode layer 32 is formed. By applying a waterproofing treatment, it is possible to suppress the leaching of metal components constituting the conductive substrates 21 and 31 from the conductive substrates. This makes it possible to suppress the generation of noise currents caused by leached metal components, and an improvement in sensing accuracy can be expected. If a conductive substrate with a coating film formed on its surface using a coating film-forming composition described later is used, the waterproofing treatment should be applied to the coating film.

[0029] Known materials and processing methods can be used for waterproofing. Examples of such methods include applying commercially available waterproof tape to the target area on a conductive substrate, or applying a waterproof coating by spraying a waterproof spray. Commercially available waterproof tapes are not particularly limited as long as they have a certain degree of function to suppress moisture penetration into the tape after application, but examples include Piolan Tape manufactured by Diatex Co., Ltd. Commercially available waterproof sprays are not particularly limited as long as the sprayed surface exhibits water repellency after drying and provides a certain level of waterproofing, but examples include Scotchgard manufactured by Sumitomo 3M.

[0030] The separator or spacer 4 is positioned between the working electrode layer 22 of the working electrode 2 and the counter electrode layer 32 of the counter electrode 3, with the two facing each other via the separator or spacer 4. When the biosensor 1 is used, the sample solution containing the target to be detected is injected into the separator or spacer 4.

[0031] The separator or spacer 4 is intended to prevent the working electrode 2 and the counter electrode 3 from making direct electrical contact in the sample solution. For example, it is provided to prevent electrons derived from an enzymatic reaction from moving to the counter electrode through enzymes or redox mediators eluted or added to the sample solution. Commercially available insulating or porous sheets can be used as the separator. Specific examples include cellulose-based separators, filter paper, paper, cellophane, proton-conducting membranes, ion-exchange membranes, and ultrafiltration membranes.

[0032] The shape of the separator or spacer 4 is not particularly limited as long as it can prevent the electrical contact between the working electrode layer 22 and the counter electrode layer 32, and it may be appropriately set according to the shape of the working electrode 2 and the counter electrode 3, etc. When the substance contained in the working electrode migrates to the counter electrode via a medium such as a test liquid, or vice versa, if it does not pose a problem in detecting the target substance, or if there are measures to prevent it from becoming a problem, a spacer may be used instead of the separator. As the spacer, there is no particular limitation as long as the material constituting it is insoluble in water or the test liquid and has electrical insulation properties. Specific examples include fine particles, beads, a sheet or a three-dimensional structure having a space for holding the test liquid, etc.

[0033] In the biosensor 1 shown in FIGS. 1 and 2, in addition to the working electrode 2 and the counter electrode 3, a reference electrode serving as a reference for accurate potential measurement during the measurement of the sample may be included (not shown).

[0034] Also, when the test liquid comes into contact with a portion of the conductive substrate 21 where the working electrode layer 22 is not formed, or a portion of the conductive substrate 31 where the counter electrode layer 32 is not formed during use, a noise current may be generated due to elution of metal components from the conductive substrates 21 and 31, etc. Therefore, if necessary, a partition (not shown) or the like for preventing the test liquid from entering the above portions may be provided at an appropriate location around the working electrode layer 22 and the counter electrode layer 32.

[0035] The shape, size, arrangement method, etc. of each part such as the support, the working electrode, the counter electrode, and the separator may be appropriately changed according to the shape of the biosensor to be manufactured. Also, a case, a test liquid conducting material, a protective film, etc. that can be other device components can be appropriately selected from known materials and used.

[0036] Hereinafter, the details of the working electrode layer 22 of the working electrode 2 and the counter electrode layer 32 of the counter electrode 3 will be described.

[0037] [2] Working electrode layer [2-1] Components of the working electrode layer From the perspective of enhancing the sensitivity of the biosensor of the present invention, the working electrode layer contains a redox mediator (RM) having electron transfer ability. Further, if necessary, it may further contain a carbon material, a dispersant / binder, an enzyme, an enzyme crosslinking agent, and the like.

[0038] <Redox mediator>A redox mediator (RM) can reversibly become an oxidized form and a reduced form and mediates the transfer of electrons between substances. Examples of RM include metal complexes such as osmium complexes, ruthenium complexes, and iron complexes; quinone compounds such as benzoquinone, naphthoquinone, phenanthrenequinone, phenanthrolinequinone, anthraquinone, and their derivatives; phenazine compounds; viologen compounds; phenothiazine compounds; and phenol compounds. In the present invention, from the perspectives of reactivity with an enzyme and reactivity with an electrode, a quinone compound is preferable, a naphthoquinone and its derivatives are more preferable, and 1,2-naphthoquinone, 1,4-naphthoquinone, and sodium 1,2-naphthoquinone-4-sulfonate are even more preferable. The above RM may be used alone or in combination of two or more.

[0039] When the working electrode layer contains RM, from the perspectives of enabling self-driven operation and enhancing sensing sensitivity, etc., the redox potential of RM is preferably the same as or more negative than the redox potential of the redox active substance contained in the counter electrode. In order to increase the voltage obtained during sensing, perform voltage detection with high precision, and obtain sufficient power to transmit a sensing signal independently, it is preferable that the potential difference is larger.

[0040] In the above working electrode layer, the areal density of RM is preferably 0.01 to 1,000 μg / cm 2 from the perspective of enhancing the sensitivity of sensing, more preferably 0.1 to 100 μg / cm 2 . In the present invention, the areal density of the redox mediator means the content per unit area of RM in the working electrode layer.

[0041] Furthermore, when RM is included, its content is usually 100% by mass of the solid content, but if other components are included, it can be the remainder after excluding those components.

[0042] <Carbon Material> The working electrode layer may contain a carbon material from the viewpoint of improving the conductivity of RM. Examples of the carbon material include carbon nanotubes (CNTs), activated carbon, carbon black, Ketjenblack, acetylene black, carbon whiskers, carbon fibers, natural graphite, and artificial graphite. In the present invention, carbon nanotubes (CNTs) are preferred from the viewpoint of improving the conductivity and sensitivity of the resulting working electrode, reducing noise current, and ensuring uniformity of the coating film thickness. The carbon material may be used alone or in combination of two or more types.

[0043] Carbon nanotubes (CNTs) are generally produced by methods such as arc discharge, chemical vapor deposition (CVD), and laser ablation, but the CNTs used in this invention may be obtained by any of these methods. Furthermore, CNTs include single-walled CNTs (hereinafter abbreviated as SWCNTs), in which one carbon film (graphene sheet) is wound into a cylindrical shape; double-walled CNTs (hereinafter abbreviated as DWCNTs), in which two graphene sheets are wound concentrically; and multi-walled CNTs (MWCNTs), in which multiple graphene sheets are wound concentrically. In this invention, SWCNTs, DWCNTs, and MWCNTs can be used individually or in combination.

[0044] When producing SWCNTs, DWCNTs, or MWCNTs using the methods described above, catalytic metals such as nickel, iron, cobalt, and yttrium may remain, requiring purification to remove these impurities. Acid treatment with nitric acid, sulfuric acid, etc., along with ultrasonic treatment, are effective for removing impurities. However, acid treatment with nitric acid, sulfuric acid, etc., can destroy the π-conjugated system that constitutes the CNTs, potentially impairing the CNTs' inherent properties. Therefore, it is desirable to purify them under appropriate conditions before use.

[0045] Specific examples of CNTs usable in this invention include Supergrowth CNTs [manufactured by the New Energy and Industrial Technology Development Organization (NEDO)], eDIPS-CNTs [manufactured by the New Energy and Industrial Technology Development Organization (NEDO)], SWNT series [manufactured by Meijo Nanocarbon Co., Ltd.: product name], VGCF series [manufactured by Showa Denko K.K.: product name], FloTube series [manufactured by CNano Technology Inc.: product name], AMC [manufactured by Ube Industries, Ltd.: product name], and NANOCYL NC7000 series [manufactured by NanoCyl S.A. Examples include Bayer's products (product name), Bayer's products (product name), GRAPHISTRENGTH (product name), MWNT7 (product name), Hyperion CNT (product name), Hyperion Catalytics International (product name), and the TC series (product name), etc.

[0046] Examples of activated carbon include coconut shell activated carbon and fibrous activated carbon. The activated carbon is not particularly limited by its activation method, and in this invention, activated carbon obtained by methods such as steam activation or chemical activation can be used.

[0047] When the working electrode layer contains carbon material, its content is preferably 0.1 to 1,000, and more preferably 1 to 100, in mass ratio with respect to RM1, considering the conductivity and uniformity of the resulting working electrode.

[0048] <Dispersant and Binder> The working electrode may contain a dispersant and binder from the viewpoint of improving the dispersibility and binding of the redox mediator and carbon material, improving adhesion to the conductive substrate, and improving the strength and water resistance of the working electrode. The dispersant and binder can be appropriately selected from known materials and is not particularly limited, but specific examples include carboxymethylcellulose (CMC) and its salts (sodium salt, ammonium salt, etc.), styrene-butadiene rubber (SBR), polyacrylic acid and its salts (sodium salt, ammonium salt, etc.), polyvinylidene fluoride, polytetrafluoroethylene, polyvinylpyrrolidone, and oxazoline group-containing polymers described later (hereinafter sometimes referred to as "oxazoline polymers"). Furthermore, the dispersant and binder may be crosslinked during film formation with a carboxyl group-containing polymer having carboxyl groups that can react with the oxazoline groups of the oxazoline polymer. In the present invention, CMC, SBR, polyvinylpyrrolidone, oxazoline polymer, and crosslinked products of oxazoline polymer and carboxyl group-containing polymer are preferred. Furthermore, when CNT is used as the carbon material, oxazoline polymer and polyvinylpyrrolidone are more preferred, and when activated carbon is used, CMC and SBR are more preferred. The above dispersant and binder may be used individually or in combination of two or more types.

[0049] <Oxazoline Group-Containing Polymers> Oxazoline group-containing polymers (hereinafter sometimes referred to as oxazoline polymers) function as dispersants, and when used in combination with carboxyl group-containing polymers described later, they function as binders. Furthermore, when a crosslinked structure is formed with carboxyl group-containing polymers described later, they have the function of improving the strength and water resistance (solvent resistance) of the counter electrode layer. In particular, when a carbon material described later is included, they are suitable as dispersants for stably dispersing the carbon material in the composition.

[0050] The above-mentioned oxazoline polymer is not particularly limited as long as it is a polymer in which an oxazoline group is bonded directly to the repeating unit constituting the main chain or via a spacer group such as an alkylene group. Specifically, it is preferable to use a polymer obtained by radical polymerization of an oxazoline monomer having a polymerizable carbon-carbon double bond-containing group at the 2-position as shown in formula (1), which has repeating units bonded to the polymer main chain or a spacer group at the 2-position of the oxazoline ring.

[0051]

[0052] In the formula, X represents a polymerizable carbon-carbon double bond-containing group, and R 1 ~R 4 These independently represent a hydrogen atom, a halogen atom, an alkyl group having 1 to 5 carbon atoms which may have a branched structure, an aryl group having 6 to 20 carbon atoms, or an aralkyl group having 7 to 20 carbon atoms. The polymerizable carbon-carbon double bond-containing group of the oxazoline monomer is not particularly limited as long as it contains a polymerizable carbon-carbon double bond, but a chain-like hydrocarbon group containing a polymerizable carbon-carbon double bond is preferred, for example, an alkenyl group having 2 to 8 carbon atoms such as a vinyl group, an allyl group, or an isopropenyl group is preferred. Examples of halogen atoms include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Specific examples of alkyl groups having 1 to 5 carbon atoms which may have a branched structure include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, a tert-butyl group, and an n-pentyl group. Specific examples of aryl groups having 6 to 20 carbon atoms include a phenyl group, a xylyl group, a tolyl group, a biphenyl group, and a naphthyl group. Specific examples of aralkyl groups having 7 to 20 carbon atoms include the benzyl group, phenylethyl group, and phenylcyclohexyl group.

[0053] Specific examples of oxazoline monomers having a polymerizable carbon-carbon double bond-containing group at the 2-position, as shown in formula (1), include 2-vinyl-2-oxazoline, 2-vinyl-4-methyl-2-oxazoline, 2-vinyl-4-ethyl-2-oxazoline, 2-vinyl-4-propyl-2-oxazoline, 2-vinyl-4-butyl-2-oxazoline, 2-vinyl-5-methyl-2-oxazoline, 2-vinyl-5-ethyl-2-oxazoline, 2-vinyl-5-propyl-2-oxazoline, 2-vinyl-5-butyl-2-oxazoline, and 2-isopropenyl-2-oxazoline. Examples include sazolin, 2-isopropenyl-4-methyl-2-oxazoline, 2-isopropenyl-4-ethyl-2-oxazoline, 2-isopropenyl-4-propyl-2-oxazoline, 2-isopropenyl-4-butyl-2-oxazoline, 2-isopropenyl-5-methyl-2-oxazoline, 2-isopropenyl-5-ethyl-2-oxazoline, 2-isopropenyl-5-propyl-2-oxazoline, and 2-isopropenyl-5-butyl-2-oxazoline, but 2-isopropenyl-2-oxazoline is preferred from the standpoint of ease of acquisition.

[0054] Furthermore, when carbon materials such as CNTs are included, considering that the dispersion or composition containing these materials is prepared using water (and, if necessary, an aqueous solvent described later), it is preferable that the oxazoline polymer is water-soluble. Such a water-soluble oxazoline polymer may be a homopolymer of the oxazoline monomer represented by formula (1) above, but in order to further enhance its solubility in water, it is preferable that it is obtained by radical polymerization of at least two monomers, the oxazoline monomer and a (meth)acrylic acid ester monomer having a hydrophilic functional group.

[0055] Specific examples of (meth)acrylic monomers having hydrophilic functional groups include (meth)acrylic acid, 2-hydroxyethyl acrylate, methoxypolyethylene glycol acrylate, monoesters of acrylic acid and polyethylene glycol, 2-aminoethyl acrylate and its salts, 2-hydroxyethyl methacrylate, methoxypolyethylene glycol methacrylate, monoesters of methacrylic acid and polyethylene glycol, 2-aminoethyl methacrylate and its salts, sodium (meth)acrylate, ammonium (meth)acrylate, (meth)acrylonitrile, (meth)acrylamide, N-methylol(meth)acrylamide, N-(2-hydroxyethyl)(meth)acrylamide, sodium styrene sulfonate, etc. These may be used individually or in combination of two or more. Among these, methoxypolyethylene glycol (meth)acrylate and monoesters of (meth)acrylic acid and polyethylene glycol are preferred.

[0056] Furthermore, in the present invention, other monomers other than the oxazoline monomer and (meth)acrylic monomers having hydrophilic functional groups can be used in combination, as long as they do not adversely affect the carbon material dispersibility of the obtained oxazoline polymer. Specific examples of other monomers include (meth)acrylic acid ester monomers such as methyl (meth)acrylate, ethyl (meth)acrylate, butyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, stearyl (meth)acrylate, perfluoroethyl (meth)acrylate, and phenyl (meth)acrylate; α-olefin monomers such as ethylene, propylene, butene, and pentene; haloolefin monomers such as vinyl chloride, vinylidene chloride, and vinyl fluoride; styrene monomers such as styrene and α-methylstyrene; vinyl carboxylate ester monomers such as vinyl acetate and vinyl propionate; and vinyl ether monomers such as methyl vinyl ether and ethyl vinyl ether. These can be used individually or in combination of two or more.

[0057] In the monomer component used to produce the oxazoline polymer used in the present invention, the content of oxazoline monomer is preferably 10% by mass or more, more preferably 20% by mass or more, and even more preferably 30% by mass or more, from the viewpoint of further enhancing the carbon material dispersion ability of the obtained oxazoline polymer. The upper limit of the content of oxazoline monomer in the monomer component is 100% by mass, in which case a homopolymer of oxazoline monomer is obtained.

[0058] On the other hand, in order to further enhance the water solubility of the resulting oxazoline polymer, the content of (meth)acrylic monomers having hydrophilic functional groups in the monomer component is preferably 10% by mass or more, more preferably 20% by mass or more, and even more preferably 30% by mass or more. Furthermore, as mentioned above, the content of other monomers in the monomer component is within a range that does not affect the carbon material dispersibility of the resulting oxazoline polymer and varies depending on the type of monomer, so it cannot be determined in general terms, but it can be appropriately set in the range of 5 to 95% by mass or less, preferably 10 to 90% by mass or less.

[0059] The average molecular weight of the oxazoline polymer is not particularly limited, but it is preferable that the weight-average molecular weight is between 1,000 and 2,000,000. If the weight-average molecular weight of the polymer is less than 1,000, the dispersibility of carbon materials may be significantly reduced or may not be exhibited at all. On the other hand, if the weight-average molecular weight exceeds 2,000,000, handling in the dispersion process may become extremely difficult. Oxazoline polymers with a weight-average molecular weight of 2,000 to 1,000,000 are more preferable. Note that the weight-average molecular weight in this invention is a measurement value (polystyrene equivalent) obtained by gel permeation chromatography.

[0060] The oxazoline polymer used in the present invention can be produced by polymerizing the various monomers described above using known radical polymerization methods, such as those described in Japanese Patent Publication No. 6-32844 and Japanese Patent Publication No. 2013-72002. Furthermore, oxazoline polymers usable in the present invention can also be obtained as commercially available products. Examples of such commercially available products include Epocross WS-300 (manufactured by Nippon Shokubai Co., Ltd., solid content concentration 10% by mass, aqueous solution), Epocross WS-700 (manufactured by Nippon Shokubai Co., Ltd., solid content concentration 25% by mass, aqueous solution), Epocross WS-500 (manufactured by Nippon Shokubai Co., Ltd., solid content concentration 39% by mass, water / 1-methoxy-2-propanol solution), Poly(2-ethyl-2-oxazoline) (Aldrich), Poly(2-ethyl-2-oxazoline) (AlfaAesar), Poly(2-ethyl-2-oxazoline) (VWR International, LLC), etc. When commercially available as a solution, it can be used as is, or the solvent may be replaced to create the desired solvent system.

[0061] The above-mentioned oxazoline polymers may be used individually or in combination of two or more types.

[0062] Films obtained by drying oxazoline polymers on a conductive substrate typically lack water resistance and may elute in sample solutions, leading to unstable sensing performance. In such cases, water resistance can be imparted by using a carboxyl group-containing polymer in combination and performing heat drying to promote a crosslinking reaction.

[0063] <Carboxyloid-containing polymer> The carboxyloid-containing polymer is not particularly limited as long as it is a polymer having at least one carboxyloid group that can react with the oxazoline group of the oxazoline-containing polymer described above.

[0064] Furthermore, any functional group that can react with an oxazoline group may be a functional group other than a carboxyl group, and specific examples of such groups include aromatic hydroxyl groups and aromatic thiol groups.

[0065] When considering the purpose of crosslinking the oxazoline polymer to enhance the strength and water resistance (solvent resistance) of the resulting coating film, a carboxyl group-containing polymer having at least two functional groups capable of reacting with oxazoline groups, including at least one carboxyl group, is preferred, and a polymer having two or more carboxyl groups is more preferred. (Hereinafter, a carboxyl group-containing polymer having two or more functional groups capable of reacting with oxazoline groups and capable of crosslinking two or more molecules of oxazoline polymer may be referred to as a "crosslinking agent.")

[0066] Furthermore, the carboxyl group-containing polymer may have a functional group that reacts with the oxazoline group upon heating during coating film formation or in the presence of an acid catalyst, for example, a carboxylic acid (carboxyl group), a sodium salt, potassium salt, lithium salt, ammonium salt, or amine salt of a carboxylic acid. In the present invention, a polymer having an ammonium salt or amine salt of a carboxylic acid is preferred in order to improve the stability of the dispersion and to improve the efficiency of the crosslinking reaction.

[0067] Specific examples of carboxyl group-containing polymers include synthetic polymers such as polyacrylic acid and its copolymers, and metal salts of natural polymers such as carboxymethylcellulose and alginic acid, which exhibit crosslinking reactivity in the presence of an acid catalyst, and ammonium salts of the above synthetic polymers and natural polymers, which exhibit crosslinking reactivity upon heating. In particular, polyacrylic acid, sodium polyacrylate, lithium polyacrylate, ammonium polyacrylate, polyacrylateamine, carboxymethylcellulose, sodium carboxymethylcellulose, lithium carboxymethylcellulose, and ammonium carboxymethylcellulose, which exhibit crosslinking reactivity in the presence of an acid catalyst or under heating conditions, are preferred, with ammonium polyacrylate and polyacrylateamine being more preferred.

[0068] The above-mentioned carboxyl group-containing polymers can also be obtained as commercial products. Examples of such commercial products include sodium polyacrylate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., degree of polymerization 2,700 to 7,500), sodium carboxymethylcellulose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), sodium alginate (manufactured by Kanto Chemical Co., Ltd., Grade 1), Aron A-30 (ammonium polyacrylate, manufactured by Toagosei Co., Ltd., solid content concentration 32% by mass, aqueous solution), DN-800H (ammonium carboxymethylcellulose, manufactured by Daicel Finechem Co., Ltd.), ammonium alginate (manufactured by Kimika Co., Ltd.), and others.

[0069] Alternatively, polyacrylate amine may be prepared by mixing commercially available polyacrylic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., molecular weight 5,000 to 1,000,000) with commercially available amines such as diethylamine (manufactured by Tokyo Chemical Industries, Ltd.), triethanolamine (manufactured by Tokyo Chemical Industries, Ltd.), and N,N-dimethylethanolamine (manufactured by Tokyo Chemical Industries, Ltd.) in a solvent.

[0070] The above-mentioned carboxyl group-containing polymer may be used individually or in combination of two or more types.

[0071] The content of the carboxyl group-containing polymer varies depending on the solvent used, the conductive substrate used, the required viscosity and film shape, etc., but is preferably 2.0 to 0.1, more preferably 1.0 to 0.2, and even more preferably 0.5 to 0.3 per 1 oxazoline polymer by mass ratio.

[0072] Furthermore, when the above-mentioned oxazoline polymer and carboxyl group-containing polymer are used in combination, a thermoacid generator such as 2,4,4,6-tetrabromocyclohexadienone, benzoin tosylate, 2-nitrobenzyl tosylate, or an alkyl organic sulfonate may be included as a catalyst to promote the crosslinking reaction between the two.

[0073] When the above catalyst is included, its content is usually 0.0001 to 20% by mass, preferably 0.0005 to 10% by mass, and more preferably 0.001 to 3% by mass, relative to the mass of the oxazoline polymer.

[0074] If the above-mentioned active layer contains a dispersant and binder, the amount is preferably 0.1 to 99 parts by mass, more preferably 0.5 to 50 parts by mass, and even more preferably 1 to 10 parts by mass, based on 100 parts by mass calculated by totaling the above-mentioned RM and carbon material.

[0075] In particular, when using CNTs or activated carbon as the carbon material, the content of the dispersant and binder is preferably within the following range from the viewpoint of more efficiently dispersing the carbon material.

[0076] When the carbon material is CNT, the content of the dispersant and binder is preferably 0.1 to 9, and more preferably 0.3 to 3, in mass ratio with respect to 1 unit of the carbon material.

[0077] When the carbon material is activated carbon, the content of the dispersant and binder is preferably 0.01 to 0.5 by mass ratio, and more preferably 0.05 to 0.2 per 1 unit of the carbon material.

[0078] Furthermore, if the dispersant and binder include oxazoline polymer and carboxyl group-containing polymer, their content should be adjusted so that the total amount of oxazoline polymer and carboxyl group-containing polymer falls within the above range.

[0079] <Enzymes> The above-mentioned working electrode can also be used as an enzyme electrode by incorporating a predetermined enzyme. The enzymes that can be used include oxidases and dehydrogenases, and are appropriately selected depending on the substrate to be detected.

[0080] Specific examples of oxidases include glucose oxidase, lactate oxidase, pyruvate oxidase, cholesterol oxidase, amino acid oxidase, glutamate oxidase, fructosyl amino acid oxidase, alcohol oxidase, ascorbate oxidase, fructosyl peptide oxidase, bilirubin oxidase, and aldehyde oxidase.

[0081] Specific examples of dehydrogenases include glucose dehydrogenase (e.g., flavin-bound glucose dehydrogenase (FAD-GDH)), lactate dehydrogenase, pyruvate dehydrogenase, amino acid dehydrogenase, glutamate dehydrogenase, 3-hydroxybutyrate dehydrogenase, alcohol dehydrogenase, and aldehyde dehydrogenase.

[0082] The biosensor of the present invention can be suitably used as a glucose sensor by using glucose dehydrogenase in particular as the enzyme applied to the working electrode. The amount of enzyme used can be appropriately set according to the target concentration to be detected, etc.

[0083] <Enzyme Crosslinking Agent> If the product contains enzymes, it may also contain an enzyme crosslinking agent that crosslinks the enzymes together. By crosslinking the enzymes together, the elution of enzymes from the working electrode layer can be suppressed. Examples of the above-mentioned enzyme crosslinking agents include epoxy crosslinking agents, glutaraldehyde, isocyanate derivatives, etc.

[0084] Specific examples of the epoxy crosslinking agents mentioned above include sorbitol polyglycidyl ether, glycerol polyglycidyl ether, trimethylolpropane polyglycidyl ether, diglycerol polyglycidyl ether, polyglycerol polyglycidyl ether, ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, resorcinol glycidyl ether, neopentyl glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, 1,6-hexanediol diglycidyl ether, 1,4-butanediol diglycidyl ether, hydrogenated bisphenol A diglycidyl ether, phthalate diglycidyl ester, and polypropylene glycol diglycidyl ether.

[0085] Specific examples of the above-mentioned isocyanate derivatives include diphenylmethane diisocyanate, hexamethylene diisocyanate, toluene diisocyanate, and isophorone diisocyanate.

[0086] When the above-mentioned enzyme crosslinking agent is included, its content is preferably 0.01 to 1000 by mass ratio per 1 unit of enzyme, and more preferably 0.1 to 100.

[0087] The thickness of the working electrode layer 22 is not particularly limited, but considering the reduction of internal resistance and the low cost of manufacturing electrodes, it is preferably 0.05 to 10 μm, and more preferably 0.10 to 3 μm. Furthermore, when CNTs are used as the carbon material, a thickness of about 0.2 to 1 μm is preferable.

[0088] The above-mentioned working electrode layer can be obtained, for example, by applying a working electrode composition containing the above-mentioned RM to all or part of the conductive substrate described above and heating and drying it to form the working electrode layer, or by applying a predetermined coating film-forming composition that does not contain RM to all or part of the conductive substrate and heating and drying it to form a coating film, and then impregnating or laminating the working electrode composition containing the above-mentioned RM into the coating film to form the working electrode layer.

[0089] The following describes the composition for the working electrode and the composition for forming the coating film.

[0090] [2-2] Working electrode composition The working electrode composition is a composition comprising RM, and optionally a carbon material, a dispersant / binder, an enzyme, an enzyme crosslinking agent, and a solvent. The RM, carbon material, dispersant / binder, enzyme, and enzyme crosslinking agent are as described above. Furthermore, the content of each component in the working electrode composition is not particularly limited, as long as the content of each component in the resulting working electrode layer can be within the range described above, taking into consideration coating properties and the like. Preferred embodiments of the working electrode composition will be described below.

[0091] <Redox Mediator> The redox mediator (RM) is described in accordance with the description of the working electrode above.

[0092] <Carbon Material> When the above working electrode composition contains a carbon material, the amount of carbon material used will vary depending on the type of carbon material used, but the range is preferably 0.01 to 10,000 by mass ratio relative to RM1, more preferably 0.1 to 1,000, and even more preferably 1 to 100.

[0093] In particular, when using CNTs or activated carbon as the carbon material, the content is preferably within the following range.

[0094] When the carbon material is CNT, its content is preferably 0.01 to 10,000, more preferably 0.1 to 1,000, and even more preferably 0.5 to 100, relative to RM1 by mass ratio.

[0095] <Dispersant and Binder> If the above active electrode composition contains a dispersant and binder, the amount thereof is preferably 0.1 to 99 parts by mass, more preferably 0.5 to 50 parts by mass, and even more preferably 1 to 10 parts by mass, based on 100 parts by mass calculated by totaling the redox active substance and carbon material.

[0096] In particular, when using CNTs or activated carbon as the carbon material, the content of the dispersant and binder is preferably within the following range from the viewpoint of more efficiently dispersing the carbon material.

[0097] When the carbon material is CNT, the content of the dispersant and binder is preferably 0.1 to 9, and more preferably 0.3 to 3, in mass ratio with respect to 1 unit of the carbon material.

[0098] Furthermore, if an oxazoline polymer is included as both a dispersant and binder, its content should be adjusted so that the total amount of the oxazoline polymer and the carboxyl group-containing polymer falls within the above range. Also, if a catalyst is included, its content should conform to the range described in the description of the counter electrode.

[0099] <Enzymes> The enzymes are as described above for the working electrode.

[0100] <Enzyme Crosslinking Agent> The enzyme crosslinking agent is as described above for the working electrode.

[0101] <Solvent> The above working electrode composition may contain a solvent. The solvent is not particularly limited as long as it can dissolve the above RM, and examples include water, ethers such as tetrahydrofuran (THF), diethyl ether, and 1,2-dimethoxyethane (DME); amides such as N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), and N-methyl-2-pyrrolidone (NMP); ketones such as acetone and methyl ethyl ketone; alcohols such as methanol, ethanol, n-propanol, and 2-propanol; glycol ethers such as ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, and propylene glycol monomethyl ether; glycols such as ethylene glycol and propylene glycol; and organic solvents such as acetonitrile. In the present invention, among these solvents, water, THF, DMF, NMP, methanol, ethanol, 2-propanol, and acetonitrile are preferred, and THF, NMP, and acetonitrile are more preferred, considering the solubility of the quinone compound, the dispersibility of the carbon material, and the coating properties of the composition. These solvents may be used individually or in combination of two or more types.

[0102] In addition to the solvents mentioned above, phosphate buffers and phosphate-buffered saline can also be used to dissolve RM. Commercially available phosphate buffers can be used, such as the phosphate buffer manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0103] When the above-mentioned working electrode composition contains a solvent, the solid content concentration is set appropriately considering the basis weight of RM, the coating properties of the composition, and the thickness of the thin film (working electrode layer) to be formed, but is usually about 0.0001 to 50% by mass, preferably about 0.001 to 20% by mass, and more preferably about 0.01 to 10% by mass.

[0104] Furthermore, if the working electrode composition contains a solvent, the concentration of RM is preferably 0.01 to 1,000 mmol / L, and more preferably 0.1 to 100 mmol / L, from the viewpoint of increasing sensing sensitivity.

[0105] [2-3] Preparation of the Working Electrode Composition The working electrode composition may be prepared by using RM as is, or by dissolving it in a suitable solvent as needed. In this case, the carbon material, dispersant / binder, and enzyme can be mixed, dispersed, and dissolved at any time.

[0106] In particular, from the viewpoint of the coating properties of the composition, it is preferable to dissolve the above-mentioned RM in a solvent and use it as an RM solution. As the solvent, it can be appropriately selected from the above-mentioned solvents depending on the type of RM, and water, tetrahydrofuran, N,N-dimethylformamide, N-methylpyrrolidone, ethanol, 2-propanol, phosphate buffer, and phosphate-buffered physiological saline are preferred, with phosphate buffer being more preferred from the viewpoint of increasing the solubility of the RM in the sample solution.

[0107] The concentration of RM in an RM solution is not particularly limited, but is usually around 0.1 to 100 mM (molecule / L).

[0108] When carbon material is included, it is preferable to disperse the resulting mixture, which can further improve the dispersion ratio of the carbon material. Dispersion treatments include mechanical treatments such as wet treatment using ball mills, bead mills, and jet mills, and ultrasonic treatment using bath-type or probe-type sonicators. The duration of the dispersion treatment is arbitrary, but it is preferably from 1 minute to 10 hours, and more preferably from 1 minute to 5 hours. In addition, heat treatment may be applied as needed.

[0109] Furthermore, it is preferable to use the above-mentioned enzyme as an enzyme solution in a solvent containing water. The solvent can be appropriately selected depending on the type of enzyme, and specific examples include water, physiological saline, phosphate buffer, phosphate-buffered physiological saline, ethanol, 2-propanol, etc. Two or more of these solvents or solutions may also be used in mixture form.

[0110] The enzyme concentration in the enzyme solution is not particularly limited, but is usually around 0.2 to 5.0% by mass. The enzyme activity concentration is not particularly limited, but is usually around 5,000 to 100,000 units / mL.

[0111] [2-4] Composition for forming a coating film The composition for forming a coating film is a composition comprising a carbon material and a solvent, and optionally an optional component such as a dispersant and binder. The carbon material, solvent, and dispersant and binder are as described above. Furthermore, the content of each component in the composition for forming a coating film is not particularly limited, as long as it can be within the range described above in the counter electrode layer obtained, taking into consideration the coating properties, etc. Preferred embodiments of the composition for forming a coating film will be described below.

[0112] <Carbon Material> The carbon material content usually includes optional components such as dispersants and binders described later, but may be 100% by mass of the solid content. When optional components such as dispersants and binders described later are included, the carbon material content varies depending on the type of carbon material used, but is preferably 0.1 to 98% by mass of the solid content, and more preferably 0.5 to 95% by mass. In the present invention, solid content means components other than the solvent that constitute the composition (the same applies hereinafter).

[0113] In particular, when an optional component is included and CNTs or activated carbon are used as the carbon material, the content is preferably within the following range, taking into consideration their respective dispersibility and coating uniformity.

[0114] Furthermore, when the carbon material is CNT, its content is preferably about 1 to 98% by mass of the solid content, more preferably about 10 to 95% by mass, and even more preferably about 20 to 80% by mass.

[0115] When the carbon material is activated carbon, its content is preferably about 1 to 98% by mass of the solid content, more preferably about 30 to 95% by mass, and even more preferably about 60 to 95% by mass.

[0116] In this invention, the carbon material only needs to be present in the counter electrode in the desired amount at the stage when the counter electrode layer is formed. Therefore, the carbon material may be included in either the biosensor counter electrode composition or the coating film forming composition.

[0117] <Solvent> The above-mentioned coating film-forming composition contains a solvent, the ratio of which is set appropriately considering the coating properties of the composition and the thickness of the thin film (counter electrode layer) to be formed, etc., but is usually about 5 to 99.99% by mass, preferably about 10 to 99.9% by mass, and more preferably about 20 to 99.5% by mass.

[0118] In particular, when using CNTs or activated carbon as the carbon material, the solvent ratio is preferably within the following range, taking into consideration their respective dispersibility and coating uniformity.

[0119] Furthermore, when the carbon material includes CNTs, the solvent ratio of the composition is preferably about 90 to 99.8% by mass, more preferably about 92 to 99.5% by mass, and even more preferably about 95 to 99% by mass.

[0120] When the carbon material includes activated carbon, the solvent ratio of the composition is preferably about 10 to 50% by mass, more preferably about 30 to 95% by mass, and even more preferably about 10 to 50% by mass.

[0121] <Dispersant and Binder> When the above dispersant and binder is included, the amount will vary depending on the type of carbon material used, but for example, it is preferably 0.1 to 99 parts by mass, more preferably 0.5 to 50 parts by mass, and even more preferably 1 to 10 parts by mass per 100 parts by mass of the above carbon material.

[0122] In particular, when using CNTs or activated carbon as the carbon material, the content of the dispersant and binder is preferably within the following range from the viewpoint of more efficiently dispersing the carbon material.

[0123] When the carbon material is CNT, the content of the dispersant and binder is preferably 0.01 to 9, and more preferably 0.1 to 2, in mass ratio with respect to 1 unit of the carbon material.

[0124] When the carbon material is activated carbon, the content of the dispersant and binder is preferably 0.01 to 0.5 by mass ratio, and more preferably 0.05 to 0.2 per 1 unit of the carbon material.

[0125] [2-5] Preparation of the coating film-forming composition The above coating film-forming composition can be prepared by dispersing a carbon material in a suitable solvent. In addition, a dispersant and binder can be mixed, dispersed, and dissolved at any time.

[0126] When preparing the composition, it is preferable to disperse the carbon material, as this treatment can further improve the dispersion ratio of the carbon material. Examples of dispersion treatments include mechanical treatments such as wet treatment using ball mills, bead mills, and jet mills, and ultrasonic treatment using bath-type or probe-type sonicators. The duration of the dispersion treatment is arbitrary, but it is preferably from 1 minute to 10 hours, and more preferably from 1 minute to 1 hour. In addition, heat treatment may be applied as needed.

[0127] [2-6] Formation of the working electrode layer The working electrode layer can be obtained, for example, by applying the working electrode composition containing the above RM to all or part of a conductive substrate and heating and drying it to form the working electrode layer, or by applying a predetermined coating film forming composition to all or part of a conductive substrate and heating and drying it to form a coating film, and then impregnating or laminating the working electrode composition containing the above RM onto the coating film to form the working electrode layer.

[0128] Examples of application methods for the working electrode composition and the coating film-forming composition include spin coating, dip coating, flow coating, inkjet coating, spray coating, bar coating, gravure coating, slit coating, roll coating, flexographic printing, transfer printing, brush coating, blade coating, and air knife coating. However, from the standpoint of work efficiency, inkjet coating, casting, dip coating, bar coating, blade coating, roll coating, gravure coating, flexographic printing, and spray coating are preferred.

[0129] The temperature for heating and drying is arbitrary as long as it can dry the solvent containing at least water, but is preferably around 50 to 200°C, and more preferably around 80 to 150°C considering the reactivity of the oxazoline polymer and the carboxyl group-containing polymer. The drying time is not particularly limited as long as the solvent can be evaporated, but is usually preferably around 1 to 30 minutes. Heating and drying can be carried out using suitable equipment such as a hot plate, oven, or vacuum oven.

[0130] The basis weight of the working electrode layer or coating film is not particularly limited as long as it satisfies the thickness of the working electrode layer as described above, however, for working electrodes containing CNTs as the carbon material, it is 1 to 1,000 μg / cm³. 2 Preferably, and more preferably, 10 to 500 μg / cm³ 2 That is the case.

[0131] The basis weight and film thickness of RM in the working electrode layer can be adjusted by known methods. For example, they can be adjusted by changing the solid content concentration of the working electrode composition and the coating film forming composition, the coating conditions, the number of coats, etc. To increase the basis weight and film thickness, the device conditions can be changed in a direction that makes the working electrode layer or coating film thicker, or the solid content concentration can be increased, or the number of coats can be increased. To decrease the basis weight and film thickness, the device conditions can be changed in a direction that makes the working electrode layer or coating film thinner, or the solid content concentration can be decreased, the number of coats can be decreased, or the clearance can be reduced.

[0132] Furthermore, if a coating film is formed, the performance can also be adjusted by controlling the solid content concentration and impregnation amount of the working electrode composition used to impregnate the formed coating film.

[0133] When using the above-mentioned RM and enzyme, for example, a working electrode can be obtained by dropping a solution containing RM or enzyme onto the thin film (working electrode) and drying it, thereby laminating these components onto the thin film. When using both RM and enzyme, it is preferable to drop a solution containing RM onto the thin film, dry it, and then drop a solution containing the enzyme onto it and dry it.

[0134] The drying conditions for RM should be such that the solvent evaporates efficiently and the contained components do not volatilize or deteriorate, but 20 to 150°C is preferred, 20 to 80°C is more preferred, and 20 to 50°C is even more preferred. The drying time is not particularly limited, but is usually about 1 to 30 minutes.

[0135] The drying conditions for the enzyme solution should be such that the solvent evaporates efficiently and the components do not volatilize or deteriorate. However, 20 to 60°C is preferred, 20 to 50°C is more preferred, and 20 to 40°C is even more preferred. The drying time is not particularly limited, but is usually about 1 to 30 minutes.

[0136] [3] Counter electrode layer [3-1] Components of the counter electrode layer The counter electrode layer contains a redox active substance. It may also contain optional components such as carbon material, binder and dispersant as needed.

[0137] <Redox Active Substances> Redox active substances are substances whose potential changes through the transfer of electrons, hydrogen atoms, or hydride ions, and in this invention, they are reversible. In this invention, by including such a redox active substance in the counter electrode layer, a self-driving biosensor can be created.

[0138] Examples of redox active substances include organic redox active substances and inorganic redox active substances. However, from the viewpoint of further improving sensing sensitivity, organic redox active substances are preferred, quinone compounds and nitroxyl radical-containing compounds are more preferred, and quinone compounds are even more preferred.

[0139] Specific examples of quinone compounds include chloranil, bromanil, 1,2-naphthoquinone, 1,4-naphthoquinone, dichlorodicyanobenzoquinone, and polymers containing these as functional groups, with chloranil being preferred. The above redox active substances may be used individually or in combination of two or more.

[0140] In the counter electrode layer described above, the basis amount of the redox active substance is not particularly limited as long as it is sufficiently large compared to the amount of the sample (target substance) detected by the sensing process, and may be varied depending on the target detection concentration. However, from the viewpoint of increasing the sensitivity of sensing, it is recommended to use 1 to 1,000 μg / cm³. 2 Preferably, and more preferably, 5 to 500 μg / cm³ 2In this invention, the basis weight of the redox active substance refers to the amount of the redox active substance per unit area in the counter electrode (or counter electrode layer).

[0141] <Carbon Material> The counter electrode layer may contain a carbon material from the viewpoint of improving the conductivity of the redox active substance. Examples of carbon materials are the same as those exemplified in the description of the working electrode layer above. In the present invention, carbon nanotubes and activated carbon are preferred from the viewpoint of conductivity, reaction rate, sensitivity, sensing power, and sensing current density, and activated carbon is more preferred from the viewpoint of high specific surface area, suppression of enzymatic reactions in the counter electrode layer, and suppression of reactions between the redox mediator eluted in the sample solution and the counter electrode layer. The carbon material may be used alone or in combination of two or more types.

[0142] When the counter electrode layer contains a carbon material, the amount varies depending on the type of carbon material used. However, from the viewpoint of conductivity and uniformity of the resulting counter electrode, the mass ratio is preferably 0.01 to 10,000, more preferably 0.1 to 1,000, and even more preferably 1 to 100, relative to 1 unit of the redox active substance.

[0143] In particular, when using CNTs or activated carbon as the carbon material, the content is preferably within the following range.

[0144] When the carbon material is CNT, its content is preferably 0.001 to 1,000 by mass ratio, more preferably 0.01 to 100, and even more preferably 0.1 to 10, per 1 unit of the redox active substance.

[0145] When the carbon material is activated carbon, its content is preferably 0.01 to 10,000, more preferably 0.1 to 1,000, and even more preferably 1 to 100, in mass ratio with respect to 1 unit of the redox active substance.

[0146] <Dispersant and Binder> The counter electrode may contain a dispersant and binder from the viewpoint of improving the dispersibility and binding of the redox active substance and carbon material, improving adhesion to the conductive substrate, and improving the strength and water resistance of the counter electrode. Examples of dispersants and binders are the same as those exemplified in the description of the working electrode layer above. In the present invention, CMC, SBR, polyvinylpyrrolidone, oxazoline polymer, and crosslinked products of oxazoline polymer and carboxyl group-containing polymer are preferred. Furthermore, when CNT is used as the carbon material, oxazoline polymer and polyvinylpyrrolidone are more preferred, and when activated carbon is used, CMC and SBR are more preferred. The above dispersant and binder may be used alone or in combination of two or more types.

[0147] If the counter electrode layer contains a dispersant and binder, the amount thereof is preferably 0.001 to 1,000 parts by mass, more preferably 0.01 to 100 parts by mass, and even more preferably 0.1 to 10 parts by mass, based on 100 parts by mass calculated by totaling the redox active substance and carbon material, from the viewpoint of the strength and water resistance of the resulting counter electrode layer.

[0148] In particular, when using CNTs or activated carbon as the carbon material, the content of the dispersant and binder is preferably within the following range from the viewpoint of more efficiently dispersing the carbon material.

[0149] When the carbon material is CNT, the content of the dispersant and binder is preferably 0.1 to 9, and more preferably 0.3 to 3, in mass ratio with respect to 1 unit of the carbon material.

[0150] When the carbon material is activated carbon, the content of the dispersant and binder is preferably 0.01 to 0.5 by mass ratio, and more preferably 0.05 to 0.2 per 1 unit of the carbon material.

[0151] Furthermore, if the dispersant and binder include oxazoline polymer and carboxyl group-containing polymer, their content should be adjusted so that the total amount of oxazoline polymer and carboxyl group-containing polymer falls within the above range.

[0152] The thickness of the counter electrode layer is not particularly limited, but considering the reduction of internal resistance and the low cost of manufacturing electrodes, it is preferably 0.05 to 200 μm, and more preferably 0.10 to 100 μm. Furthermore, when CNTs are used as the carbon material, a thickness of about 0.2 to 1 μm is suitable, and when activated carbon is used, a thickness of about 20 to 100 μm is suitable.

[0153] The counter electrode layer can be obtained, for example, by applying the counter electrode composition containing the redox active substance to all or part of the conductive substrate described above and heating and drying it to form the counter electrode layer, or by applying a predetermined coating film-forming composition that does not contain a redox active substance to all or part of the conductive substrate and heating and drying it to form a coating film, and then impregnating or laminating the counter electrode composition containing the redox active substance into the coating film to form the counter electrode layer.

[0154] The following describes the composition for the counter electrode and the composition for forming the coating film.

[0155] [3-2] Counter Electrode Composition The counter electrode composition is a composition comprising a redox active substance and, if necessary, a carbon material, a dispersant / binder, and a solvent. The redox active substance, carbon material, and dispersant / binder are as described above. Furthermore, the content of each component in the counter electrode composition is not particularly limited, as long as the content of each component in the resulting counter electrode layer can be within the range described above, taking into consideration coating properties, etc. Preferred embodiments of the counter electrode composition will be described below.

[0156] <Redox Active Substances> Redox active substances are described in accordance with the explanation of the opposite side above.

[0157] <Carbon Material> When the above counter electrode composition contains a carbon material, the amount of carbon material used will vary depending on the type of carbon material used, but the range is preferably 0.001 to 10,000 by mass ratio, more preferably 0.01 to 1,000, and even more preferably 0.1 to 100, relative to 1 unit of the redox active substance.

[0158] In particular, when using CNTs or activated carbon as the carbon material, the content is preferably within the following range.

[0159] When the carbon material is CNT, its content is preferably 0.001 to 1,000 by mass ratio, more preferably 0.01 to 100, and even more preferably 0.1 to 10, per 1 unit of the redox active substance.

[0160] When the carbon material is activated carbon, its content is preferably 0.01 to 10,000, more preferably 0.1 to 1,000, and even more preferably 1 to 100, in mass ratio with respect to 1 unit of the redox active substance.

[0161] <Dispersant and Binder> If the above counter electrode composition contains a dispersant and binder, the amount thereof is preferably 0.1 to 99 parts by mass, more preferably 0.5 to 50 parts by mass, and even more preferably 1 to 10 parts by mass, based on 100 parts by mass calculated by totaling the redox active substance and carbon material.

[0162] In particular, when using CNTs or activated carbon as the carbon material, the content of the dispersant and binder is preferably within the following range from the viewpoint of more efficiently dispersing the carbon material.

[0163] When the carbon material is CNT, the content of the dispersant and binder is preferably 0.1 to 9, and more preferably 0.3 to 3, in mass ratio with respect to 1 unit of the carbon material.

[0164] When the carbon material is activated carbon, the content of the dispersant and binder is preferably 0.01 to 0.5 by mass ratio, and more preferably 0.05 to 0.2 per 1 unit of the carbon material.

[0165] Furthermore, if an oxazoline polymer is included as both a dispersant and binder, its content should be adjusted so that the total amount of the oxazoline polymer and the carboxyl group-containing polymer falls within the above range. Also, if a catalyst is included, its content should conform to the range described in the description of the counter electrode layer.

[0166] <Solvent> The above counter electrode composition may contain a solvent. The solvent is not particularly limited as long as it can dissolve the above redox active substance. Examples include water, ethers such as tetrahydrofuran (THF), diethyl ether, and 1,2-dimethoxyethane (DME); amides such as N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), and N-methyl-2-pyrrolidone (NMP); ketones such as acetone and methyl ethyl ketone; alcohols such as methanol, ethanol, n-propanol, and 2-propanol; glycol ethers such as ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, and propylene glycol monomethyl ether; glycols such as ethylene glycol and propylene glycol; and organic solvents such as acetonitrile. In the present invention, among these solvents, water, THF, DMF, NMP, methanol, ethanol, 2-propanol, and acetonitrile are preferred, and THF, NMP, and acetonitrile are more preferred, considering the solubility of the quinone compound, the dispersibility of the carbon material, and the coating properties of the composition. These solvents may be used individually or in combination of two or more types.

[0167] When the above counter electrode composition contains a solvent, the solid content concentration is set appropriately considering the basis amount of the redox active substance, the coating properties of the composition, and the thickness of the thin film (counter electrode) to be formed, but is usually about 0.0001 to 50% by mass, preferably about 0.001 to 20% by mass, and more preferably about 0.01 to 10% by mass.

[0168] Furthermore, when the counter electrode composition contains a solvent, the concentration of the redox active substance is preferably 0.01 to 1,000 mmol / L, more preferably 0.1 to 500 mmol / L, and even more preferably 1 to 100 mmol / L, from the viewpoint of supplying a sufficient amount to the counter electrode for sample detection, as well as the uniformity of the coating film and dispersibility in carbon materials, etc.

[0169] [3-3] Preparation of Counter Electrode Composition The above counter electrode composition may be prepared by using the redox active substance as is, or by dissolving it in a suitable solvent as needed. In this case, the carbon material and the dispersant / binder can be mixed, dispersed, and dissolved at any time.

[0170] When carbon material is included, it is preferable to disperse the resulting mixture, which can further improve the dispersion ratio of the carbon material. Dispersion treatments include mechanical treatments such as wet treatment using ball mills, bead mills, and jet mills, and ultrasonic treatment using bath-type or probe-type sonicators. The duration of the dispersion treatment is arbitrary, but is preferably from 1 minute to 10 hours, and more preferably from 1 minute to 1 hour. Heat treatment may also be applied as needed.

[0171] [3-4] Composition for forming a coating film The composition for forming a coating film is a composition containing a carbon material and a solvent, and optionally an optional component such as a dispersant and binder. Specific examples of each component and their content are the same as those described in the description of the working layer.

[0172] [3-5] Preparation of the counter electrode The counter electrode layer can be obtained, for example, by applying the counter electrode composition containing the redox active substance to all or part of a conductive substrate and heating and drying it to form the counter electrode layer, or by applying a predetermined coating film-forming composition to all or part of a conductive substrate and heating and drying it to form a coating film, and then impregnating or laminating the biosensor counter electrode composition containing the redox active substance into the coating film to form the counter electrode layer.

[0173] Furthermore, if the counter electrode contains a carbon material, the redox active substance may be pre-supported on activated carbon, or a dispersion containing the redox active substance and the carbon material may be used for coating, or the redox active substance may be impregnated (supported) into a coating film that does not contain the redox active substance afterward.

[0174] As methods for applying the composition for the counter electrode and the composition for forming a coating film, for example, spin coating method, dip coating method, flow coating method, inkjet method, spray coating method, bar coating method, gravure coating method, slit coating method, roll coating method, flexographic printing method, transfer printing method, brush coating, blade coating method, air knife coating method, etc. can be mentioned. From the viewpoint of work efficiency, etc., the inkjet method, casting method, dip coating method, bar coating method, blade coating method, roll coating method, gravure coating method, flexographic printing method, spray coating method are preferable.

[0175] The temperature for heat drying is arbitrary as long as it is at least in the range where the solvent can be dried, but about 40 to 200 °C is preferable. Also, the drying time is not particularly limited as long as the solvent can be volatilized, but usually about 10 seconds to 30 minutes is preferable. Heat drying can be carried out using an appropriate device such as a hot plate, oven, vacuum oven, etc.

[0176] Further, when the composition for forming a coating film is applied onto a conductive substrate and heat dried to form a coating film on the conductive substrate, the obtained coating film (thin film) can be impregnated with the above composition for the counter electrode and left for a predetermined time to form a counter electrode layer. As the conditions for leaving, it is sufficient that the redox active substance can be uniformly distributed in the coating film, and heat treatment may be carried out as necessary. As the conditions, for example, it can be set at room temperature (23 °C) to 120 °C for 10 seconds to 24 hours.

[0177] The method for impregnating the coating film with the composition for the counter electrode is not particularly limited as long as the redox active substance can be made to have a predetermined basis weight. Examples of the method include a method of dropping the composition for the counter electrode onto the coating film and a method of immersing the coating film in the composition for the counter electrode.

[0178] The basis weight of the counter electrode layer or the coating film is not particularly limited, but when activated carbon is included as the carbon material, about 0.1 to 10 mg / cm 2 is preferable, when CNT is included as the carbon material, about 1 to 1,000 μg / cm 2 is preferable, and when no carbon material is used, 1 to 1,000 μg / cm2 A certain degree is desirable.

[0179] The basis weight and film thickness of the redox active material in the counter electrode layer can be adjusted by known methods. For example, they can be adjusted by changing the solid content concentration of the counter electrode composition and the coating film-forming composition described above, as well as the coating conditions and the number of coats. To increase the basis weight and film thickness, the device conditions can be changed to make the counter electrode layer or coating film thicker, or the solid content concentration can be increased, or the number of coats can be increased. To decrease the basis weight and film thickness, the device conditions can be changed to make the counter electrode layer or coating film thinner, or the solid content concentration can be decreased, the number of coats can be decreased, or the clearance can be reduced.

[0180] Furthermore, if a coating film is formed, the characteristics can also be adjusted by adjusting the solid content concentration and impregnation amount of the counter electrode composition impregnated into the formed coating film.

[0181] [4] Use of the Biosensor The biosensor 1 of the present invention can perform sensing by connecting the wiring of the working electrode 2 and the counter electrode 3 to the terminals of an ammeter, dropping a sample solution, for example, a glucose / phosphate buffer solution of a predetermined concentration, onto a separator, setting the voltage between the working electrode 2 and the counter electrode 3 to 0V, and measuring the current. This makes it possible to identify the concentration of the target substance in the sample solution. Furthermore, when the sample solution comes into contact with the working electrode 2 (working electrode layer 22) and the counter electrode 3 (counter electrode layer 32), if there is a potential difference between the two electrodes, a short-circuit current unrelated to the sensing current originating from the sample may be detected. In such cases, the sensing current can be accurately detected by first impregnating both electrodes (working electrode layer 22 and counter electrode layer 32) with a liquid containing phosphate buffer, and then adding the sample solution after the current value has stabilized.

[0182] The biosensor of the present invention will be described below with more specific examples, but the present invention is not limited to the following examples.

[0183] (1) Preparation of a composition for forming a coating film for biosensor electrodes, and production of aluminum foil with a carbon-containing coating film [Production Example 1-1] 0.350 g of BT-1003M (manufactured by LG Energy Solutions, multi-walled carbon nanotube), 54.69 g of pure water, 1.40 g of Epocross WS-700 (manufactured by Nippon Shokubai Co., Ltd., aqueous solution of oxazoline group-containing polymer with a solid content of 25% by mass) (solid content 0.350 g), and 10.48 g of 2-propanol were sequentially added to a 100 mL polypropylene bottle container and stirred to obtain a black mixture. This black mixture was irradiated with ultrasound at 500 W for 3 minutes using a probe-type ultrasonic irradiation device UIP1000hd (manufactured by Hielsher) while stirring under ice cooling with a magnetic stirrer. After irradiation, the container was removed and the liquid was shaken to homogenize it. This ultrasonic treatment was repeated 5 times to obtain a black homogeneous dispersion. To this black homogeneous dispersion, 3.08 g (solid content 0.154 g) of a 5.00 mass% aqueous solution of ammonium polyacrylate (Aron A-30, manufactured by Toagosei Co., Ltd., a homogeneous aqueous solution obtained by diluting a 31.6 mass% aqueous solution of ammonium polyacrylate with pure water and stirring) was added and stirred to obtain black homogeneous dispersion A-1 (solid content concentration 1.22 mass%). Dispersion A-1 was spread onto aluminum foil (manufactured by UACJ Foil Co., Ltd., model number 1N30, thickness 15 μm) using a wire bar coater (OSP-30, maximum wet film thickness 30 μm, coating speed 3 m / min). Then, it was dried in an oven at 120°C for 10 minutes to obtain aluminum foil B-1 with a CNT-containing coating film.

[0184] (2) Preparation of the biosensor working electrode (enzyme-containing coating film and aluminum foil with RM-containing coating film) A working electrode 2 was prepared as shown in Figures 3(A) and 5(A) and (B). The working electrode 2 is a laminate in which a working electrode layer 22 is laminated over a predetermined area at one end of the coating film c formed on a conductive substrate 21. Although not specifically shown in Figures 3 and 5, the portion of the conductive substrate 21 where the working electrode layer 22 is not formed is waterproofed with waterproof tape or waterproof spray. The method for preparing the working electrode 2 will be described in detail below.

[0185] [Production Example 2-1] 4.0 mg of 1,2-naphthoquinone (manufactured by Tokyo Chemical Industry Co., Ltd.) and 1.0 mL of acetonitrile (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., special grade) were added to a 1.5 mL Eppendorf tube, stirred, and dissolved to obtain a pale orange transparent solution C-1 (molar concentration of 1,2-naphthoquinone 25 mmol / L), which is an RM-containing coating solution. Coated aluminum foil B-1 was cut to a size of 0.5 cm × 2.5 cm, and a 0.5 cm × 2.0 cm waterproof tape (manufactured by Diatex Co., Ltd., KM-30) was applied to the coated surface, excluding the 0.5 cm × 0.5 cm coated surface at one end which would be the reaction electrode area. Waterproof spray (manufactured by Sumitomo 3M Limited, SG-P345i) was sprayed onto the uncoated back surface of the aluminum foil, and it was dried at room temperature.

[0186] In B-1, 10 μL (0.25 μmol) of RM-containing coating solution C-1 was dropped onto a 0.5 cm x 0.5 cm coated area of ​​the reaction electrode where waterproof tape was not applied, ensuring uniform contact across the entire surface. The area was then left in MacDry (Arkray, Inc., MCU-201A) at room temperature for 1 hour to prepare aluminum foil D-1 with an RM-containing coating film for the working electrode. The basis weight of 1,2-naphthoquinone in aluminum foil D-1 was 158 μg / cm². 2 (1.00μmol / cm 2 ) can be calculated. 2 μL of FAD glucose dehydrogenase solution (GDH-1 manufactured by Ikeda Sugar Refining Co., Ltd., activity concentration 25,000 Units / mL, 2% protein solution) was then dropped onto the RM-containing coated surface as described above, ensuring uniform contact across the entire surface. The mixture was left in a MacDry at room temperature for 24 hours to produce an enzyme-containing coated film for the working electrode and an aluminum foil E-1 with an RM-containing coated film.

[0187] [Production Example 2-2] 2 μL of FAD glucose dehydrogenase and 0.2 μL (0.2 mg by mass) of sorbitol polyglycidyl ether (Nagase ChemteX Corporation, Denacol EX-614B) were sequentially added to a 1.5 mL Eppendorf tube. The mixture was stirred in a vortex mixer for 20 seconds, left at room temperature for 1 hour, and stirred again in a vortex mixer for 20 seconds to obtain cross-linked FAD glucose dehydrogenase solution F-1. 2.2 μL of cross-linked FAD glucose dehydrogenase solution F-1 was dropped onto the RM-containing coated surface of aluminum foil D-1, which was prepared using the same method as in Production Example 2-1, ensuring uniform contact across the entire surface. The mixture was left in a MacDry at room temperature for 24 hours to prepare the enzyme-containing coated film for the working electrode and aluminum foil E-2 with an RM-containing coated film.

[0188] (3) Preparation of the biosensor counter electrode (aluminum foil with a coating film containing redox active substance) A counter electrode 3 was prepared as shown in Figures 4(A) and 5(A) and (B). The counter electrode 3 is a laminate in which a counter electrode layer 32 is laminated over a predetermined area at one end of the coating film c formed on a conductive substrate 31. Although not specifically shown in Figures 4 and 5, the portion of the conductive substrate 31 where the counter electrode layer 32 is not formed is waterproofed with waterproof tape or waterproof spray. The method for preparing the counter electrode 3 will be described in detail below.

[0189] [Manufacturing Example 3-1] Coated aluminum foil B-1 was cut to a size of 0.5 cm x 2.5 cm. A 0.5 cm x 2.0 cm waterproof tape was applied to the coated surface, excluding the 0.5 cm x 0.5 cm coated surface at one end which would be the reaction electrode. Waterproof spray was sprayed onto the uncoated back surface of the aluminum foil and dried at room temperature. 10 μL (0.25 μmol) of C-1, used here as a redox active substance coating solution for the counter electrode, was dropped onto the 0.5 cm x 0.5 cm coated surface of B-1 where the waterproof tape was not applied. The liquid was uniformly applied to the entire surface, and the foil was left at room temperature for 1 hour in a MacDry to prepare aluminum foil H-1 with a redox active substance coating for the counter electrode. The basis weight of 1,2-naphthoquinone in aluminum foil H-1 was 158 μg / cm². 2(1.00μmol / cm 2 ) can be calculated as follows.

[0190] [Production Example 3-2] 6.2 mg of chloranil (manufactured by Wako Pure Chemical Industries, Ltd.) and 1.0 mL of N,N-dimethylformamide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to a 1.5 mL Eppendorf tube and stirred to dissolve, obtaining a simple yellow transparent solution C-2 (molar concentration of chloranil 25 mmol / L), which is a redox active substance solution for the biosensor counter electrode. 40 mg of MgOC (manufactured by Toyo Tanso Co., Ltd., Knobel MJ(4)150) with a particle size of 150 nm was added to the 1.5 mL Eppendorf tube. Next, 200 μL of a 5% by mass polyvinylidene fluoride (PVDF) / NMP solution (Kureha Corporation, Kureha KF Polymer L#9305) was added to the 1.5 mL Eppendorf tube and dispersed for 1 minute using a homogenizer (SMT Corporation, UH-50). Furthermore, 400 μL of NMP (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the 1.5 mL Eppendorf tube and dispersed for 1 minute using a homogenizer to obtain black dispersion I-1 (MgOC concentration 67 mg / mL), which is a redox active material support layer dispersion for biosensor counter electrodes.

[0191] A coated aluminum foil B-1 was cut to a size of 0.5 cm x 2.5 cm. A 0.5 cm x 2.0 cm waterproof tape was applied to the coated surface, excluding the 0.5 cm x 0.5 cm coated surface at one end which would be the reaction electrode. A waterproof spray was then applied to the uncoated back surface of the aluminum foil and dried at room temperature. 2 μL (134 μg) of RM-containing coating solution I-1 was dropped onto the 0.5 cm x 0.5 cm coated surface of B-1, which was the reaction electrode area without the waterproof tape, ensuring uniform contact across the entire surface. The foil was then dried in an oven (AS ONE Corporation, EO-300B) at 60°C for 12 hours. Subsequently, 2 μL (0.05 μmol) of redox active substance solution C-2 was dropped onto the foil, ensuring uniform contact across the entire surface. The foil was left in a MacDry at room temperature for 1 hour to prepare a counter electrode coated aluminum foil H-2 containing a redox active substance. The basis weight of chloranil in aluminum foil H-2 is 49 μg / cm³. 2 (0.2 μmol / cm 2 ) can be calculated as follows.

[0192] (4) Preparation and Measurement of Biosensors [Example 1] <Preparation of Glucose / Phosphate Buffer Solution> 12.193 g (0.07 mol) of anhydrous dipotassium hydrogen phosphate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 700 mL of pure water. 500 mL of pure water was dissolved in anhydrous potassium hydrogen phosphate (0.05 mol). 400 mL of the above potassium hydrogen phosphate solution was added to 600 mL of the above dipotassium hydrogen phosphate aqueous solution and stirred. The pH was confirmed to be 7.00 using a pH meter to obtain a 100 mM phosphate buffer solution (pH 7.0) (hereinafter, the phosphate buffer solution may be referred to as "PB").

[0193] 720.6 mg of D-glucose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in the above 100 mM phosphate buffer (pH 7.0), and then 100 mM phosphate buffer was added until the volume reached 40 mL. The mixture was stirred at room temperature to obtain a colorless, transparent 100 mM D-glucose / PB solution. 50 mM, 25 mM, 10 mM, 5 mM, 1 mM, and 0.5 mM D-glucose / PB solutions were obtained by the same method.

[0194] <Fabrication of the Biosensor> Figures 6 to 8 show the structure of the biosensor fabricated in this embodiment. A biosensor counter electrode H-1 (counter electrode 3, in which a coating film c and a counter electrode layer 32 are laminated on a conductive substrate 31) and a cellophane film (manufactured by Futamura Chemical Co., Ltd., PTP5-1#500) immersed in 100 mM phosphate buffer for 30 minutes as a separator 4 are sequentially layered on an acrylic plate (25 mm x 25 mm, 5 mm thick, support n), and each is fixed with waterproof tape (separator) w (see Figure 7(A) in particular). Next, the biosensor working electrode E-1 (working electrode 2, in which a coating film c and a working electrode layer 22 are laminated on a conductive substrate 21) is sequentially layered together with the support n, and each is fixed with waterproof tape (separator) w (see Figure 7(B) in particular. Note that the support m is not shown here for clarity of the structure). The coated electrode portion (working electrode layer 22) of the working electrode 2 and the coated electrode portion (counter electrode layer 32) of the counter electrode 3 are stacked so that the coated electrode portions face each other and completely overlap when viewed from above, with the opposite ends aligned in a straight line in alternating directions. The separator 4 is inserted between the working electrode layer 22 of the working electrode 2 and the counter electrode layer 32 of the counter electrode 3, completely covering both electrode surfaces and preventing contact between them. Note that in Figures 6 and 7, the coating film c is not shown for clarity of the structure.

[0195] The aluminum foil (wiring section) on the opposite side of the coated electrode surfaces of working electrode 2 and counter electrode 3 was connected to the terminals of an ammeter. 10 μL of glucose / PB solution of the above concentrations was dropped onto a separator, and glucose sensing was performed by measuring the current. The obtained peak current values ​​(maximum current values) are shown in Table 1 and Figure 9. <Measurement conditions> Measurement device: Non-resistive ammeter AM-06 (manufactured by Toho Giken Co., Ltd.) Measurement time: 120 seconds

[0196] [Example 2] The measurement was performed using the same method as in Example 1, except that the working electrode was changed from E-1 to E-2 and the counter electrode was changed to H-2. The obtained peak current values ​​(maximum current values) are shown in Table 1 and Figure 10.

[0197]

[0198] Table 1, Figure 9, and Figure 10 show that the peak current value correlates with glucose concentration, and it is possible to estimate glucose concentration from the peak current value by setting appropriate biosensor conditions, measurement conditions, and creating a calibration curve. The biosensor of the present invention enables quantitative glucose sensing without applying an external voltage. Since an external voltage is not required, sensing can be performed using only an ammeter and without a power supply. This allows for sensing in a low-cost system and does not include noise currents caused by factors other than the sample due to voltage application, such as currents derived from redox mediators reduced by some factor, or currents due to electric double-layer capacitance. Furthermore, since the background current value can be kept low, sensing can be performed with high accuracy and a high signal-to-noise ratio.

[0199] 1. Biosensor 2. Working electrode 21. Conductive substrate 22. Working electrode layer 3. Counter electrode 31. Conductive substrate 32. Counter electrode layer 4. Separator or spacer m. First support n. Second support c. Coating film w. Waterproof tape (separator)

Claims

1. A biosensor comprising a working electrode formed on a first support, a counter electrode formed on a second support, and a separator or spacer, wherein the working electrode and the counter electrode are arranged facing each other via the separator or spacer, and the working electrode, the separator or spacer, and the counter electrode are stacked so that all or part of them overlap.

2. The biosensor according to claim 1, which is self-driving.

3. The biosensor according to claim 1, wherein the working electrode is a laminate comprising a conductive substrate and a working electrode layer laminated on all or part of the conductive substrate.

4. The biosensor according to claim 3, wherein the above-mentioned action zone contains a redox mediator and an enzyme.

5. The biosensor according to claim 3, wherein the above-mentioned working electrode layer contains a carbon material.

6. The biosensor according to claim 4, further comprising an enzyme crosslinking agent.

7. The biosensor according to claim 3, wherein the conductive substrate is a metal foil.

8. The biosensor according to claim 7, wherein the portion of the conductive substrate in which the working electrode layer is not formed is waterproofed.

9. The biosensor according to claim 1, wherein the counter electrode is a laminate comprising a conductive substrate and a counter electrode layer laminated on all or part of the conductive substrate.

10. The biosensor according to claim 9, wherein the counter electrode layer contains a redox active substance.

11. The biosensor according to claim 9, wherein the counter electrode layer contains a carbon material.

12. The biosensor according to claim 9, wherein the conductive substrate is a metal foil.

13. The biosensor according to claim 12, wherein the portion of the conductive substrate in which the counter electrode layer is not formed is waterproofed.

14. The biosensor according to claim 1, wherein the separator is a cellulose-based separator or cellophane.

15. The biosensor according to claim 1, which is a glucose sensor.

16. A sensing method using a biosensor according to any one of claims 1 to 15, wherein the voltage between the counter electrode and the working electrode is set to 0V, and sensing is performed by current measurement.

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