Biosensor and sensing method using biosensor

Abstract

The present invention provides a novel biosensor provided with a counter electrode and a working electrode, the biosensor being simpler, having more stable performance, and being self-powered. The counter electrode includes a redox active substance.

Description

Biosensor and sensing method using a biosensor

[0001] The present invention relates to a biosensor and a sensing method using 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, as well as a sensing method using the biosensor.

[0007] The inventors, through diligent research to achieve the above objective, have discovered that by using a biosensor counter electrode containing a redox active substance in a biosensor comprising a counter electrode and a working electrode, it is possible to accurately and sensitively receive electrons from the working electrode without applying an external voltage during sensing, in a low-cost and simple configuration. In particular, the above counter electrode was more effective because it became self-driving by applying a substance having an oxidation-reduction potential equivalent to or higher than that of the redox mediator functioning in 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. In other words, the inventors have found that the biosensor equipped with the counter electrode of the present invention exhibits stable and advanced sensing performance as a self-driving electrochemical biosensor, thus completing the present invention.

[0008] In other words, the present invention provides the following biosensors and sensing methods using the biosensors: 1. A biosensor comprising a counter electrode and a working electrode, wherein the counter electrode contains a redox active substance. 2. The biosensor of 1 which is self-driving. 3. The biosensor of 1 or 2 wherein the redox active substance contains an organic redox active substance. 4. The biosensor of 3 wherein the redox active substance is a quinone compound. 5. The biosensor of any of 1 to 4 wherein the counter electrode further contains a carbon material. 6. The biosensor of 5 wherein the carbon material is activated carbon. 7. The biosensor of any of 1 to 6 wherein the counter electrode further contains a dispersant and binder. 8. The biosensor of 1 wherein the counter electrode is a laminate comprising a substrate and a counter electrode layer formed on the substrate, wherein the substrate is aluminum foil and the counter electrode layer contains a redox active substance. 9. The biosensor of any of 1 to 8 wherein the working electrode contains a redox mediator. 10. 9. Biosensor 9 in which the redox mediator is a quinone compound. 11. Biosensor 9 or 10 in which the redox mediator's oxidation-reduction potential is less negative than the redox active substance of the counter electrode. 12. Biosensor 9 to 11 in which the working electrode further comprises a carbon material. 13. Biosensor 12 in which the carbon material is a carbon nanotube. 14. Biosensor 9 to 13 in which the working electrode further comprises an enzyme. 15. Biosensor 14 in which the enzyme is glucose dehydrogenase. 16. Biosensor 9 to 15 in which the working electrode further comprises a dispersant and binder. 17. Biosensor 9 in which the working electrode is a laminate comprising a substrate and a working electrode layer formed on the substrate, wherein the substrate is aluminum foil and the working electrode layer comprises a redox mediator. 18. Biosensor 1 to 17 in which is a glucose sensor. 19. A sensing method using one of the biosensors 1 to 18, in which the applied voltage between the counter electrode and the working electrode is set to 0V and sensing is performed by current measurement. 20. A sensing method using one of the biosensors 1 to 18, in which the counter electrode and the working electrode are immersed in a liquid containing a phosphate buffer, and then a sample solution containing the substance to be detected is added and sensing is performed.21. A sensing method using 20 biosensors containing enzymes in the above-mentioned sample solution.

[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. The counter electrode of the biosensor of the present invention does not contain expensive precious metals, so it can be manufactured at low cost and can be applied to small to large areas depending on the purpose. Furthermore, the counter electrode of the biosensor of the present invention can contain metal foil and has low volume resistance, so it can be used as wiring in addition to electrode applications. In addition, by imparting water resistance to each electrode of the biosensor of the present invention, it can operate stably for a long time even in sample liquid and can be applied to 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 voltage application from outside the sensor and no inflow of external current, noise current peaks such as those originating from electric double layer capacitance or oxidation currents to redox mediators reduced by some factor do not occur. Therefore, reaction current values ​​can be measured and sensed with high sensitivity, high accuracy, and 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 graph plots the peak current measured in the biosensor cells of Examples 3-1 to 3-4 against the glucose concentration.

[0012] The present invention will be described in more detail below. The biosensor of the present invention is a biosensor comprising a counter electrode and a working electrode, wherein the counter electrode contains a redox active substance.

[0013] [1] Counter electrode [1-1] Counter electrode The counter electrode (hereinafter sometimes referred to as the "biosensor counter electrode") contains a redox active substance. It may also contain optional components such as carbon material, binder and dispersant as needed.

[0014] <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, a self-driving biosensor can be fabricated by creating a biosensor counter electrode using a composition containing such a redox active substance.

[0015] 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.

[0016] 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 can be used individually or in combination of two or more.

[0017] In contrast to the above, the estimated amount of redox active substance is not particularly limited as long as it is sufficiently large relative to the amount of substance 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 sensing sensitivity, it is recommended to use 1 to 1,000 μg / cm³. 2Preferably, and more preferably, 5 to 500 μg / cm³ 2 Furthermore, in this invention, the basis amount 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).

[0018] <Carbon Material> The counter electrode may contain a carbon material from the viewpoint of improving the conductivity of the redox active substance. 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. From the viewpoint of conductivity, reaction rate, sensitivity, sensing power, and sensing current density, carbon nanotubes and activated carbon are preferred, and activated carbon is even more preferred from the viewpoint of high specific surface area, suppression of enzymatic reactions at the counter electrode, and suppression of reactions between the redox mediator eluted in the sample solution and the counter electrode. The carbon material may be used alone or in combination of two or more types.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] When the counter electrode 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.

[0024] In particular, when using CNTs or activated carbon as the carbon material, the content is preferably within the following range.

[0025] 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.

[0026] 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.

[0027] <Dispersant and Binder> The counter electrode may contain a dispersant and binder from the viewpoint of improving the dispersibility and binding of redox active substances and carbon materials, improving adhesion to the substrate, and improving the strength and water resistance of the counter 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.

[0028] <Oxazoline group-containing polymer> The oxazoline group-containing polymer (hereinafter sometimes referred to as oxazoline polymer) functions as a dispersant, and also functions as a binder when used in combination with a carboxy group-containing polymer described later. Further, when a crosslinked structure is formed with the carboxy group-containing polymer described later, it has a function of improving the strength and water resistance (solvent resistance) of the counter electrode layer. Particularly, when containing a carbon material described later, it is suitable as a dispersant for stably dispersing the carbon material in the composition.

[0029] The above oxazoline polymer is not particularly limited as long as it is a polymer in which an oxazoline group is bonded directly or via a spacer group such as an alkylene group to a repeating unit constituting the main chain. Specifically, it is preferably a polymer having a repeating unit bonded to the polymer main chain or a spacer group at the 2-position of the oxazoline ring, 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).

[0030]

[0031] In the formula, X represents a polymerizable carbon-carbon double bond-containing group, and R 1 ~R 4These 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.

[0032] Specific examples of the oxazoline monomer having a polymerizable carbon-carbon double bond-containing group at the 2-position represented by the 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, 2-isopropenyl-2-oxazoline, 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, 2-isopropenyl-5-butyl-2-oxazoline, etc. From the viewpoint of easy availability, etc., 2-isopropenyl-2-oxazoline is preferable.

[0033] Further, when a carbon material such as CNT is included, considering that a dispersion or composition in which these are dispersed 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 the above formula (1), but in order to further enhance the solubility in water, it is preferably obtained by radical polymerization of at least two kinds of monomers including the above oxazoline monomer and a (meth)acrylic acid ester-based monomer having a hydrophilic functional group.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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 Application Publication No. 6-32844 and Japanese Patent Application 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.

[0040] The above-mentioned oxazoline polymers may be used individually or in combination of two or more types.

[0041] Films obtained by drying oxazoline polymers on a 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.

[0042] <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.

[0043] 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.

[0044] 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.")

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] The above-mentioned carboxyl group-containing polymer may be used individually or in combination of two or more types.

[0050] The content of the carboxyl group-containing polymer varies depending on the solvent used, the 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.

[0051] 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.

[0052] 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.

[0053] If the counter electrode 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 (counter electrode layer).

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] The counter electrode described above can be any structure appropriate to the structure of the biosensor being manufactured, and is not particularly limited. Examples of such structures include a laminate comprising a base material and a counter electrode layer laminated on all or part of the base material, or a self-supporting film obtained by peeling the counter electrode layer from the base material. Furthermore, the counter electrode may be cut to the desired size and shape as needed. The biosensor counter electrode can be cut by known methods such as punching or slitting.

[0059] 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.

[0060] The above-mentioned substrate can be appropriately selected from those conventionally used as electrode substrates. 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 above-mentioned substrate is not particularly limited, but in the present invention, 1 to 100 μm is preferred.

[0061] Furthermore, when the above-mentioned laminate is used as the counter electrode, it is preferable to use metal foil as the base material, 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 as the base material, after cutting the base material, the parts other than the electrode (the parts where the counter electrode layer is not formed) can be used as conductors, so that a biosensor counter electrode with wiring can be easily manufactured, and an improvement in the productivity of biosensor counter electrodes can be expected. In addition, since the shape of the counter electrode layer is stable due to the above-mentioned base material, it is also easy to cut the counter electrode layer together with the base material into a shape corresponding to the shape of the wiring.

[0062] The above-mentioned counter electrode can be obtained, for example, by applying a biosensor counter electrode composition containing the above-mentioned redox active substance (hereinafter sometimes referred to as "counter electrode composition") to all or part of a substrate and heating and drying it to form a 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 a substrate and heating and drying it to form a coating film, and then impregnating the coating film with the above-mentioned counter electrode composition containing the redox active substance to form a counter electrode layer. The counter electrode composition and the coating film-forming composition will be described below.

[0063] [1-2] 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.

[0064] <Redox Active Substances> Redox active substances are described in accordance with the explanation of the opposite side above.

[0065] <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.

[0066] In particular, when using CNTs or activated carbon as the carbon material, the content is preferably within the following range.

[0067] 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.

[0068] 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.

[0069] <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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] <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.

[0075] 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.

[0076] 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.

[0077] [1-3] 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.

[0078] 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.

[0079] [1-4] 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.

[0080] <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).

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] <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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] <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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] [1-5] 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.

[0094] 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.

[0095] [1-6] Preparation of the counter electrode The counter electrode can be obtained, for example, by applying the counter electrode composition containing the redox active substance to all or part of a substrate and heating and drying it to form a counter electrode layer, or by applying a predetermined coating film-forming composition to all or part of a substrate and heating and drying it to form a coating film, and then impregnating the coating film with the biosensor counter electrode composition containing the redox active substance to form a counter electrode layer.

[0096] 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.

[0097] Examples of coating methods for the counter 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.

[0098] The temperature for heating and drying is arbitrary as long as it can dry the solvent, but it is preferably around 40 to 200°C. The drying time is not particularly limited as long as the solvent can be evaporated, but it is usually preferably around 10 seconds to 30 minutes. Heating and drying can be carried out using suitable equipment such as a hot plate, oven, or vacuum oven.

[0099] When a counter electrode composition is applied to all or part of a substrate and heated and dried to form a counter electrode layer on the substrate, the resulting counter electrode layer may be peeled off from the substrate and used as a self-supporting film, or it may be used as a laminate with the counter electrode layer laminated on the substrate, as described above.

[0100] Furthermore, when a coating film is formed on a substrate by applying the coating film-forming composition to the substrate and heating and drying it, a counter electrode layer can be formed by impregnating the resulting coating film (thin film) with the counter electrode composition and leaving it for a predetermined time. The conditions for leaving it are sufficient to ensure that the redox active substance is uniformly distributed in the coating film, and heat treatment may be performed as needed. For example, the conditions can be 10 seconds to 24 hours at room temperature (23°C) to 120°C.

[0101] The method for impregnating the coating film with the counter electrode composition is not particularly limited, as long as the amount of redox active substance can be set to a predetermined basis amount. Examples of such methods include dropping the counter electrode composition onto the coating film or immersing the coating film in the counter electrode composition.

[0102] The basis weight of the counter electrode layer or coating film is not particularly limited, but if activated carbon is included as the carbon material, it should be 0.1 to 10 mg / cm². 2 A certain degree is preferable, and if the carbon material contains CNTs, then 1 to 1,000 μg / cm³. 2 A certain degree is preferable, and if carbon material is not used, 1 to 1,000 μg / cm³ is preferred. 2 A certain degree is desirable.

[0103] 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.

[0104] 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.

[0105] [2] Working electrode [2-1] Working electrode As the working electrode, electrodes known as biosensors can be used and are not particularly limited, but in the biosensor of the present invention, it is preferable to include a redox mediator (RM) having electron transfer ability in order to increase its sensitivity. In addition, if necessary, carbon material, dispersant / binder and enzyme, etc. may be further included.

[0106] <Redox Mediators> Redox mediators (RMs) can reversibly become oxidized and reduced forms and mediate the transfer of electrons between substances. Examples of RMs 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, quinone compounds are preferred from the viewpoint of reactivity with enzymes and electrodes, naphthoquinone and its derivatives are more preferred, and 1,2-naphthoquinone, 1,4-naphthoquinone, and sodium 1,2-naphthoquinone-4-sulfonate are even more preferred. The above RMs may be used individually or in combination of two or more.

[0107] When the working electrode contains RM, from the viewpoints of enabling self-driven operation, enhancing sensing sensitivity, etc., it is preferable that the redox potential of RM is the same as or lower 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, obtain sufficient power to transmit a sensing signal independently, etc., a larger potential difference is preferable.

[0108] In the above working electrode, from the viewpoint of enhancing the sensing sensitivity, the areal density of RM is preferably 0.01 to 1,000 μg / cm 2 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 (or working electrode layer).

[0109] Also, when containing RM, the content is usually 100% by mass in the solid content, but when other components are included, it can be the remainder excluding these.

[0110] <Carbon material> From the viewpoint of improving the conductivity of RM, the above working electrode may contain a carbon material. Examples of the carbon material include the same ones as those exemplified in the description of the above counter electrode. In the present invention, from the viewpoints of the conductivity of the obtained working electrode, improvement in sensitivity, reduction of noise current, film thickness uniformity of the coating film, etc., carbon nanotubes (CNT) are preferable. The above carbon material may be used alone or in combination of two or more kinds.

[0111] When the above working electrode contains a carbon material, considering the conductivity and uniformity of the obtained working electrode, the content is preferably 0.1 to 1,000, more preferably 1 to 100, in terms of mass ratio with respect to the above RM1.

[0112] <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 substrate, and improving the strength and water resistance of the working electrode. Examples of dispersants and binders are the same as those exemplified in the description of the counter electrode 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 as the carbon material, CMC and SBR are more preferred. The above binder may be used alone or in combination of two or more types.

[0113] If the working electrode 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 RM and carbon material.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] <Enzymes> The above 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.

[0119] 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.

[0120] 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.

[0121] The working electrode described above can be any structure appropriate to the structure of the biosensor being manufactured, and is not particularly limited. Examples of such structures include a laminate comprising a base material and a working electrode layer laminated on all or part of the base material, or a self-supporting film obtained by peeling the working electrode layer from the base material. Furthermore, the working electrode may be cut to the desired size and shape as needed. The biosensor working electrode can be cut by known methods such as punching or slitting. By using a metal foil such as aluminum foil as the base material, the parts other than the electrode (the parts where the working electrode layer is not formed) can be used as conductors after cutting the base material.

[0122] The thickness of the working electrode layer described above 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.

[0123] The above-mentioned substrate can be appropriately selected from those conventionally used as electrode substrates, and specific examples include those exemplified in the description of the counter electrode above. The thickness of the above-mentioned substrate is not particularly limited, but in the present invention, 1 to 100 μm is preferred.

[0124] Furthermore, when the above-mentioned laminate is used as the working electrode, it is preferable to use a metal foil as the base material, and it is even more preferable 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 a working electrode with wiring can be easily manufactured, and an improvement in the productivity of the working electrode can be expected. In addition, since the shape of the working electrode layer is stable due to the above-mentioned base material, it is easy to cut the working electrode layer together with the base material into a shape corresponding to the shape of the wiring.

[0125] The above-mentioned working electrode can be obtained, for example, by applying a biosensor working electrode composition containing the above-mentioned RM, etc. (hereinafter sometimes referred to as "working electrode composition") to all or part of a substrate and heating and drying it to form a working electrode layer, or by applying a predetermined coating film-forming composition that does not contain RM to all or part of a substrate and heating and drying it to form a coating film, and then impregnating the coating film with the above-mentioned working electrode composition containing RM to form a working electrode layer. The working electrode composition and the coating film-forming composition will be described below.

[0126] [2-2] Working electrode composition The working electrode composition is a composition comprising RM, and optionally a carbon material, a dispersant / binder, and a solvent. The RM, carbon material, binder, and dispersant 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, etc. Preferred embodiments of the working electrode composition will be described below.

[0127] <Redox Mediator> The redox mediator (RM) is described in accordance with the description of the working electrode above.

[0128] <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.

[0129] In particular, when using CNTs or activated carbon as the carbon material, the content is preferably within the following range.

[0130] 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.

[0131] <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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] <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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] [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.

[0140] 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.

[0141] The concentration of RM in an RM solution is not particularly limited, but is usually around 0.1 to 100 mM (molecule / L).

[0142] 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.

[0143] 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.

[0144] 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.

[0145] [2-4] 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 opposite.

[0146] [2-5] Preparation of the working electrode The working electrode can be obtained, for example, by applying the working electrode composition containing the above RM to all or part of a substrate and heating and drying it to form a working electrode layer, or by applying a predetermined coating film forming composition to all or part of a substrate and heating and drying it to form a coating film, and then impregnating the working electrode composition containing the above RM with the coating film to form a counter electrode layer.

[0147] Examples of coating methods for the working electrode 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.

[0148] 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.

[0149] The basis weight of the working electrode or coating film is not particularly limited as long as it satisfies the film thickness of the working electrode 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] [3] Detectable substance (sample), sample solution The sample is the detectable substance, and any substance that exerts an electrical effect on the working electrode by reacting with an enzyme or binding to an antigen may be used. In this biosensor, preferably, electrons contained in the sample are transferred in the order of enzyme, redox mediator (RM), and working electrode, and further, these electrons move to the counter electrode under voltage-free conditions or under applied voltage, are detected as a sensing current, and the concentration of the sample is calculated.

[0156] The sample solution contains the sample substance and a solvent to dissolve or disperse it. Furthermore, if enzymes and RM are not included in the working electrode, they should be added to this sample solution before use. Since the sample substances targeted for biosensing are usually bio-related substances such as biological substances, substances that function on living organisms, and substances metabolized by living organisms, water is mainly used as the solvent. In addition, hydrophilic solvents such as THF, DMF, DMSO, 2-propanol, and PGME may be added and used, as long as they do not impair the function of the sample substance or the physical properties required for sensing. Phosphate buffer may also be added and used.

[0157] [4] Biosensor and its use The biosensor of the present invention is characterized by using a counter electrode containing the redox active substance described above in the electrode system. Therefore, other device components such as separators, sample liquid guides, and protective films can be appropriately selected from known materials.

[0158] A separator is used to prevent the working electrode and the counter electrode from making direct electrical contact in the sample solution. For example, it is used to prevent electrons 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 separators, and specific examples include cellulose-based separators, filter paper, and proton-conducting membranes. However, if the biosensor structure is designed so that the working electrode and the counter electrode do not make direct contact in the sample solution, a separator may not be necessary.

[0159] Furthermore, the electrode system may include a reference electrode to ensure accurate measurement of the potential during the measurement of the sample.

[0160] The biosensor of the present invention is not particularly limited in form, as long as it uses the above-mentioned counter electrode in its electrode system. For example, it can be a configuration in which the working electrode, counter electrode, and reference electrode are arranged on the same substrate, or a configuration in which the working electrode and counter electrode are arranged opposite each other with a separator in between.

[0161] When constructing the biosensor of the present invention, any of the methods described above can be used, but as a simpler method, it can be constructed by, for example, the following method.

[0162] A biosensor can be constructed by adding a sample solution containing glucose, for example, as the target substance, prepared by a conventional method, to a glass cell VB2-1 (manufactured by EC Frontier Co., Ltd.), and then immersing the counter electrode and working electrode, prepared by the above method, in the sample solution. Subsequently, the glucose concentration can be determined by measuring the current using a non-resistive ammeter or by measuring the current using an electrochemical measuring device under conditions where the applied voltage is 0V. When the sample solution comes into contact with the working electrode and counter electrode, 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. However, by first immersing both electrodes in a liquid containing phosphate buffer and adding the sample solution after the current value has stabilized, the sensing current can be detected with high accuracy.

[0163] 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.

[0164] The present invention provides the following sensing methods using the biosensor described above: (1) A sensing method using the biosensor described above, in which the applied voltage between the counter electrode and the working electrode is set to 0V and sensing is performed by current measurement. (2) A sensing method using the biosensor described above, in which the counter electrode and the working electrode are immersed in a liquid containing a phosphate buffer, and then a sample solution containing the substance to be detected is added and sensing is performed. (3) A sensing method using the biosensor described above, in which the sample solution in (2) further contains an enzyme.

[0165] (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 1-1-1] 0.350 g of BT-1003M (manufactured by LG Energy Solutions, multi-walled carbon nanotubes), 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 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% by mass aqueous solution of ammonium polyacrylate (Aron A-30 manufactured by Toagosei Co., Ltd., a 31.6% by mass aqueous solution of ammonium polyacrylate diluted with pure water and stirred to obtain a homogeneous aqueous solution of 5.00% by mass) was added and stirred to obtain black homogeneous dispersion A-1 (solid content concentration 1.22% by mass).

[0166] 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-1-1 with a CNT-containing coating film.

[0167] [Manufacturing Example 1-1-2] Dispersion A-1 prepared by the method described in Manufacturing Example 1-1-1 was spread onto aluminum foil (manufactured by UACJ Foil Co., Ltd., model number 1N30, thickness 15 μm) using a wire bar coater (OSP-100, maximum wet film thickness 100 μm, coating speed 3 m / min). Then, it was dried in an oven at 120°C for 10 minutes to obtain aluminum foil B-1-2 with a CNT-containing coating film.

[0168] [Production Example 1-2] Except that the amount of BT-1003M used was changed to 0.500 g, the amount of pure water used was changed to 54.36 g, the amount of Epocross WS-300 used was changed to 2.48 g (solid content 0.248 g), and the amount of 5.00% by mass ammonium polyacrylate aqueous solution used was changed to 2.19 g (solid content 0.110 g), a black homogeneous dispersion A-2 (solid content concentration 1.22% by mass) was obtained using the same method as in Production Example 1-1-1. The obtained A-2 was coated and dried using the same method as in Production Example 1-1-2 to obtain aluminum foil B-2 with a CNT-containing coating film.

[0169] [Manufacturing Example 1-3] In a 100 mL polypropylene bottle, 0.350 g of BT-1003M, 54.17 g of pure water, 0.175 g of Pitzcol K-30 (manufactured by Daiichi Kogyo Seiyaku Co., Ltd., polyvinylpyrrolidone, weight-average molecular weight 45,000), 1.75 g of Epocross WS-300 (solid content 0.175 g), and 10.48 g of 2-propanol were sequentially added 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 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 of a 5.00% by mass aqueous solution of ammonium polyacrylate was added and stirred to obtain black homogeneous dispersion A-3 (solid content concentration 1.22% by mass). The obtained A-3 was coated and dried using the same method as in Production Example 1-1-2 to obtain aluminum foil B-3 with a CNT-containing coating film.

[0170] [Production Example 1-4] To 11.83 g of activated carbon (YP-50F, manufactured by Kuraray Co., Ltd.), 0.69 g of acetylene black Li-100 (manufactured by Denka Co., Ltd.), 18.33 g of a 1.5% by mass aqueous solution of CMC ammonium salt (DN-800H, manufactured by Daicel Mirise Co., Ltd.), and 22.17 g of pure water were sequentially added while stirring. The resulting black mixture was stirred at 1,400 rpm for 1 minute and at 3,500 rpm for 1 minute using a homomixer (T.K. Robomix T.K. Homomix MARK II 2.5 stirring section, manufactured by Primix Co., Ltd.), and then stirred at a peripheral speed of 20 m / sec for 1 minute using a thin-film swirling high-speed mixer (Filmix 40-40 type, manufactured by Primix Co., Ltd.) to obtain 45.18 g of a uniform black slurry. To the obtained black homogeneous slurry, 1.69 g of a 48.5% by mass aqueous dispersion of styrene-butadiene rubber (SBR) (manufactured by JSR Corporation, TRD2001) was added, and the mixture was stirred at 2,000 rpm for 2 minutes using an Awatori Rentaro (manufactured by Thinky Co., Ltd., ARE-310) to obtain activated carbon slurry A-4. The obtained activated carbon slurry A-4 was spread onto aluminum foil (manufactured by UACJ Foil Co., Ltd., model number 1N30, thickness 15 μm) at a speed of 3 m / min using an applicator set to a gap of 160 μm. Next, it was dried at 80°C for 30 minutes, and then at 120°C for 30 minutes to form an activated carbon-containing coating film with a thickness of 50 μm, obtaining activated carbon-containing coated aluminum foil B-4.

[0171] (2) Preparation of the counter electrode composition [Preparation Example 1-1] 61.5 mg of chloranil (manufactured by Tokyo Chemical Industry Co., Ltd.) and 10 mL of tetrahydrofuran (manufactured by Kanto Chemical Co., Ltd., special grade) were added to a 20 mL glass vial and stirred to dissolve, obtaining a simple yellow transparent solution C-1 (molar concentration of chloranil 25 mmol / L), which is a redox active substance solution for the biosensor counter electrode.

[0172] (3) Preparation of Biosensor Counter Electrode (Aluminum Foil with Redox Active Substance-Containing Coating) [Production Example 2-1] Aluminum foil B-1-1 with a coating was punched out with a 10 mm diameter hand punch, and placed in a glass petri dish with the coating surface facing upwards. 8 μL of solution C-1 was dropped onto the coating surface, and the petri dish was gently shaken to allow the solution to come into contact with the entire surface of the aluminum foil B-1-1. The dish was left at room temperature for 1 hour to prepare aluminum foil D-1 with a redox active substance-containing coating. The coating surface of aluminum foil D-1 was visually uniform. Assuming that chloranil is uniformly distributed on the coating surface of aluminum foil D-1, the basis weight of chloranil is 62.6 μg / cm². 2 (0.255μmol / cm 2 ) can be calculated as follows.

[0173] [Manufacturing Example 2-2] Aluminum foil D-2 with a chloranil-containing coating was prepared using the same method as in Example 2-1, except that coated aluminum foil B-1-1 was changed to B-4. The coating surface of aluminum foil D-2 was visually uniform. Assuming that chloranil is uniformly distributed on the coating surface of aluminum foil D-2, the basis weight of chloranil in aluminum foil D-2 is 62.6 μg / cm². 2 (0.255μmol / cm 2 ) can be calculated as follows.

[0174] The compositions of the counter electrodes prepared as described above are summarized in Table 1.

[0175]

[0176] (4) Preparation of biosensor working electrode (aluminum foil with RM-containing coating) [Production Example 3-1-1] 4.0 mg of 1,2-naphthoquinone (manufactured by Combi-Blocks) and 10 mL of tetrahydrofuran (manufactured by Kanto Chemical Co., Ltd., special grade) were added to a 20 mL glass vial, and the mixture was stirred to dissolve and obtain a pale orange transparent solution E-1 (molar concentration of 1,2-naphthoquinone 2.5 mmol / L).

[0177] Aluminum foil B-1-1 with a coating was punched out with a 10 mm diameter hand punch, and the coated surface was placed facing upwards in a glass petri dish. 8 μL of solution E-1 was dropped onto the coated surface, and the glass petri dish was gently shaken to allow the solution to come into contact with the entire surface of B-1-1. The dish was then left at room temperature for 1 hour to prepare aluminum foil F-1-1 with an RM-containing coating for the working electrode. The coated surface of aluminum foil F-1-1 was visually uniform. Assuming that 1,2-naphthoquinone is uniformly distributed on the aluminum foil F-1-1, the basis weight of 1,2-naphthoquinone in aluminum foil F-1-1 is 4.0 μg / cm². 2 (0.025μmol / cm 2 ) can be calculated as follows.

[0178] [Manufacturing Example 3-1-2] Aluminum foil F-1-2 with an RM-containing coating for the working electrode was prepared using the same method as in Manufacturing Example 3-1-1, except that coated aluminum foil B-1-1 was replaced with coated aluminum foil B-1-2. The coating surface of aluminum foil F-1-2 was visually uniform. Assuming that 1,2-naphthoquinone is uniformly distributed on the aluminum foil F-1-2, the basis weight of 1,2-naphthoquinone in aluminum foil F-1-2 is 4.0 μg / cm². 2 (0.025μmol / cm 2 ) can be calculated as follows.

[0179] [Manufacturing Example 3-2] Aluminum foil F-2 with an RM-containing coating for the working electrode was prepared using the same method as in Manufacturing Example 3-1-1, except that coated aluminum foil B-1-1 was replaced with coated aluminum foil B-2. The coating surface of aluminum foil F-2 was visually uniform. Assuming that 1,2-naphthoquinone is uniformly distributed on the aluminum foil F-2, the basis weight of 1,2-naphthoquinone in aluminum foil F-2 is 4.0 μg / cm². 2 (0.025μmol / cm 2 ) can be calculated as follows.

[0180] [Manufacturing Example 3-3] Aluminum foil F-3 with an RM-containing coating for the working electrode was prepared using the same method as in Manufacturing Example 3-1-1, except that coated aluminum foil B-1-1 was replaced with coated aluminum foil B-3. The coating surface of aluminum foil F-3 was visually uniform. Assuming that 1,2-naphthoquinone is uniformly distributed on the aluminum foil F-3, the basis weight of 1,2-naphthoquinone in aluminum foil F-3 is 4.0 μg / cm². 2 (0.025μmol / cm 2 ) can be calculated as follows.

[0181] [Manufacturing Example 3-4] Aluminum foil with coating B-1-1 is used with a glassy carbon electrode (GC, GC-6355 manufactured by EC Frontier Co., Ltd., electrode diameter 3 mm, electrode area 0.0707 cm²). 2 Except for changing the amount of solution E-1 added to 2 μL, aluminum foil F-4 with an RM-containing coating for the working electrode was prepared using the same method as in Production Example 3-1-1. The coating surface of aluminum foil F-3 was visually uniform. Assuming that 1,2-naphthoquinone is uniformly distributed on the aluminum foil F-3, the basis weight of 1,2-naphthoquinone in aluminum foil F-4 is 11.2 μg / cm². 2 (0.071 μmol / cm 2 ) can be calculated as follows.

[0182] The composition of the working electrode prepared as described above is summarized in Table 2.

[0183]

[0184] (5) Preparation and Measurement of Biosensors [Example 1-1-1] <Preparation of Glucose / Phosphate Buffer Solution> 720.6 mg of D-glucose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 100 mM phosphate buffer (pH 7.0, hereinafter sometimes abbreviated as "PB"), and then 100 mM phosphate buffer was added until the volume reached 40 mL, and the mixture was stirred at room temperature to obtain a colorless, transparent solution, 100 mM D-glucose / PB solution G-1. 10 mM D-glucose / PB solution G-2 and 1 mM D-glucose / PB solution G-3 were obtained by the same method.

[0185] <Setting up the counter electrode> The aluminum foil D-1 with a redox active substance coating, manufactured in Manufacturing Example 2-1, was fixed to the electrode holder AE9-2 with the coated surface facing the opening. The opening area of ​​AE9-2 is circular with a diameter of 5 mm, and the area of ​​the counter electrode is 0.785 cm². 2 That is the case.

[0186] <Setting of the Working Electrode> The RM-containing coated aluminum foil F-1-1, manufactured in Manufacturing Example 3-1-1, was fixed to the electrode holder AE9-2 with the coated surface facing the opening. The opening area of ​​AE9-2 is circular with a diameter of 5 mm, and the working electrode area is 0.785 cm². 2 That is the case.

[0187] <Preparation of sample solution, fabrication and measurement of biosensor cell> 6.0 mL of PB was added to a glass cell of the compact analytical cell VB2-S (manufactured by EC Frontier Co., Ltd.), and 42 μL of FAD glucose dehydrogenase (GDH-1, manufactured by Ikeda Sugar Refining Co., Ltd., activity concentration 25,000 units / mL) was added and mixed to obtain a glucose-free sample solution.

[0188] The working electrode and counter electrode, set as described above, were fixed to the cell cap of VB2-S. Immediately after starting the current measurement under the measurement conditions described below, the working electrode and counter electrode were simultaneously immersed in the glucose-free sample solution described above and stirred. Twenty seconds after immersion of both electrodes, 667 μL of the 100 mM D-glucose / PB solution described above was added dropwise to the glucose-free sample solution described above and stirred to obtain sample solution K-1. The glucose concentration of sample solution K-1 is 10 mM. After that, it was left to stand until the end of the measurement time, and the current was measured under the conditions described below, where the potential difference between the working electrode and the counter electrode was 0V, i.e., under the same conditions as for short-circuit current measurement. In this measurement, a potentiostat was used for convenience, but a non-resistive ammeter may also be used.

[0189] Almost no current was measured before dropping the glucose / PB solution. After dropping the glucose / PB solution, a current thought to originate from glucose sensing was measured. The maximum value of the current obtained at that time was called the peak current, and this, along with the peak current density calculated from the working electrode area, is shown in Table 3 and Figure 1. <Measurement conditions> Measurement device: ALS-1000C (BAS Corporation) Measurement mode: Amperometric i-t Curve Working electrode-counter electrode voltage: 0V → Measurement time: 60-90 seconds Reference electrode: None, terminal connected to the counter electrode

[0190] [Example 1-1-2] Measurements were performed using the same method as in Example 1-1-1, except that the working electrode was changed to F-1-2. Almost no current was measured before the glucose / PB solution was added, and after the glucose / PB solution was added, a current thought to be from glucose sensing was measured. Table 3 shows the peak current and peak current density after glucose addition and stirring.

[0191] [Example 1-2] Sample solution K-2 was prepared using the same method as in Example 1-1-1, except that the 100 mM D-glucose / PB solution was changed to a 10 mM D-glucose / PB solution during sample solution preparation. The glucose concentration of sample solution K-2 was 1 mM. The solution was then allowed to stand until the end of the measurement time, and the current measurement was completed. Almost no current was measured before dropping the glucose / PB solution, and after dropping the glucose / PB solution, a current thought to be derived from glucose sensing was measured. The peak current and peak current density after glucose dropping and stirring are shown in Table 3 and Figure 1.

[0192] [Example 1-3] Sample solution K-3 was prepared using the same method as in Example 1-1-1, except that the 100 mM D-glucose / PB solution was changed to a 1 mM D-glucose / PB solution. The glucose concentration of sample solution K-3 was 100 μM. The solution was then allowed to stand until the end of the measurement time, and the current measurement was completed. Almost no current was measured before dropping the glucose / PB solution, and after dropping the glucose / PB solution, a current thought to be derived from glucose sensing was measured. The peak current and peak current density after glucose dropping and stirring are shown in Table 3 and Figure 1.

[0193] [Example 1-4] Sample solution K-4 was prepared using the same method as in Example 1-1-1, except that 667 μL of 100 mM D-glucose / PB solution was changed to 60.6 μL of 1 mM D-glucose / PB solution in the preparation of the sample solution. The glucose concentration of sample solution K-3 was 10 μM. The solution was then allowed to stand until the end of the measurement time, and the current measurement was completed. Almost no current was measured before dropping the glucose / PB solution, and after dropping the glucose / PB solution, a current thought to be derived from glucose sensing was measured. The peak current and peak current density after glucose dropping and stirring are shown in Table 3 and Figure 1.

[0194] [Example 1-5] The counter electrode and working electrode were fixed to the electrode holder using the same method as in Example 1-1-1, except that the working electrode was changed from aluminum foil F-1-1 with an RM-containing coating to F-2. Sample solution K-2, which is a 1 mM glucose solution, was prepared using the same method as in Example 1-2. Almost no current was measured before dropping the glucose / PB solution, and after dropping the glucose / PB solution, the current thought to be from glucose sensing was measured. The peak current and peak current density after glucose dropping and stirring are shown in Table 3.

[0195] [Example 1-6] The counter electrode and working electrode were fixed to the electrode holder using the same method as in Example 1-1-1, except that the working electrode was changed from aluminum foil F-1-1 with an RM-containing coating to F-3. Sample solution K-2, which is a 1 mM glucose solution, was prepared using the same method as in Example 1-2, and glucose sensing was performed. Almost no current was measured before dropping the glucose / PB solution, and after dropping the glucose / PB solution, a current thought to be from glucose sensing was measured. The peak current and peak current density after glucose dropping and stirring are shown in Table 3.

[0196] [Example 1-7] The counter electrode and working electrode were fixed to the electrode holder using the same method as in Example 1-1-1, except that the working electrode was changed from aluminum foil F-1-1 with an RM-containing coating to F-4. Sample solution K-1, which is a 10 mM glucose solution, was prepared using the same method as in Example 1-1-1, and glucose sensing was performed. Almost no current was measured before dropping the glucose / PB solution, and after dropping the glucose / PB solution, a current thought to be derived from glucose sensing was measured. The peak current and peak current density after glucose dropping and stirring are shown in Table 3.

[0197] [Example 1-8] The counter electrode and working electrode were fixed to the electrode holder using the same method as in Example 1-1-1, except that aluminum foil D-1 with a redox active substance-containing coating film was changed to D-2 as the counter electrode. Sample solution K-1, which is a 10 mM glucose solution, was prepared using the same method as in Example 1-1-1, and glucose sensing was performed. Almost no current was measured before dropping the glucose / PB solution, and after dropping the glucose / PB solution, a current thought to be from glucose sensing was measured. The peak current and peak current density after glucose dropping and stirring are shown in Table 3.

[0198] [Comparative Example 1-1] The counter electrode and working electrode were fixed to the electrode holder using the same method as in Example 1-1-1, except that aluminum foil D-1 with a coating containing a redox active substance was changed to aluminum foil B-1-1 with a coating that does not contain chloranil as the counter electrode. Sample solution K-1, which is a 10 mM glucose solution, was prepared using the same method as in Example 3-1-1, and glucose sensing was performed. Almost no current was measured before dropping the glucose / PB solution, and after dropping the glucose / PB solution, only a weak current was measured compared to Example 1-1-1, where the conditions were the same except for the counter electrode. The peak current and peak current density after glucose dropping and stirring are shown in Table 3.

[0199] [Comparative Example 1-2] As a counter electrode, aluminum foil D-1 with a redox active substance-containing coating film was used, and a chloranil-free glassy carbon electrode (GC, GC-6355 manufactured by EC Frontier Co., Ltd., electrode diameter 3 mm, electrode area 0.0707 cm²) was used.2 Except for the change made to the counter electrode and working electrode, the counter electrode and working electrode were fixed to the electrode holder using the same method as in Example 1-1-1. Sample solution K-1, which is a 10 mM glucose solution, was prepared using the same method as in Example 1-1-1, and glucose sensing was performed. Almost no current was measured before dropping the glucose / PB solution, and even after dropping the glucose / PB solution, only a weak current was measured compared to Example 1-1-1, where the conditions were the same except for the counter electrode. The peak current and peak current density after glucose dropping and stirring are shown in Table 3.

[0200] [Comparative Example 1-3] As a counter electrode, aluminum foil D-1 with a redox active substance-containing coating film was used, and a chloranil-free gold electrode (AU, AU-6355 manufactured by EC Frontier Co., Ltd., electrode diameter 3 mm, electrode area 0.0707 cm²) was used. 2 Except for the change made to the counter electrode and working electrode, the counter electrode and working electrode were fixed to the electrode holder using the same method as in Example 1-1-1. Sample solution K-1, which is a 10 mM glucose solution, was prepared using the same method as in Example 1-1-1, and glucose sensing was performed. Almost no current was measured before dropping the glucose / PB solution, and even after dropping the glucose / PB solution, only a weak current was measured compared to Example 1-1-1, where the conditions were the same except for the counter electrode. The peak current and peak current density after glucose dropping and stirring are shown in Table 3.

[0201]

[0202] Table 3 and Figure 1 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 and 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.

Claims

1. A biosensor comprising a counter electrode and a working electrode, wherein the counter electrode contains a redox active substance.

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

3. The biosensor according to claim 1, wherein the redox active substance includes an organic redox active substance.

4. The biosensor according to claim 3, wherein the redox active substance is a quinone compound.

5. The biosensor according to claim 1, wherein the counter electrode further comprises a carbon material.

6. The biosensor according to claim 5, wherein the carbon material is activated carbon.

7. The biosensor according to claim 1, wherein the counter electrode further comprises a dispersant and binder.

8. The biosensor according to claim 1, wherein the counter electrode is a laminate comprising a substrate and a counter electrode layer formed on the substrate, wherein the substrate is aluminum foil and the counter electrode layer contains a redox active substance.

9. The biosensor according to claim 1, wherein the working electrode includes a redox mediator.

10. The biosensor according to claim 9, wherein the redox mediator is a quinone compound.

11. The biosensor according to claim 9, wherein the oxidation-reduction potential of the redox mediator is less negative than the oxidation-reduction potential of the counter redox active substance.

12. The biosensor according to claim 9, wherein the working electrode further comprises a carbon material.

13. The biosensor according to claim 12, wherein the carbon material is a carbon nanotube.

14. The biosensor according to claim 9, wherein the working electrode further comprises an enzyme.

15. The biosensor according to claim 14, wherein the enzyme is glucose dehygrogenase.

16. The biosensor according to claim 9, wherein the working electrode further comprises a dispersant and binder.

17. The biosensor according to claim 9, wherein the working electrode is a laminate comprising a substrate and a working electrode layer formed on the substrate, the substrate being aluminum foil, and the working electrode layer comprising a redox mediator.

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

19. A sensing method using a biosensor according to any one of claims 1 to 18, wherein the applied voltage between the counter electrode and the working electrode is set to 0V, and sensing is performed by current measurement.

20. A sensing method using a biosensor according to any one of claims 1 to 18, wherein the counter electrode and the working electrode are immersed in a liquid containing a phosphate buffer, and then a sample solution containing the substance to be detected is added to perform sensing.

21. A sensing method using the biosensor according to claim 20, wherein the sample solution contains an enzyme.

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