Biosensor electrode-forming composition, biosensor electrode, and biosensor

Abstract

The purpose of the present invention is to provide, as a biosensor electrode-forming composition that can be suitably used to form an electrochemical biosensor electrode, a biosensor electrode-forming composition comprising carbon nanotubes, an oxazoline group-containing polymer, a carboxyl group-containing polymer, and water.

Description

Composition for forming biosensor electrodes, biosensor electrodes, and biosensors

[0001] The present invention relates to a composition for forming biosensor electrodes, a biosensor electrode, and 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] While metals such as platinum, palladium, gold, and silver are commonly used as materials for biosensor electrodes, the high cost of the metals themselves and the process of forming conductive wires through metal mask deposition has led to an increasing trend in recent years towards the use of carbon materials to reduce costs. However, when using carbon materials, problems remain in terms of processability and conductivity compared to the use of the aforementioned metals. For example, when using carbon materials alone, such as glassy carbon, processing for forming wiring and electrodes is difficult. In contrast, when forming electrodes using a composition of carbon material mixed with an organic binder, while it offers good processability and ease of fabrication, the resulting electrodes suffer from high electronic resistance, resulting in reduced detection current values ​​and decreased sensitivity under low voltage conditions. Furthermore, if unevenness occurs in the coating film during patterning, it can lead to variations in electrode area and fluctuations in detection current values. Finally, conductive wire formation by resist patterning is expensive.

[0005] Ann. NY Acad Sci. 102, 1962, p. 29-45.Nature. 214, 1967, p. 986.Anal. Cham. 1984, 56, 667-671

[0006] The present invention has been made in view of the above circumstances, and aims to provide a biosensor electrode formation composition that can be suitably used for forming electrochemical biosensor electrodes, a biosensor electrode made using the biosensor electrode formation composition, and a biosensor.

[0007] As a result of diligent research to achieve the above objectives, the inventors have found that when a thin film (coated film) obtained from a homogeneous dispersion containing carbon nanotubes, oxazoline group-containing polymers, and carboxyl group-containing polymers is used as a biosensor electrode, it exhibits a high detection current value, high sensitivity under low voltage conditions, and stable current detection due to its high water resistance, making it suitable for applications requiring durability such as continuous monitoring.

[0008] In other words, the present invention provides the following biosensor electrode formation compositions, biosensor electrodes, and biosensors: 1. A biosensor electrode formation composition comprising carbon nanotubes, an oxazoline group-containing polymer, a carboxyl group-containing polymer, and water. 2. The biosensor electrode formation composition of 1, which is a homogeneous dispersion. 3. The biosensor electrode formation composition of 1 or 2, wherein the carboxyl group-containing polymer is an ammonium polyacrylate salt or an amine salt. 4. Any of the biosensor electrode formation compositions of 1 to 3, further comprising polyvinylpyrrolidone. 5. The biosensor electrode formation composition of 4, wherein the weight-average molecular weight of the polyvinylpyrrolidone is 50,000 or less. 6. Any of the biosensor electrode formation compositions of 1 to 5, wherein the carbon nanotube content is 30% by mass or more in solid content. 7. Any of the biosensor electrode formation compositions of 1 to 6, further comprising at least one selected from the group consisting of ethanol and 2-propanol. 8. A thin film obtained from any of the biosensor electrode formation compositions of 1 to 7. 9. 8. A thin film that is water-resistant. 10. Basis weight is 0.5 g / m 2The following are the thin films of 8 or 9. 11. A biosensor electrode comprising any of the thin films of 8 to 11. 12. A biosensor electrode of 11 in which the thin film further comprises a redox mediator. 13. A biosensor electrode of 12 in which the redox mediator comprises a quinone compound. 14. A biosensor electrode of 11 to 13 in which the thin film further comprises an enzyme. 15. A biosensor electrode of 14 in which the enzyme is glucose dehydrogenase. 16. A biosensor electrode of 11 to 15 comprising a substrate and the thin film laminated on the substrate. 17. A biosensor electrode of 16 in which the substrate is metal foil. 18. A biosensor electrode of 17 in which the substrate is aluminum foil. 19. A biosensor comprising any of the biosensor electrodes of 11 to 18. 20. A method for manufacturing a biosensor electrode, comprising the steps of applying any of the biosensor electrode forming compositions of 1 to 7 onto a substrate and heating and drying the resulting coating film. 21. A method for manufacturing a biosensor electrode, comprising the step of applying a composition containing a redox mediator and a phosphate buffer onto the above-mentioned coating film and drying it.

[0009] By using the biosensor electrode formation composition of the present invention, it is possible to form a biosensor electrode that can obtain a sensing signal with high sensitivity even at low external voltages, or that can obtain a sensing signal with a high maximum current density, and a high-performance biosensor can be obtained by using this electrode. In particular, when an electrode is formed by forming a thin film on a metal foil, the wire resistance is reduced and the current signal is increased, and the amount of coating film formation composition (biosensor electrode formation composition) used is reduced, making it easier to reduce costs. Furthermore, the obtained electrode is easy to process by cutting, punching, etc., and the shape of the electrode and wire can be set easily and at low cost. In addition, the biosensor electrode formation composition of the present invention can form a thinner film than compositions using other carbon materials other than carbon nanotubes, and further high functionality of the biosensor can be expected. Furthermore, since the thin film adsorbs redox mediators and enzymes well, a biosensor electrode using these in combination is expected to exhibit high sensing performance as an enzyme electrode for biosensors.

[0010] The present invention will be described in more detail below. [1] Composition for forming biosensor electrodes The composition for forming biosensor electrodes according to the present invention comprises carbon nanotubes, an oxazoline group-containing polymer, a carboxyl group-containing polymer, and water.

[0011] <Carbon Nanotubes> 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.

[0012] 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.

[0013] 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.

[0014] From the viewpoint of the conductivity of the resulting biosensor electrode, the CNT content is preferably 30% by mass or more, more preferably 35% by mass or more, and even more preferably 40% by mass or more, in the solid content. Furthermore, there is no particular upper limit to the CNT content, but considering dispersibility and coating uniformity, it is preferably 90% by mass or less, more preferably 80% by mass or less, and even more preferably 70% by mass or less, in the solid content.

[0015] <Oxazoline Group-Containing Polymer> The oxazoline group-containing polymer (hereinafter sometimes referred to as oxazoline polymer) functions as a dispersant that stably disperses the above-mentioned CNTs in the composition, and when a crosslinked structure is formed with the carboxyl group-containing polymer described later during the formation of a thin film, it has the function of improving the strength and water resistance (solvent resistance) of the thin film.

[0016] The above-mentioned oxazoline polymer is not particularly limited as long as it is a polymer in which an oxazoline group is bonded directly to the repeating unit constituting the main chain or via a spacer group such as an alkylene group. Specifically, it is preferable to use a polymer obtained by radical polymerization of an oxazoline monomer having a polymerizable carbon-carbon double bond-containing group at the 2-position as shown in formula (1), which has repeating units bonded to the polymer main chain or a spacer group at the 2-position of the oxazoline ring.

[0017]

[0018] In the formula, X represents a polymerizable carbon-carbon double bond-containing group, and R 1 ~R 4 These independently represent a hydrogen atom, a halogen atom, an alkyl group having 1 to 5 carbon atoms which may have a branched structure, an aryl group having 6 to 20 carbon atoms, or an aralkyl group having 7 to 20 carbon atoms. The polymerizable carbon-carbon double bond-containing group of the oxazoline monomer is not particularly limited as long as it contains a polymerizable carbon-carbon double bond, but a chain-like hydrocarbon group containing a polymerizable carbon-carbon double bond is preferred, for example, an alkenyl group having 2 to 8 carbon atoms such as a vinyl group, an allyl group, or an isopropenyl group is preferred. Examples of halogen atoms include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Specific examples of alkyl groups having 1 to 5 carbon atoms which may have a branched structure include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, a tert-butyl group, and an n-pentyl group. Specific examples of aryl groups having 6 to 20 carbon atoms include a phenyl group, a xylyl group, a tolyl group, a biphenyl group, and a naphthyl group. Specific examples of aralkyl groups having 7 to 20 carbon atoms include the benzyl group, phenylethyl group, and phenylcyclohexyl group.

[0019] Specific examples of oxazoline monomers having a polymerizable carbon-carbon double bond-containing group at the 2-position, as shown in formula (1), include 2-vinyl-2-oxazoline, 2-vinyl-4-methyl-2-oxazoline, 2-vinyl-4-ethyl-2-oxazoline, 2-vinyl-4-propyl-2-oxazoline, 2-vinyl-4-butyl-2-oxazoline, 2-vinyl-5-methyl-2-oxazoline, 2-vinyl-5-ethyl-2-oxazoline, 2-vinyl-5-propyl-2-oxazoline, 2-vinyl-5-butyl-2-oxazoline, and 2-isopropenyl-2-oxazoline. Examples include sazolin, 2-isopropenyl-4-methyl-2-oxazoline, 2-isopropenyl-4-ethyl-2-oxazoline, 2-isopropenyl-4-propyl-2-oxazoline, 2-isopropenyl-4-butyl-2-oxazoline, 2-isopropenyl-5-methyl-2-oxazoline, 2-isopropenyl-5-ethyl-2-oxazoline, 2-isopropenyl-5-propyl-2-oxazoline, and 2-isopropenyl-5-butyl-2-oxazoline, but 2-isopropenyl-2-oxazoline is preferred from the standpoint of ease of acquisition.

[0020] Furthermore, considering that the dispersion or composition containing dispersed CNTs is prepared using water (and, if necessary, an aqueous solvent described later), it is preferable that the oxazoline polymer is water-soluble. Such a water-soluble oxazoline polymer may be a homopolymer of the oxazoline monomer represented by formula (1) above, but in order to further enhance its solubility in water, it is preferable that it is obtained by radical polymerization of at least two monomers, the oxazoline monomer and a (meth)acrylic acid ester monomer having a hydrophilic functional group.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] The oxazoline polymer used in the present invention can be produced by polymerizing the above-described various monomers by a known radical polymerization method described in, for example, JP-A-6-32844 and JP-A-2013-72002. The oxazoline polymer that can be used in the present invention can also be obtained as a commercially available product. Examples of such commercially available products include Epocros WS-300 (manufactured by Nippon Shokubai Co., Ltd., solid content concentration: 10% by mass, aqueous solution), Epocros WS-700 (manufactured by Nippon Shokubai Co., Ltd., solid content concentration: 25% by mass, aqueous solution), Epocros 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) (Alfa Aesar), Poly(2-ethyl-2-oxazoline) (VWR International, LLC), and the like. When commercially available as a solution, it may be used as it is or the solvent may be replaced with the desired solvent system.

[0027] The above oxazoline polymer may be used alone or in combination of two or more.

[0028] The film obtained by drying the oxazoline polymer on a substrate usually has no water resistance and may elute in the test liquid, resulting in unstable sensing performance. In such a case, a carboxy group-containing polymer or the like can be used in combination, and a crosslinking reaction can be advanced by performing heat drying to impart water resistance.

[0029] From the viewpoint of the dispersibility of the carbon nanotubes, the content of the oxazoline polymer is preferably 1.4 to 0.05, more preferably 1.2 to 0.2, and even more preferably 1.1 to 0.4 in terms of mass ratio with respect to 1 of the carbon nanotubes.

[0030] <Carboxy Group-Containing Polymer> The composition for forming a biosensor electrode of the present invention contains a carboxy group-containing polymer. The carboxy group-containing polymer is not particularly limited as long as it is a polymer having at least one carboxy group capable of reacting with the oxazoline group of the above oxazoline group-containing polymer.

[0031] Further, as long as it is a functional group capable of reacting with an oxazoline group, it may have a functional group other than a carboxy group, and specific examples thereof include an aromatic hydroxyl group, an aromatic thiol group, and the like.

[0032] Considering that the carboxy group-containing polymer crosslinks the above oxazoline polymer to enhance the strength and water resistance (solvent resistance) of the resulting thin film, a polymer having two or more functional groups capable of reacting with an oxazoline group, including at least one carboxy group, is preferable, and a polymer having two or more carboxy groups is more preferable.

[0033] Further, the carboxy group-containing polymer may have a functional group that generates the above functional group in the presence of heat during thin film formation or in the presence of an acid catalyst and reacts with an oxazoline group, for example, a carboxylic acid (carboxy group), a sodium salt, potassium salt, lithium salt, ammonium salt, amine salt, etc. of a carboxylic acid. In the present invention, for improving the stability of the dispersion liquid and for improving the efficiency of the crosslinking reaction, those having an ammonium salt or amine salt of a carboxylic acid are preferable.

[0034] Specific examples of the carboxy group-containing polymer include synthetic polymers such as polyacrylic acid and its copolymers, and metal salts of natural polymers such as carboxymethyl cellulose 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 by heating. In particular, polyacrylic acid, sodium polyacrylate, lithium polyacrylate, ammonium polyacrylate, polyacrylic acid amine, carboxymethyl cellulose, sodium carboxymethyl cellulose, lithium carboxymethyl cellulose, ammonium carboxymethyl cellulose, etc., which exhibit crosslinking reactivity in the presence of an acid catalyst or under heating conditions, are preferable, and ammonium polyacrylate and polyacrylic acid amine are more preferable.

[0035] 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.

[0036] 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.

[0037] The above-mentioned carboxyl group-containing polymer may be used individually or in combination of two or more types.

[0038] 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.

[0039] Furthermore, the biosensor electrode formation composition of the present invention may contain a thermoacid generator such as 2,4,4,6-tetrabromocyclohexadienone, benzoin tosylate, 2-nitrobenzyl tosylate, or an alkyl organic sulfonate as a catalyst to promote the crosslinking reaction.

[0040] 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.

[0041] <Water and Other Solvents> The biosensor electrode formation composition of the present invention uses water as a solvent, but other solvents may be included as long as they do not impair the effects of the present invention. Other solvents that are hydrophilic are preferred, and examples include 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. Among these solvents, in the present invention, THF, DMF, NMP, methanol, ethanol, 2-propanol, and acetonitrile are preferred, and ethanol and 2-propanol are more preferred, considering the dispersibility of carbon nanotubes and the coating properties of the composition. These solvents may be used individually or in combination of two or more.

[0042] If other solvents are included as described above, their content is preferably 50% by mass or less of the total solvent used in the composition, more preferably 40% by mass or less, and even more preferably 30% by mass or less. The lower limit is not particularly limited, but 10% by mass or more is preferred.

[0043] <Polyvinylpyrrolidone> The biosensor electrode formation composition of the present invention may further contain polyvinylpyrrolidone. By including the above polyvinylpyrrolidone, the affinity with enzymes in the resulting thin film is improved, and an improvement in the sensitivity of the enzyme-containing biosensor electrode (hereinafter sometimes referred to as "enzyme electrode") described later is expected.

[0044] The weight-average molecular weight of polyvinylpyrrolidone is preferably 50,000 or less, more preferably 47,000 or less, and even more preferably 45,000 or less, from the viewpoint of affinity with the enzyme. The lower limit of the above weight-average molecular weight is not particularly limited, but is preferably 5,000 or more. The weight-average molecular weight is the polystyrene equivalent value obtained by gel permeation chromatography (GPC).

[0045] The polyvinylpyrrolidone content is preferably 1.0 to 0, more preferably 0.5 to 0, and even more preferably 0.3 to 0, in terms of mass ratio, per carbon nanotube 1.

[0046] <Other Carbon Materials> The compositions of the present invention may contain other carbon materials other than the CNTs described above, to the extent that they do not impair the effects of the present invention. Other carbon materials can be appropriately selected and used from known carbon materials such as carbon black, Ketjenblack, acetylene black, carbon whiskers, carbon fibers, natural graphite, artificial graphite, and porous carbon.

[0047] <Preparation of Biosensor Electrode Formation Composition> The method for preparing the biosensor electrode formation composition of the present invention is arbitrary. A dispersion can be prepared by mixing CNTs, oxazoline polymer (dispersant), carboxyl group-containing polymer, and water (solvent) in any order, and a homogeneous dispersion is preferred. In this case, it is preferable to disperse the mixture consisting of CNTs, oxazoline polymer, carboxyl group-containing polymer, and water, and this treatment can further improve the dispersion ratio of carbon nanotubes. 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 5 hours. The oxazoline polymer used in the present invention has excellent CNT dispersion ability, so a composition in which CNTs are stably dispersed can be obtained without heat treatment before the dispersion treatment, but heat treatment may be applied if necessary.

[0048] Furthermore, the concentration of the oxazoline polymer in the composition is not particularly limited as long as it is a concentration that can disperse the CNTs in the solvent, but it is preferably about 0.001 to 30% by mass in the dispersion, and more preferably about 0.002 to 20% by mass.

[0049] Furthermore, the concentration of CNTs in this composition varies depending on the mechanical, electrical, and thermal properties required of the thin film, and is arbitrary as long as at least a portion of the CNTs are dispersed in isolation, but it is preferably about 0.0001 to 30% by mass in the dispersion, more preferably about 0.001 to 20% by mass, and even more preferably about 0.001 to 10% by mass.

[0050] The solid content concentration of the biosensor electrode formation composition of the present invention is set appropriately considering the coating properties of the composition and the thickness of the thin film (electrode) to be formed, but is usually about 0.1 to 10% by mass, preferably about 0.3 to 5% by mass, and more preferably about 0.5 to 2.5% by mass. In the present invention, solid content refers to components other than the solvent that constitute the composition (the same applies hereinafter).

[0051] [2] Biosensor Electrode The biosensor electrode of the present invention can be obtained by coating all or part of the above-described biosensor electrode forming composition onto a substrate and heating and drying it. During heating and drying (when a thin film is formed), the oxazoline groups of the oxazoline polymer react with the carboxyl groups (and other functional groups that react with the oxazoline groups) of the carboxyl group-containing polymer. In this case, if the oxazoline polymer and the carboxyl group-containing polymer form a crosslinked structure, the strength and water resistance (solvent resistance) of the resulting thin film can be further increased.

[0052] 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.

[0053] Examples of methods for applying the 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.

[0054] 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.

[0055] The basis weight of the thin film is not particularly limited as long as it satisfies the above film thickness requirement, but is 0.5 g / m². 2 The following is preferred, and more preferably, 0.4 g / m 2 The following applies. On the other hand, the lower limit of the above basis weight is not particularly limited, but from the viewpoint of obtaining a biosensor with excellent characteristics that ensures the function as an electrode with good reproducibility, 0.1 g / m 2 The above is preferable, and more preferably 0.2 g / m 2 That concludes the explanation. Furthermore, while the thickness of the thin film is not particularly limited, considering the reduction of internal resistance and the low cost of manufacturing electrodes, a thickness of 0.05 to 1 μm is preferred, and a thickness of 0.10 to 0.50 μm is more preferred.

[0056] The basis weight and film thickness can be adjusted by known methods. For example, they can be adjusted by changing the solid content concentration, coating conditions, and number of coats of the biosensor electrode formation composition. To increase the basis weight and film thickness, the device conditions should be changed to make the coating thicker, or the solid content concentration should be increased, or the number of coats should be increased. To decrease the basis weight and film thickness, the device conditions should be changed to make the coating thinner, or the solid content concentration should be decreased, or the number of coats should be reduced, or the clearance should be reduced.

[0057] The biosensor electrode of the present invention may be used as a self-supporting film with the thin film (coating film) peeled off from the substrate, or as a laminate with thin films laminated on the substrate, depending on the structure of the biosensor being manufactured. Furthermore, the biosensor electrode may be cut to the desired size and shape as needed. In this invention, it is preferable to cut the thin film together with the substrate at the laminate stage, since the shape of the thin film is stable due to the substrate. The biosensor electrode may be cut by known methods such as punching.

[0058] When the above-mentioned laminate is used as an electrode, it is preferable to use a metal foil as the substrate, 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 substrate, after cutting the substrate, the parts other than the electrode (the parts where the thin film is not formed) can be used as conductors, so that a biosensor electrode with wiring can be easily manufactured, and an improvement in the productivity of biosensor electrodes can be expected. Furthermore, since the shape of the thin film is stable due to the above-mentioned substrate, it is easy to cut the thin film together with the substrate into a shape corresponding to the shape of the wiring.

[0059] The biosensor electrode of the present invention can be used as the working electrode, counter electrode, or reference electrode of a biosensor, but is particularly suitable for use as the working electrode. To enhance its sensitivity, the biosensor electrode preferably further contains a redox mediator (RM) having electron transfer capability in a thin film.

[0060] 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 phenolic compounds. In the present invention, from the viewpoint of reactivity with enzymes and electrodes, quinone compounds are preferred, 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.

[0061] Furthermore, it is preferable to dissolve the above-mentioned RM in a solvent and use it as an RM solution. The solvent can be appropriately selected depending on the type of RM, and specific examples include water, phosphate buffer, phosphate-buffered saline, acetonitrile, tetrahydrofuran, ethanol, 2-propanol, N,N-dimethylformamide, N-methylpyrrolidone, etc. These solvents and solutions may be used in combination.

[0062] Furthermore, the RM solution preferably contains a phosphate buffer to enhance the solubility of RM in the sample solution. A commercially available phosphate buffer can be used as the phosphate buffer, for example, a phosphate buffer manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0063] The concentration of RM in an RM solution is not particularly limited, but is usually around 0.1 to 100 mM (molecule / L).

[0064] The biosensor electrode of the present invention can also be used as a so-called enzyme electrode by incorporating a predetermined enzyme. The enzyme can be an oxidase, a dehydrogenase, or the like, and can be appropriately selected depending on the substrate to be detected.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] When using the above-mentioned RM and enzyme, for example, a biosensor electrode can be obtained by dropping a solution containing RM or enzyme onto the thin film and drying it, thereby stacking them on 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 enzyme onto it and dry it. The above-mentioned RM and enzyme can also be added to the sample solution, but in actual use, it is often not possible to effectively add RM or enzyme to the sample solution, so it is preferable to drop them onto the thin film and dry them.

[0070] 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.

[0071] 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.

[0072] [3] Biosensor The biosensor of the present invention is equipped with the biosensor electrodes described above, and more specifically, is configured to include an electrode system that includes at least a working electrode and a counter electrode, wherein at least one of the working electrode and the counter electrode is made of the biosensor electrodes described above.

[0073] This biosensor is characterized by the use of the aforementioned biosensor electrodes in its electrode system; therefore, other device components such as separators, sample fluid guides, and protective films can be appropriately selected from known materials.

[0074] 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.

[0075] Furthermore, the electrode system may include a reference electrode to ensure accurate measurement of the potential during the measurement of the sample.

[0076] The biosensor of the present invention is not particularly limited in form, as long as it uses the above-mentioned biosensor electrode in any of the electrode systems. 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. However, the present invention is suitably applicable to the latter configuration in which the working electrode and counter electrode are arranged opposite each other.

[0077] When constructing a biosensor using the biosensor electrode of the present invention as the working electrode, any of the methods described above can be used, but as a simpler method, it can be constructed by, for example, the following method.

[0078] A biosensor can be constructed by preparing a sample solution containing glucose as the target substance using a standard method and adding it to a glass cell VB2-1 (manufactured by EC Frontier Co., Ltd.), and then immersing the working electrode and platinum spiral counter electrode (manufactured by EC Frontier Co., Ltd.), prepared by the above method, in the sample solution. Subsequently, the glucose concentration can be determined by performing cyclic voltammetry measurements using an electrochemical measuring device with the prepared biosensor and measuring the current value at a specified voltage.

[0079] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples.

[0080] (1) Preparation of composition for biosensor electrode (working electrode) [Example 1-1] 0.350 g of BT-1003M (manufactured by LG Energy Solutions, multi-walled carbon nanotube), 54.69 g of pure water, 1.40 g of Epocross WS-700 (manufactured by Nippon Shokubai Co., Ltd., aqueous solution of oxazoline group-containing polymer with a solid content of 25% by mass) (solid content 0.350 g), and 10.48 g of 2-propanol were sequentially added to a 100 mL polypropylene bottle 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 (a homogeneous aqueous solution obtained by diluting a 31.6% by mass aqueous solution of ammonium polyacrylate (Aron A-30 manufactured by Toagosei Co., Ltd.) with pure water and stirring) was added and stirred to obtain black homogeneous dispersion A-1 (solid content concentration 1.22% by mass).

[0081] [Example 1-2] Except that 1.40 g of Epocross WS-700 was replaced with 3.50 g of Epocross WS-300 (manufactured by Nippon Shokubai Co., Ltd., 10% by mass solid content aqueous solution of oxazoline group-containing polymer) (0.350 g solid content), and the amount of pure water used was changed to 52.59 g, a black homogeneous dispersion A-2 (solid content concentration 1.22% by mass) was obtained using the same method as in Example 1-1.

[0082] [Example 1-3] 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-3 (solid content concentration 1.22% by mass) was obtained using the same method as in Example 1-2.

[0083] [Examples 1-4] 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-4 (solid content concentration 1.22% by mass).

[0084] [Comparative Example 1-1] A black homogeneous dispersion B-1 (solid content concentration 5.00% by mass) was obtained using the same method as in Example 1-1, except that 0.350 g of BT-1003M was replaced with 1.75 g of Li-435 (Acetylene Black, manufactured by Denka Co., Ltd.), the amount of pure water used was changed to 39.69 g, the amount of Epocross WS-700 used was changed to 3.89 g (solid content 0.973 g), the amount of 2-propanol used was changed to 22.17 g, and 3.08 g of a 5.00% by mass aqueous solution of ammonium polyacrylate was replaced with 2.51 g of a 31.6% by mass aqueous solution of ammonium polyacrylate (Aron A-30, manufactured by Toagosei Co., Ltd.) (solid content 0.778 g).

[0085] [Comparative Example 1-2] Except that the amount of BT-1003M used was changed to 0.214 g, the amount of pure water used was changed to 49.91 g, the amount of Epocross WS-700 used was changed to 0.854 g (solid content 0.214 g), and the amount of 5.00% by mass ammonium polyacrylate aqueous solution used was changed to 8.54 g (solid content 0.427 g), a black homogeneous dispersion B-2 (solid content concentration 1.22% by mass) was obtained using the same method as in Example 1-1.

[0086] [Comparative Examples 1-3] In a 100 mL polypropylene bottle container, 0.350 g of BT-1003M, 55.44 g of pure water, 0.350 g of Pitts coal K-30, and 13.86 g of 2-propanol were sequentially added and stirred to obtain a black mixed solution. With respect to this black mixed solution, while stirring with a magnetic stirrer under ice cooling, ultrasonic waves were irradiated at 500 W for 3 minutes using a probe type ultrasonic irradiation device UIP1000hd (manufactured by Hielscher). After irradiation, the container was taken out and the liquid was shaken to be homogenized. This ultrasonic treatment was repeated 5 times to obtain a black homogeneous dispersion B-3 (solid content concentration: 1.00% by mass).

[0087] [Comparative Example 1-4] A black homogeneous dispersion B-4 (solid content concentration: 1.22% by mass) was obtained in the same manner as in Example 1-4, except that Pitts coal K-30 was changed to Pitts coal K-90 (manufactured by Daiichi Kogyo Seiyaku Co., Ltd., polyvinylpyrrolidone, weight average molecular weight 1,200,000).

[0088] (2) Fabrication of Biosensor Electrode (Working Electrode) [Example 2-1] Dispersion A-1 was spread on an aluminum foil (manufactured by UACJ Co., Ltd., foil type No. 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 at 120 °C for 10 minutes using an oven to obtain a biosensor electrode (working electrode) C-1. The coated film surface of the working electrode C-1 was uniform by visual observation. The coated film surface weight of the working electrode C-1 was estimated to be 0.36 g / m from the maximum wet film thickness, the solvent specific gravity (0.99 g / cm 3 ) of Dispersion A-1, and the solid content concentration of the composition. 2 It is presumed that.

[0089] [Example 2-2] A biosensor electrode (working electrode) C-2 was obtained in the same manner as in Example 2-1, except that Dispersion A-1 was changed to A-2. The coated film surface of the working electrode C-2 was uniform by visual observation. The coated film surface weight of the working electrode C-2 was estimated to be 0.36 g / m from the maximum wet film thickness, the solvent specific gravity (0.99 g / cm 3 ) of Dispersion A-2, and the solid content concentration. 2 It is presumed that.

[0090] [Example 2-3] A biosensor electrode (working electrode) C-3 was obtained using the same method as in Example 2-1, except that dispersion A-1 was changed to A-3. The coated surface of working electrode C-3 was uniform upon visual observation. The basis weight of the coated surface of working electrode C-3 was the maximum wet film thickness and the solvent specific gravity of dispersion A-3 (0.99 g / cm³). 3 ) and the solid content concentration is 0.36 g / m 2 It is presumed that...

[0091] [Example 2-4] A biosensor electrode (working electrode) C-4 was obtained using the same method as in Example 2-1, except that dispersion A-1 was changed to A-4. The coated surface of working electrode C-4 was uniform upon visual inspection. The basis weight of the coated surface of working electrode C-4 was the maximum wet film thickness and the solvent specific gravity of dispersion A-4 (0.99 g / cm³). 3 ) and the solid content concentration is 0.36 g / m 2 It is presumed that...

[0092] [Comparative Example 2-1] A biosensor electrode (working electrode) D-1 was obtained using the same method as in Example 2-1, except that dispersion A-1 was changed to B-1. The coated surface of working electrode D-1 was uniform upon visual observation. The basis weight of the coated surface of working electrode D-1 was the maximum wet film thickness and the solvent specific gravity of dispersion C-1 (0.95 g / cm³). 3 ) and the solid content concentration is 1.4 g / m 2 It is presumed that...

[0093] [Comparative Example 2-2] A biosensor electrode (working electrode) D-2 was obtained using the same method as in Example 2-1, except that dispersion A-1 was changed to B-2. The coated surface of working electrode D-2 was uniform upon visual observation. The basis weight of the coated surface of working electrode D-2 was the maximum wet film thickness and the solvent specific gravity of dispersion B-2 (0.99 g / cm³). 3 ) and the solid content concentration is 0.36 g / m 2 It is presumed that...

[0094] [Comparative Example 2-3] A biosensor electrode (working electrode) D-3 was obtained using the same method as in Example 2-1, except that dispersion A-1 was changed to B-3. The coated surface of working electrode D-3 was uniform upon visual observation. The basis weight of the coated surface of working electrode D-3 was the maximum wet film thickness and the solvent specific gravity of dispersion B-3 (0.99 g / cm³). 3) and the solid content concentration is 0.30 g / m 2 It is presumed that...

[0095] [Comparative Example 2-4] A biosensor electrode (working electrode) D-4 was obtained using the same method as in Example 2-1, except that dispersion A-1 was changed to B-4. Visual inspection revealed unevenness and non-uniformity on the coated surface of working electrode D-4. The basis weight of the coated surface of working electrode D-4 was the maximum wet film thickness and the solvent specific gravity of dispersion B-4 (0.99 g / cm³). 3 ) and the solid content concentration is 0.36 g / m 2 It is presumed that...

[0096] The biosensor electrodes (working electrodes) prepared in Examples 2-1 to 2-4 and Comparative Examples 2-1 to 2-4 were evaluated for coating film uniformity and scratch resistance using the following method.

[0097] <Uniformity of the Coating Surface> A biosensor electrode formation composition (black dispersion) was spread onto metal foil using a wire bar coater, and the state of the coating film after drying was observed visually. The uniformity of the coating film was evaluated by visually observing the state of the coating film surface after drying, according to the following criteria. The evaluation results are shown in Table 1. <Evaluation Criteria> ○: The majority of the coating film surface is uniform as seen visually △: There are uniform pattern-like uneven surfaces on the coating film surface as seen visually (×: The entire coating surface is clearly uneven as seen visually) In the above coating film uniformity test, a "○" evaluation was given as a pass.

[0098] <Scratch Resistance (Dry Bencot Peel Test)> Contact area with coating surface: 1 cm² 2 50g / cm² of nonwoven fabric wiper (manufactured by Asahi Kasei Corporation, Bencot M-1) 2 The load was applied and the tool was moved once horizontally across the metal foil surface. The condition of the coating surface in the wiped area was visually inspected and evaluated according to the following criteria. The results are shown in Table 1. <Evaluation Criteria> ○: No scratches occurred on the coating surface. △: Linear scratches occurred on the coating surface, and the metal foil surface was exposed. (×: Surface peeling occurred on the coating surface, and the metal foil surface was exposed.) In the above scratch resistance test, a "○" or "△" evaluation was given, and the test was considered successful.

[0099] <Water resistance (water-based peel test)> Contact area with the coated film surface: 1 cm²2 50 g / cm of nonwoven wiper (Asahi Kasei Corporation, Bencot M-1) moistened with water. 2 The load was applied and the tool was moved once horizontally across the metal foil surface. The condition of the coating surface in the wiped area was visually inspected and evaluated according to the following criteria. The results are shown in Table 1. <Evaluation Criteria> ○: No scratches were made on the coating surface. △: Linear scratches were made on the coating surface and the metal foil surface was exposed. (×: Surface peeling occurred on the coating surface and the metal foil surface was exposed.) In the above water resistance test, a "○" or "△" evaluation was given to pass.

[0100]

[0101] The results in Table 1 confirm that the biosensor electrodes of the examples exhibit excellent coating film uniformity, scratch resistance, and water resistance. Since the sample solution used in biosensors is generally an aqueous solution and is highly polar, it is expected that biosensor electrodes with excellent water resistance will have stable biosensor function and excellent durability.

[0102] (3) Evaluation of the reactivity between the biosensor electrode (working electrode) and redox mediator by cyclic voltammetry (hereinafter abbreviated as CV) measurement. In electrochemical biosensors, a common method for detecting a sample involves a quaternary chain electron transfer reaction in which an enzyme oxidizes the sample (target substance), a redox mediator oxidizes the enzyme reduced by the sample, and the electrode oxidizes the redox mediator reduced by the enzyme via an external power source. In this reaction, the electrochemical oxidation of the redox mediator by the electrode is the final stage of this chain electron transfer reaction, and its reactivity is an important measure of the performance of the biosensor electrode. When the reactivity is high, the biosensor electrode and redox mediator will have high current values ​​under low voltage conditions, and the biosensor can be highly sensitive. Therefore, in this study, measurements were performed using the CV method to evaluate the reactivity between the biosensor electrode and the redox mediator.

[0103] [Example 3-1] <Preparation of the working electrode> The biosensor electrode C-1 prepared in Example 2-1 was punched out to a diameter of 10 mm, and the coated film surface was fixed with the opening side facing the opening inside a 5 mm diameter back-contact type sample holder AE9-2 (manufactured by EC Frontier Co., Ltd.) to form a working electrode with a diameter of 5 mm.

[0104] <Preparation of Redox Mediator (hereinafter abbreviated as RM) Solution> A 1 mM (mol / L) 1,2-naphthoquinone solution was prepared by the following method and designated as RM Solution E-1. 6.3 mg of 1,2-naphthoquinone was dissolved in 2.5 mL of acetonitrile by stirring. Then, 37.5 mL of 100 mM phosphate buffer (pH 7.0, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter abbreviated as PB) was added little by little while stirring to obtain RM Solution E-1, a yellow-orange transparent solution.

[0105] <CV Measurement> 10 mL of redox mediator solution E-1 was added to a glass cell VB2-1 (manufactured by EC Frontier Co., Ltd.). A biosensor electrode C-1 was used as the working electrode, a platinum spiral electrode (manufactured by EC Frontier Co., Ltd., model number CE-200) as the counter electrode, and an Ag / AgCl electrode (manufactured by EC Frontier Co., Ltd., model number RE-T2A) as the reference electrode were fixed in this solution by immersion using the starter kit VB2-S. After stirring and standing, CV measurement was performed under the following conditions: Initial voltage: 0.2V Voltage sweep range: 0.2V to -0.5V Scan speed: 0.02V / sec Voltage step: 0.001V Number of cycles: 3

[0106] The CV curve shape obtained was one oxidation current peak and one reduction current peak. From the second cycle onward, the CV curve shape stabilized, including the initial voltage, and the CV curves for the second and third cycles were almost identical. Table 2 shows the oxidation peak voltage and its current density, the reduction peak voltage and its current density, and the difference between the two peak voltages (peak separation) in the second cycle.

[0107] [Example 3-2] CV measurement was performed using the same method as in Example 3-1, except that biosensor electrode C-1 was changed to C-2. The results under the same conditions as in Example 3-1 are shown in Table 2.

[0108] [Example 3-3] CV measurement was performed using the same method as in Example 3-1, except that biosensor electrode C-1 was changed to C-3. The results under the same conditions as in Example 3-1 are shown in Table 2.

[0109] [Example 3-4] CV measurements were performed using the same method as in Example 3-1, except that the preparation methods for RM and RM solution were changed as follows. The results under the same conditions as in Example 3-1 are shown in Table 2.

[0110] <Preparation of RM Solution> A 1 mM (mol / L) sodium 1,2-naphthoquinone-4-sulfonate solution was prepared by the following method and designated as RM solution E-2. 10.4 mg of sodium 1,2-naphthoquinone-4-sulfonate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 40.0 mL of 100 mM PB by stirring, and a yellow transparent solution, redox mediator solution E-2, was obtained. CV measurement was performed using the same method as in Example 3-3, except that RM solution E-1 was changed to E-2. The results under the same conditions as in Example 3-1 are shown in Table 2.

[0111] [Example 3-5] CV measurement was performed using the same method as in Example 3-1, except that biosensor electrode C-1 was changed to C-4. The results under the same conditions as in Example 3-1 are shown in Table 2.

[0112] [Comparative Example 3-1] CV measurement was performed using the same method as in Example 3-1, except that biosensor electrode C-1 was changed to D-1. During the CV measurement, the coated film surface peeled off from the aluminum foil, and neither oxidation peaks nor reduction peaks were generated.

[0113] [Comparative Example 3-2] CV measurement was performed using the same method as in Example 3-1, except that biosensor electrode C-1 was changed to D-2. No clear oxidation or reduction peaks were observed.

[0114] [Comparative Example 3-3] CV measurement was performed using the same method as in Example 3-1, except that biosensor electrode C-1 was changed to D-3. The results under the same conditions as in Example 3-1 are shown in Table 2.

[0115] [Comparative Example 3-4] CV measurement was performed using the same method as in Example 3-1, except that the preparation methods for RM and RM solution were changed as follows. The results under the same conditions as in Example 3-1 are shown in Table 2.

[0116] <Preparation of RM Solution> A 1 mM (mol / L) potassium ferricyanide solution was prepared by the following method and designated as RM Solution E-3. 13.2 mg of potassium ferricyanide (K3 [Fe(CN)6], manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 40.0 mL of 100 mM PB by stirring, and a yellow transparent solution, Redox Mediator Solution E-3, was obtained.

[0117] [Comparative Example 3-5] CV measurement was performed using the same method as in Example 3-1, except that the biosensor electrode C-1 was changed to aluminum foil (manufactured by UACJ Foil Co., Ltd., model number 1N30, thickness 15um, denoted as "plain Al" in Table 2 below). No clear oxidation or reduction peaks were observed.

[0118] [Comparative Example 3-6] CV measurement was performed using the same method as in Example 3-1, except that the biosensor electrode C-1 was changed to a gold electrode (manufactured by EC Frontier Co., Ltd., 3 mm in diameter, model number Au-6355, denoted as Au in Table 2 below). The results under the same conditions as in Example 3-1 are shown in Table 2.

[0119] [Comparative Example 3-7] CV measurement was performed using the same method as in Comparative Example 3-6, except that the RM solution was changed from E-1 to E-2. The results under the same conditions as in Example 3-1 are shown in Table 2.

[0120] [Comparative Example 3-8] CV measurement was performed using the same method as in Comparative Example 3-7, except that the biosensor electrode was changed from a gold electrode to a glassy carbon electrode (manufactured by EC Frontier Co., Ltd., 3 mm in diameter, model number GC-6355, denoted as GC in Table 2 below). The results under the same conditions as in Example 3-1 are shown in Table 2.

[0121]

[0122] Under these conditions, the current density of the oxidation current peak is 150 μA / cm². 2 In summary, the current density at the reduction current peak was -150 μA / cm².2 The following conditions were met: the peak voltage difference must be 0.2V or less; if all of these conditions were met, the product was considered acceptable.

[0123] (4) Evaluation of glucose sensing characteristics of biosensor electrode (working electrode) by CV measurement [Example 4-1] <Fabrication of working electrode> The working electrode was fabricated using the same method as in Example 3-1 <Fabrication of working electrode>.

[0124] <Preparation of RM and enzyme-containing sample solution> Glucose was set as the sample to be sensed, and RM and enzyme-containing sample solution F-1 was prepared by adding RM and enzyme to it using the following method. 720.6 mg of D-glucose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 100 mM PB, and then 100 mM PB was added until the volume reached 40 mL to prepare a 100 mM D-glucose / PB solution. 42 μL of GDH-1 (manufactured by Ikeda Sugar Refining Co., Ltd., flavin-bound glucose dehydrogenase (FAD-GDH), activity concentration 25,000 Units / mL) and 667 μL of RM solution E-1 were sequentially added to 6.0 mL of the obtained 100 mM D-glucose / PB solution and mixed to obtain RM and enzyme-containing sample solution F-1, which is a 90 mM D-glucose solution.

[0125] <Electrochemical Cell Setup and CV Measurement> 6.0 mL of RM and enzyme-containing sample solution F-1 was added to a glass cell VB2-1 (manufactured by EC Frontier Co., Ltd.). A biosensor electrode C-1 was used as the working electrode, a platinum spiral electrode (manufactured by EC Frontier Co., Ltd., model CE-200) as the counter electrode, and an Ag / AgCl electrode (manufactured by EC Frontier Co., Ltd., model RE-T2A) as the reference electrode were fixed in this solution by immersion using the starter kit VB2-S. After stirring and standing, CV measurement was performed under the following conditions: Initial voltage: -0.2V Voltage sweep range: -0.2V to 0.5V Scan speed: 0.01V / sec Voltage step: 0.001V Number of cycles: 2

[0126] The CV curves for the first and second cycles were almost identical, but the second cycle had a slightly lower current density due to the decrease in glucose concentration near the working electrode interface caused by the glucose oxidation reaction in the first cycle. Table 3 shows the current densities at Ag / AgCl ratios of 0.00 V and 0.10 V in the first cycle.

[0127] [Example 4-2] CV measurement was performed using the same method as in Example 4-1, except that the biosensor electrode used as the working electrode was changed from C-1 to C-3. The results under the same conditions as in Example 4-1 are shown in Table 3.

[0128] [Example 4-3] CV measurement was performed using the same method as in Example 4-1, except that the biosensor electrode used as the working electrode was changed from C-1 to C-4. The results under the same conditions as in Example 4-1 are shown in Table 3.

[0129] [Example 4-4] CV measurement was performed using the same method as in Example 4-1, except that the RM solution used in the RM and enzyme-containing sample solution was changed from E-1 to E-2. The results under the same conditions as in Example 4-1 are shown in Table 3.

[0130] [Example 4-5] CV measurement was performed using the same method as in Example 4-4, except that the biosensor electrode used as the working electrode was changed from C-1 to C-3. The results under the same conditions as in Example 4-1 are shown in Table 3.

[0131] [Example 4-6] CV measurement was performed using the same method as in Example 4-4, except that the biosensor electrode used as the working electrode was changed from C-1 to C-4. The results under the same conditions as in Example 4-1 are shown in Table 3.

[0132] [Comparative Example 4-1] CV measurement was performed using the same method as in Example 4-1, except that the biosensor electrode used as the working electrode was changed from C-1 to a gold electrode (manufactured by EC Frontier Co., Ltd., 3 mm in diameter, model number Au-6355, denoted as Au in Table 3 below). The results under the same conditions as in Example 4-1 are shown in Table 3.

[0133] [Comparative Example 4-2] CV measurement was performed using the same method as in Example 4-4, except that the biosensor electrode used as the working electrode was changed from C-1 to a gold electrode (manufactured by EC Frontier Co., Ltd., 3 mm in diameter, model number Au-6355, denoted as Au in Table 3 below). The results under the same conditions as in Example 4-1 are shown in Table 3.

[0134] [Comparative Example 4-3] CV measurement was performed using the same method as in Example 4-4, except that the biosensor electrode used as the working electrode was changed from C-1 to a glassy carbon electrode (manufactured by EC Frontier Co., Ltd., 3 mm in diameter, model number GC-6355, denoted as GC in Table 3 below). The results under the same conditions as in Example 4-1 are shown in Table 3.

[0135]

[0136] Table 3 shows that, comparing the results of Examples 4-1 to 4-6 with those of Comparative Examples 4-1 to 4-3, the former shows higher values ​​for the glucose-derived current signal under low voltage conditions, indicating greater sensitivity.

[0137] (5) Evaluation of glucose sensing characteristics of enzyme electrode (working electrode) by CV measurement [Example 5-1] In commercially available biosensors, RM and enzyme are pre-attached to the biosensor electrode to form an enzyme electrode. When the sample solution comes into contact with this enzyme electrode, the RM and enzyme dissolve from the electrode, and a chain-type electron transfer reaction between the sample, enzyme, RM, and electrode is carried out electrochemically in the sample solution to perform detection. An enzyme electrode was prepared by the following method and biosensing evaluation was performed.

[0138] <Preparation of RM Solution> A 100 mM 1,2-naphthoquinone-4-sulfonate sodium / PB solution was prepared by the following method and designated as RM solution E-4. 130 mg of 1,2-naphthoquinone-4-sulfonate sodium was mixed with 4.870 g of 100 mM PB and stirred to dissolve, obtaining RM solution E-4, which is a brown, transparent solution.

[0139] <Preparation of Enzyme Electrode> The biosensor electrode C-1 prepared in Example 2-1 was punched out to a diameter of 10 mm, and 4.0 μL of RM solution E-4 was dropped onto the center of the coated film side. It was left to stand on a hot plate at 50°C for 10 minutes to dry. 10.5 μL of enzyme GDH-1 was then dropped onto the top of the dried RM and dried on a hot plate at 40°C for 10 minutes to prepare enzyme electrode F-1. After drying, it was visually confirmed that the RM and enzyme were dry within a central diameter area of ​​5 mm.

[0140] <Preparation of the working electrode> An enzyme electrode F-1 was fixed inside a 5 mm diameter back-contact type sample holder AE9-2 (manufactured by EC Frontier Co., Ltd.) with the coated film surface facing the opening side, creating a working electrode with a diameter of 5 mm.

[0141] <Preparation of Sample Solution> Glucose was selected as the sample to be sensed, and sample solution G-1 was prepared by the following method. 720.6 mg of D-glucose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 100 mM PB, and then 100 mM PB was added until the volume reached 40 mL to prepare a 100 mM D-glucose / PB solution, which was then designated as sample solution G-1.

[0142] <Electrochemical Cell Setup and CV Measurement> RM and 6.0 mL of sample solution G-1 were added to a glass cell VB2-1 (manufactured by EC Frontier Co., Ltd.). Enzyme electrode F-1 was used as the working electrode, a platinum spiral electrode (manufactured by EC Frontier Co., Ltd., model CE-200) as the counter electrode, and an Ag / AgCl electrode (manufactured by EC Frontier Co., Ltd., model RE-T2A) as the reference electrode. These were fixed by immersion using the starter kit VB2-S, and after stirring and standing, CV measurement was performed under the following conditions: Initial voltage: -0.2V Voltage sweep range: -0.2V to 0.5V Scan speed: 0.01V / sec Voltage step: 0.001V Number of cycles: 2

[0143] The CV curves for the first and second cycles were almost identical, but the second cycle had a slightly lower current density due to the decrease in glucose concentration near the working electrode interface caused by the glucose oxidation reaction in the first cycle. Table 4 shows the current density at an Ag / AgCl ratio of 0.00 V and at 0.00–0.30 V in the first cycle.

[0144] [Comparative Example 5-1] Enzyme electrode F-2 was prepared using the same method as in Example 5-1, except that in the preparation of the enzyme electrode, a glassy carbon electrode (manufactured by EC Frontier Co., Ltd., 3 mm in diameter, model number GC-6355) was used instead of the biosensor electrode C-1, punching was not performed, and drying was carried out using an oven under the same temperature and time conditions. After drying, the RM and enzyme were visually confirmed to be dry on the GC electrode (GC part diameter 3 mm, diameter 6 mm including the outer resin part). Otherwise, CV measurement was performed using the same method as in Example 5-1. The results obtained under the same conditions are shown in Table 4.

[0145]

[0146] Table 4 shows that by using the biosensor electrode (enzyme electrode) of the present invention as the working electrode, it was confirmed that current derived from glucose sensing could be detected with an extremely high current density. This result confirms that a highly sensitive and high-performance glucose sensor can be fabricated using the biosensor electrode of the present invention. Furthermore, this method can be applied to biosensors used for other samples.

Claims

A composition for forming biosensor electrodes, comprising carbon nanotubes, an oxazoline group-containing polymer, a carboxyl group-containing polymer, and water. 　 The biosensor electrode formation composition according to claim 1, which is a homogeneous dispersion. 　 The biosensor electrode forming composition according to claim 1, wherein the carboxyl group-containing polymer is an ammonium polyacrylate salt or an amine salt. 　 Furthermore, the biosensor electrode formation composition according to claim 1 further comprises polyvinylpyrrolidone. 　 The biosensor electrode forming composition according to claim 4, wherein the weight-average molecular weight of the polyvinylpyrrolidone is 50,000 or less. 　 The biosensor electrode forming composition according to claim 1, wherein the carbon nanotube content is 30% by mass or more in the solid content. Furthermore, the biosensor electrode forming composition according to claim 1 further comprises at least one selected from the group consisting of ethanol and 2-propanol. 　 A thin film obtained from the biosensor electrode formation composition according to any one of claims 1 to 7. 　 The thin film according to claim 8, which is a water-resistant film. 　 The basis weight is 0.5 g / m 2 The thin film according to claim 8, which is as follows: 　 A biosensor electrode comprising the thin film described in claim 8. 　 The biosensor electrode according to claim 11, wherein the thin film further comprises a redox mediator. 　 The biosensor electrode according to claim 12, wherein the redox mediator comprises a quinone compound. 　 The biosensor electrode according to claim 11, wherein the thin film further comprises an enzyme. 　 The biosensor electrode according to claim 14, wherein the enzyme is glucose dehydrogenase. 　 The biosensor electrode according to claim 11, comprising a substrate and the thin film laminated on the substrate. 　 The biosensor electrode according to claim 16, wherein the substrate is a metal foil. 　 The biosensor electrode according to claim 17, wherein the substrate is aluminum foil. 　 A biosensor comprising the biosensor electrode according to claim 11. 　 A method for producing a biosensor electrode, comprising the steps of applying a biosensor electrode forming composition according to any one of claims 1 to 7 onto a substrate and heating and drying the resulting coating film. 　 A method for producing a biosensor electrode according to claim 20, comprising the step of applying a composition containing a redox mediator and a phosphate buffer onto the above-mentioned coating film and drying it.

Images