Enzyme electrode, method for producing enzyme electrode, biosensor, and biobattery

Abstract

An enzyme electrode 1 comprises an electrode base material 2, an oxidoreductase 3, a conjugate 6 of a silane coupling agent 4 and an electron mediator 5, and a sol-gel matrix 7. The oxidoreductase 3 and the conjugate 6 are fixed to the electrode base material 2 by the sol-gel matrix 7. The silane coupling agent 4 comprises a silicon atom, a reactive functional group and a hydrolyzable group, and has a structure in which the silicon atom and the reactive functional group are connected to each other by a linking group that has 4 or more carbon atoms.

Description

Enzyme electrode, method for manufacturing enzyme electrode, biosensor, and biobattery

[0001] The present invention relates to an enzyme electrode, a method for manufacturing an enzyme electrode, a biosensor, and a biobattery.

[0002] In recent years, with growing interest in energy issues, biobatteries that use bio-related substances such as sugars and alcohols as fuel have been attracting attention. Biobatteries use enzymes as electrode catalysts and can generate electricity by combining an oxidation reaction of biofuel at the anode and a reduction reaction of oxygen or the like at the cathode. Furthermore, for example, biosensors using enzyme electrodes are used in various fields, such as the medical field and clinical testing field, to measure target substances in biological samples. A redox reaction occurs between the target substance and an oxidative-reductase contained in the enzyme electrode, and the target substance, and the resulting generated current can be detected to perform the measurement.

[0003] In enzyme electrodes for biobatteries, biosensors, and other devices, a method using a substance called an electron mediator is known to efficiently transfer electrons. Furthermore, to further improve the efficiency of electron transfer, techniques have been developed for immobilizing enzymes and small molecules such as electron mediators and indicators on electrodes. The sol-gel method is known as one method for immobilizing enzymes and other small molecules, and involves comprehensively immobilizing molecules by encapsulating them in a metal oxide matrix formed in a liquid.

[0004] Regarding enzyme immobilization methods, Patent Document 1 discloses a method for immobilizing an enzyme, which comprises immobilizing an enzyme on a structural unit having an inner diameter 1.2 times or more the diameter of the enzyme and structural stability, and then forming a network structure of a gelling substance by a sol-gel method in the openings and / or internal voids of the structural unit to improve the stability of the immobilized enzyme. Patent Documents 2 and 3 disclose techniques for co-encapsulating small molecules such as electron mediators and indicators with enzymes for the purpose of sensing.

[0005] Although the techniques described in Patent Documents 1 to 3 can suppress leakage of enzymes, they have the problem of small molecules such as electron mediators leaking from the gel matrix. To address this problem, Non-Patent Document 1 discloses a method for preventing leakage of electron mediators by covalently bonding a small molecule electron mediator to a sol-gel matrix. Non-Patent Document 2 discloses making the sol-gel matrix hydrophobic to improve the stability of immobilized enzymes.

[0006] Japanese Patent Application Laid-Open No. 2001-178457 Special Publication No. 2005-529309 Special Publication No. 2006-512573

[0007] Z. Wang et al., Biosens. Bioelectron., 2012, Vol. 32, pp. 111-117D Weiser et al., Green Chem., 2017, Vol. 19, pp. 3927-3937

[0008] As described above, various techniques for immobilizing enzymes and the like have been disclosed in the past, but enzyme electrodes using conventional immobilization methods are insufficient in terms of electrocatalytic function, and there is room for improvement.

[0009] The present invention has been made in view of the above-mentioned current situation, and has as its object to provide an enzyme electrode having a superior electrocatalytic function to conventional enzyme electrodes.

[0010] The present inventors have conducted extensive research to solve the above problems, and have found that, in an enzyme electrode using an oxidoreductase and an electron mediator, excellent electrode catalytic function can be achieved by fixing a conjugate formed by bonding the electron mediator to a silane coupling agent having a structure in which a silicon atom and a reactive functional group are linked by a linking group having 4 or more carbon atoms to an electrode substrate using a sol-gel matrix. They have come to the realization that the above problems can be solved beautifully, and have arrived at the present invention.

[0011] The present invention provides an enzyme-containing electrode, which comprises an electrode substrate, an oxidoreductase, a conjugate of a silane coupling agent and an electron mediator, and a sol-gel matrix, wherein the oxidoreductase and the conjugate are fixed to the electrode substrate by the sol-gel matrix, and the silane coupling agent has a silicon atom, a reactive functional group, and a hydrolyzable group, and the silicon atom and the reactive functional group are linked by a linking group having 4 or more carbon atoms.

[0012] The enzyme electrode of the present invention has the above-mentioned structure and has excellent electrocatalytic function, and therefore can be suitably used in biobatteries, biosensors, etc.

[0013] Fig. 1 is a schematic diagram of one embodiment of the enzyme electrode of the present invention. Fig. 2 is a schematic diagram showing the interaction between a silane coupling agent and an oxidoreductase in one embodiment of the enzyme electrode of the present invention. Fig. 3 is a diagram showing voltammograms in cyclic voltammetry (CV) measurements using the enzyme electrodes obtained in Example 1 and Comparative Example 1. Fig. 4 is a diagram showing voltammograms in cyclic voltammetry (CV) measurements using the enzyme electrode obtained in Example 2. Fig. 5 is a diagram showing voltammograms in cyclic voltammetry (CV) measurements using the enzyme electrode obtained in Example 3. Fig. 6 is a diagram showing the results of SAXS analysis of the compositions applied to the electrode surfaces of the enzyme electrodes obtained in Examples 2 and 3.

[0014] The enzyme electrode, the method for manufacturing the enzyme electrode, the biosensor, and the biobattery of the present invention will be described below. However, the present invention is not limited to the following configurations, and can be appropriately modified and applied within the scope of the present invention. Note that a combination of two or more of the individual preferred configurations of the present invention described below also constitutes the present invention.

[0015] [Enzyme Electrode] The enzyme electrode of the present invention comprises an electrode substrate, an oxidoreductase, a conjugate of a silane coupling agent and an electron mediator, and a sol-gel matrix. The oxidoreductase and the conjugate are immobilized to the electrode substrate by the sol-gel matrix. The silane coupling agent has a silicon atom, a reactive functional group, and a hydrolyzable group, and the silicon atom and the reactive functional group are linked by a linking group having 4 or more carbon atoms. For example, Non-Patent Document 1 discloses a technique for covalently linking an electron mediator to a sol-gel matrix. However, although leakage of the electron mediator out of the matrix is ​​suppressed, the range of motion of the electron mediator is limited, resulting in poor reactivity. In contrast, in the present invention, the silane coupling agent has a long-chain linking group as described above, so that the electron mediator has a wide range of motion and is easily mobile even when immobilized by the sol-gel matrix, thereby enabling efficient electron transfer between enzymes and / or between enzymes and electrodes. This improves catalytic current, and when such an electrode is used in a battery, sensor, or the like, improves output.

[0016] The content of the conjugate of the silane coupling agent and the electron mediator in the enzyme electrode is preferably 1,000 mol % or more and 10,000,000 mol % or less, more preferably 10,000 mol % or more and 1,000,000 mol % or less, and even more preferably 100,000 mol % or more and 1,000,000 mol % or less, relative to 100 mol % of the oxidoreductase.

[0017] <Conjugate of silane coupling agent and electron mediator> The conjugate of the silane coupling agent and electron mediator is not particularly limited as long as the silane coupling agent and the electron mediator are bonded together, but it is preferable that the reactive functional group in the silane coupling agent and the reactive functional group in the electron mediator are bonded together via a covalent bond.

[0018] (Silane Coupling Agent) The silane coupling agent is not particularly limited as long as it has a structure having a silicon atom, a reactive functional group, and a hydrolyzable group, and the silicon atom and the reactive functional group are linked by a linking group having 4 or more carbon atoms.

[0019] The reactive functional group of the silane coupling agent is not particularly limited as long as it can bond with the electron mediator, and examples thereof include those that interact with each other through intermolecular force, hydrogen bond, Coulomb force, etc., and those that can form covalent bonds, but those that can form covalent bonds are preferred in terms of bond strength.Specific examples of the reactive functional group that can form covalent bonds include functional groups or reactive groups such as amino groups, sulfonic acid groups, sulfate groups, phosphate groups, sulfhydryl groups, carboxyl groups, and salt groups thereof, epoxy groups, thiol groups, hydroxyl groups, polymerizable unsaturated groups, azido groups, azo groups, nitro groups, nitrile groups, cyano groups, allene groups, isonitrile groups, urea groups, aldehyde groups, ketone groups, halogen groups, NHS esters, imide esters, maleimides, pyridyldithiols, allyl azides, haloacetates, isocyanates, carbodiimides, allyl azides, diazirines, hydrazides, psoralens, pyridine disulfides, and vinyl sulfones. Of these, preferred are amino groups, epoxy groups, etc. The silane coupling agent may have one or more of the reactive functional groups.

[0020] The silane coupling agent has a linking group having 4 or more carbon atoms that links the silicon atom and the reactive functional group. The linking group is not particularly limited as long as it is an organic group having 4 or more carbon atoms, but is preferably a hydrocarbon group having 4 to 30 carbon atoms, which may contain a heteroatom. The linking group having such a structure becomes hydrophobic, and can undergo hydrophobic interaction with the hydrophobic portion of the enzyme, stabilizing the enzyme. This results in excellent durability when the enzyme electrode of the present invention is used as a battery or sensor. Furthermore, when the linking group has a long-chain hydrophobic group having 4 or more carbon atoms, hydration water near the active site of the enzyme is eliminated, further improving the electron transfer reaction rate between the enzyme and the electron mediator.

[0021] The hydrocarbon group is not particularly limited, but is preferably a group obtained by removing one or more hydrogen atoms from a linear or branched alkyl group, an alkenyl group, an alkynyl group, an aryl group, etc. Examples of the linear or branched alkyl group include a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-pentyl group (amyl group), an n-hexyl group, an n-heptyl group, an n-octyl group, an n-nonyl group, an n-decyl group, an n-undecyl group, an n-dodecyl group, an n-tridecyl group, an n-tetradecyl group, an n-pentadecyl group, an n-hexadecyl group, an n-heptadecyl group, an n-octadecyl group, an n-nonadecyl group, an n-eicosanyl group, an i-propyl group, a sec-butyl group, an i-butyl group, a t-butyl group, a 1-methylbutyl group, a 1-ethylpropyl group, a 2-methyl ... Examples of alkyl groups include 1-methylbutyl group, i-amyl group, neopentyl group, 1,2-dimethylpropyl group, 1,1-dimethylpropyl group, t-amyl group, 1,3-dimethylbutyl group, 3,3-dimethylbutyl group, 2-ethylbutyl group, 2-ethyl-2-methylpropyl group, 1-methylheptyl group, 2-ethylhexyl group, 1,5-dimethylhexyl group, t-octyl group, branched nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, stearyl group, and icosyl group.

[0022] Examples of the alkenyl group include vinyl, allyl, 1-butenyl, 2-butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, dodecenyl, octadecenyl, and icosenyl groups. Examples of the alkynyl group include ethynyl, 1-propynyl, 2-propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, dodecynyl, octadecenyl, and icosenyl groups.

[0023] Examples of the aryl group include a phenyl group, a naphthyl group, a benzyl group, a methylphenyl group, an ethylphenyl group, a propylphenyl group, a butylphenyl group, a butylmethylphenyl group, a dimethylphenyl group, a diethylphenyl group, a dibutylphenyl group, a biphenyl group, a methylbiphenyl group, an ethylbiphenyl group, a methylnaphthyl group, an ethylnaphthyl group, and a cinnamyl group (Ph-CH=CHCH ２ - group), aralkyl groups such as 1-benzocyclobutenyl group, and the like.

[0024] The hydrocarbon group preferably has 5 or more and 25 or less carbon atoms, more preferably 6 or more and 20 or less carbon atoms, even more preferably 7 or more and 18 or less carbon atoms, and particularly preferably 8 or more and 15 or less carbon atoms.

[0025] The hydrocarbon group may have a heteroatom such as a nitrogen atom, a sulfur atom, an oxygen atom, a phosphorus atom, or a halogen atom, or may have a substituent containing a heteroatom such as a hydroxyl group, an alkoxy group, a carboxyl group, an acyl group, a sulfonic acid group, an amino group, or a phosphate group.

[0026] The silane coupling agent preferably has a positive or negative charge, which allows the enzyme to be more stabilized and the enzyme activity to be maintained for a longer period of time through electrostatic interaction with the positively or negatively charged site on the enzyme surface.

[0027] The silane coupling agent preferably has a functional group having a positive or negative charge, and the functional group having a positive or negative charge may be present in any part of the silane coupling agent. The silane coupling agent preferably has a positive or negative charge in the linking group and / or reactive functional group. When the reactive functional group has a positive or negative charge, it is sufficient that the structure after bonding with the electron mediator has a positive or negative charge. When the reactive functional group in the silane coupling agent has a positive or negative charge and the linking group has a hydrocarbon group with 4 or more carbon atoms, the enzyme can be further stabilized by electrostatic interaction and hydrophobic interaction.

[0028] Examples of the positively charged functional group include primary to tertiary amino groups, quaternary ammonium groups, etc. Examples of the negatively charged functional group include epoxy groups, ether groups, carboxyl groups, sulfonic acid groups, sulfate groups, phosphate groups, thiol groups, halogen groups, etc.

[0029] The silane coupling agent preferably has a structure in which a hydrolyzable group such as an alkoxy group is bonded to a silicon atom, and more preferably has a structure represented by the following formula (1): —Si(R １ ) ３－ｎ (OR ２ ) ｎ (1) (wherein, R １ , R ２ are the same or different and represent a hydrocarbon group having 1 to 5 carbon atoms. n represents an integer of 1 to 3. n is an integer of 1 to 3, preferably 2 or 3, and more preferably 3. R in the above formula (1) １ , R ２ The hydrocarbon group in R is not particularly limited, but examples thereof include alkyl groups, alkenyl groups, and aryl groups. Specific examples of these groups include those described above for the linking group. １ , R ２ The number of carbon atoms in the hydrocarbon group in R is preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2. １ , R ２ The hydrocarbon group in is preferably an alkyl group, more preferably a methyl group, an ethyl group, an n-propyl group, or an n-butyl group, and even more preferably a methyl group or an ethyl group.

[0030] Examples of the silane coupling agent include N-2-(aminoethyl)-8-aminooctyltrimethoxysilane, N-2-(aminoethyl)-8-aminooctylmethyldimethoxysilane, N-2-(aminoethyl)-8-aminooctyldimethylmethoxysilane, N-2-(aminoethyl)-8-aminooctyltriethoxysilane, N-β-(aminoethyl)-γ-aminopropyltrimethoxysilane, N-β-(aminoethyl)-γ-aminopropylmethyldimethoxysilane, N-β-(aminoethyl)-γ-aminopropyltriethoxysilane, and N-β-(aminoethyl) amino group-containing silane coupling agents such as -γ-aminopropylmethyldiethoxysilane, N-β-(aminoethyl)-γ-aminopropyltriisopropoxysilane, 4-aminobutyltrimethoxysilane, 4-aminobutyltriethoxysilane, 5-aminopentyltrimethoxysilane, 5-aminopentyltriethoxysilane, 6-aminohexyltrimethoxysilane, 6-aminohexyltriethoxysilane, 7-aminoheptyltrimethoxysilane, 7-aminoheptyltriethoxysilane, 8-aminooctyltrimethoxysilane, and 8-aminooctyltriethoxysilane;8-glycidoxyoctyltrimethoxysilane, 8-glycidoxyoctylmethyldimethoxysilane, 8-glycidoxyoctylmethyldiethoxysilane, 8-glycidoxyoctyltriethoxysilane, 7-glycidoxyheptyltrimethoxysilane, 7-glycidoxyheptylmethyldimethoxysilane, 7-glycidoxyheptylmethyldiethoxysilane, 7-glycidoxyheptyltriethoxysilane, 6-glycidoxyhexyltrimethoxysilane, 6-glycidoxyhexylmethyldimethoxysilane, 6-glycidoxyhexylmethyldiethoxysilane, 6-glycidoxyhexyltriethoxysilane, 5-glycidoxy Epoxy group-containing silane coupling agents such as pentyltrimethoxysilane, 5-glycidoxypentylmethyldiethoxysilane, 5-glycidoxypentylmethyldiethoxysilane, 5-glycidoxypentyltriethoxysilane, 4-glycidoxybutyltrimethoxysilane, 4-glycidoxybutylmethyldimethoxysilane, 4-glycidoxybutylmethyldiethoxysilane, 4-glycidoxybutyltriethoxysilane, γ-glycidoxypropyl(ethyl)dimethoxysilane, β-3,4-epoxycyclohexylethyltrimethoxysilane, and β-3,4-epoxycyclohexylethyltriethoxysilane are included;

[0031] (Electron Mediator) In the enzyme electrode of the present invention, the electron mediator bound to the silane coupling agent is not particularly limited as long as it donates and receives electrons to and from the oxidoreductase contained in the enzyme electrode of the present invention and can bond with the reactive functional group in the silane coupling agent, but is preferably one having a functional group that forms a bond with the reactive functional group of the silane coupling agent, such as an amino group, a carboxyl group, or an aldehyde group.

[0032] Examples of electron mediators include coenzymes of oxidoreductases and electron carriers other than coenzymes. Specific examples of coenzymes of oxidoreductases include vitamin coenzymes such as nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, and flavin mononucleotide; and quinone coenzymes such as pyrrolopyrroline quinone, topaquinone, tryptophan-tryptophylquinone, lysine tyrosylquinone, and cysteinyl-tryptophan quinone. In this specification, the oxidized form of nicotinamide adenine dinucleotide is also referred to as NAD, and its reduced form is also referred to as NADH, and the oxidized form of nicotinamide adenine dinucleotide phosphate is also referred to as NADP, and its reduced form is also referred to as NADPH. Furthermore, NAD(P)H means NADH or NADPH, and NAD(P) means NAD or NADP. Furthermore, NAD is NAD ＋ , NADP is NADP ＋ means.

[0033] Examples of electron carriers other than the coenzymes include metal complexes having a metal element such as Os, Fe, Ru, Co, Cu, Ni, V, Mo, Cr, Mn, Pt, or W, or an ion of such a metal as the central metal (alkali metal ferricyanides such as ferrocene, potassium ferricyanide, lithium ferricyanide, or sodium ferricyanide, or alkyl-substituted derivatives thereof (methyl-substituted, ethyl-substituted, propyl-substituted, or the like), potassium octacyanotungstate, etc.); quinones such as quinone, benzoquinone, anthraquinone, naphthoquinone, and aminonaphthoquinone; heterocyclic compounds such as toluidine blue, methylene blue, viologen, methyl viologen, benzyl viologen, phenazine methosulfate, phenazine ethosulfate, bipyridine, or a derivative thereof; and other examples include 2,6-dichlorophenolindophenol, methylene blue, and potassium β-naphthoquinone-4-sulfonate.

[0034] The metal complex is preferably a metal complex having iron as the central metal, and more preferably a ferrocene such as aminoferrocene or ferrocene carboxaldehyde.

[0035] When the enzyme electrode of the present invention contains a substrate oxidoreductase (A) described below, it is preferable to use an electron mediator (a) that is a coenzyme of the oxidoreductase (A). When the coenzyme of the substrate oxidoreductase (A) is NAD(P), that is, when the enzyme electrode of the present invention contains an NAD(P)-dependent oxidoreductase, it is preferable to use NAD(P) as the electron mediator (a).

[0036] When the enzyme electrode of the present invention further comprises an enzyme (B) that receives electrons from an electron mediator (a) such as a coenzyme and transfers the received electrons to the electrode substrate, or transfers electrons received from the electrode substrate to the electron mediator (a), it is preferable to use an electron mediator (b) that mediates electron transfer between the enzyme (B) and the electrode substrate. A preferred embodiment of the present invention is one in which the electron mediator comprises NAD(P)H and / or NAD(P) as the electron mediator (a) and an electron mediator other than NAD(P)H and NAD(P) as the electron mediator (b). The electron mediator (b) is not particularly limited, but is preferably a quinone, a ferrocene, or a heterocyclic compound having a phenothiazine skeleton such as toluidine blue. A quinone such as aminonaphthoquinone is more preferred.

[0037] A preferred embodiment of the present invention is one in which the enzyme electrode comprises a conjugate of a silane coupling agent and an electron mediator (a) such as a coenzyme (hereinafter also referred to as conjugate (α)), and a conjugate of a silane coupling agent and an electron mediator (b) (hereinafter also referred to as conjugate (β)).

[0038] <Oxidoreductase> The oxidoreductase is not particularly limited as long as it donates and receives electrons to and from an electron mediator, but the enzyme electrode of the present invention preferably contains an enzyme (A) (hereinafter also referred to as oxidoreductase (A) or simply as enzyme (A)) that oxidizes and / or reduces a substrate described below and donates and receives electrons to and from an electron mediator. The enzyme (A) is more preferably an oxidase of the substrate. More preferably, it is an NAD(P)-dependent oxidase (hereinafter also referred to as NAD(P)-dependent oxidase) that donates and receives electrons to and from NAD(P)H or NAD(P).

[0039] Preferred examples of the oxidase include glycerol dehydrogenase, glucose dehydrogenase, a series of enzymes in the electron transport system, ATP synthase, and enzymes involved in sugar metabolism (e.g., hexokinase, glucose phosphate isomerase, phosphofructokinase, fructose bisphosphate aldolase, triose phosphate isomerase, glyceraldehyde phosphate dehydrogenase, phosphoglycerate mutase, phosphopyruvate hydratase, pyruvate kinase, L-lactate dehydrogenase, D-lactate dehydrogenase, pyruvate dehydrogenase, citrate synthase, aconitase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, and malonate dehydrogenase). One or more of these may be used. Among these, glycerol dehydrogenase and glucose dehydrogenase are preferred.

[0040] The enzyme electrode of the present invention preferably further comprises an enzyme (B) (hereinafter also referred to as oxidoreductase (B) or simply enzyme (B)) that receives electrons from the electron mediator (a) and transfers the received electrons to the electrode substrate, or transfers electrons received from the electrode substrate to the electron mediator (a). The enzyme electrode of the present invention preferably comprises diaphorase as the enzyme (B). Diaphorase is an enzyme that catalyzes the oxidation-reduction reaction of an NAD(P) redox pair and can donate and receive electrons to and from the electron mediator (b) or the electrode surface. When the enzyme electrode of the present invention comprises an NAD(P)-dependent oxidase and diaphorase, electrons generated by oxidation of the substrate are received by NAD(P), and NAD(P) becomes NAD(P)H. The NAD(P)H transfers electrons to diaphorase, which then transfers electrons to the electron mediator (b), which then transfers electrons to the electrode substrate, thereby transferring the electrons extracted from the substrate to the electrode substrate.

[0041] When the oxidoreductase contains the oxidoreductase (A) and the oxidoreductase (B), the content of the oxidoreductase (B) is preferably 10 mol % or more and 1000 mol % or less, more preferably 20 mol % or more and 500 mol % or less, and even more preferably 30 mol % or more and 100 mol % or less, relative to 100 mol % of the oxidoreductase (A).

[0042] The enzyme electrode preferably contains a conjugate (α), a conjugate (β), an enzyme (A), and an enzyme (B). In this case, the content of the conjugate (α) is not particularly limited, but is preferably 1,000 mol% or more and 10,000,000 mol% or less relative to 100 mol% of the enzyme (A). It is more preferably 10,000 mol% or more and 1,000,000 mol%, and even more preferably 100,000 mol% or more and 1,000,000 mol% or less. The content of the conjugate (β) is also not particularly limited, but is preferably 1,000 mol% or more and 10,000,000 mol% or less relative to 100 mol% of the enzyme (B). It is more preferably 10,000 mol% or more and 1,000,000 mol%, and even more preferably 100,000 mol% or more and 1,000,000 mol% or less.

[0043] A preferred embodiment of the present invention is also one in which the enzyme electrode comprises the conjugate (α), the conjugate (β), an NAD-dependent oxidase, and diaphorase.

[0044] <Sol-gel matrix> The enzyme electrode of the present invention contains a sol-gel matrix. The sol-gel matrix is ​​not particularly limited as long as it can immobilize the oxidoreductase and the bond of the silane coupling agent and the electron mediator on the electrode substrate, but it is preferable that it contains a silane compound. The silane compound preferably has a structure in which a hydrolyzable group such as an alkoxy group is bonded to a silicon atom, and more preferably has a structure represented by the following formula (2): Si(R ３ ) ４－ｍ (OR ４ ) ｍ (2) (wherein, R 3 , R 4are the same or different and represent a hydrocarbon group having 1 to 5 carbon atoms; and m represents an integer of 1 to 4.

[0045] R in the above formula (2) ３ , R ４ The hydrocarbon group in R is not particularly limited, but examples thereof include alkyl groups, alkenyl groups, and aryl groups. Specific examples of these groups include those described above for the linking group. ３ , R ４ The number of carbon atoms in the hydrocarbon group in R is preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2. ３ , R ４ The hydrocarbon group in is preferably an alkyl group, more preferably a methyl group, an ethyl group, an n-propyl group, or an n-butyl group, and even more preferably a methyl group or an ethyl group.

[0046] Specific examples of compounds represented by the above formula (2) include tetrafunctional alkoxysilanes having four alkoxy groups, such as tetramethoxysilane, tetraethoxysilane, tetraisopropoxysilane, tetrabutoxysilane, and dimethoxydiethoxysilane; trifunctional alkoxysilanes having three alkoxy groups, such as methyltrimethoxysilane, methyltriethoxysilane, ethyltrimethoxysilane, and ethyltriethoxysilane; difunctional alkoxysilanes having two alkoxy groups, such as dimethyldimethoxysilane, dimethyldiethoxysilane, diethyldimethoxysilane, and diethyldiethoxysilane; and monofunctional alkoxysilanes having one alkoxy group, such as trimethylmethoxysilane, trimethylethoxysilane, triethylmethoxysilane, and triethylethoxysilane. These species or two or more species can be used. The molecular structure of the silane compound can be determined by collecting a gel applied to the electrode surface, dissolving it in deuterated sodium hydroxide, and then performing NMR (Si, H) measurement.

[0047] The sol-gel matrix preferably contains a curing catalyst to promote the hydrolysis and polycondensation reaction of the silane compound. The curing catalyst is not particularly limited, but examples include basic catalysts and acidic catalysts. Examples of basic catalysts include amines such as polyethyleneimine, N,N-diethylethanolamine, N,N-dimethylethanolamine, triethanolamine, triethylamine, and 3-morpholinopropylamine. Examples of acidic catalysts include hydrogen halides such as hydrochloric acid, nitric acid, sulfuric acid, sulfurous acid, hydrogen sulfide, perchloric acid, hydrogen peroxide, carbonic acid, and carboxylic acids such as formic acid and acetic acid. The curing catalyst is preferably a basic catalyst, more preferably an amine, and even more preferably polyethyleneimine.

[0048] The sol-gel matrix preferably further contains a photocurable material, which allows the sol-gel matrix to be cured more sufficiently. The photocurable material is not particularly limited, but examples thereof include polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, tripropylene glycol triacrylate, bispentaerythritol hexaacrylate, ethylene glycol diacrylate, 1,3-butylene glycol di(meth)acrylate, 1,4-butanediol oligoacrylate, diethylene glycol diacrylate, 1,6-hexanediol oligoacrylate, neopentyl glycol diacrylate, triethylene glycol diacrylate, tripropylene glycol diacrylate, dipropylene glycol diacrylate, cyclohexanedimethanol diacrylate, tricyclodecanedimethanol diacrylate, bisphenol A polyethoxydiacrylate, bisphenol F polyethoxydiacrylate, pentaerythritol tetraacrylate, propoxylated (2) neopentyl glycol diacrylate, and the like. Glycol diacrylate, trimethylolpropane triacrylate, tris(2-hydroxyethyl)isocyanurate triacrylate, pentaerythritol triacrylate, ethoxylated (3) trimethylolpropane triacrylate, propoxylated (3) glyceryl triacrylate, pentaerythritol tetraacrylate, ditrimethylolpropane tetraacrylate, ethoxylated (4) pentaerythritol tetraacrylate, dipentaerythritol pentaacrylate, 2-(2-ethoxyethoxy)ethyl acrylate, hexadiol diacrylate, 1,6-hexanediol diacrylate, ethoxylated 1,6-hexanediol diacrylate, polypropylene glycol diacrylate, 1,4-butanediol diacrylate, 1,9-nonanediol diacrylate, tetraethylene glycol diacrylate, 2-n-butyl-2-ethyl-1,3-propanediol diacrylate, hydroxypivalic acid neopentyl glycol diacrylate, hydroxypivalic acid trimethylolpropane triacrylate, ethoxylated phosphoric acid triacrylate, ethoxylated tripropylene glycol diacrylate, neopentyl glycol modified trimethylolpropane diacrylate, stearic acid modified pentaerythritol diacrylate, tetramethylolpropane triacrylate, tetramethylolmethane triacrylate, caprolactone modified trimethylolpropane triacrylate, propoxylated glyceryl triacrylate, tetramethylolmethane tetraacrylate, ethoxylated pentaerythritol tetraacrylate, dipentaerythritol hexaacrylate, caprolactone Examples of suitable acrylic compounds include polyfunctional acrylic compounds such as methacrylic acid-modified dipentaerythritol hexaacrylate, dipentaerythritol hydroxypentaacrylate, neopentyl glycol oligoacrylate, trimethylolpropane oligoacrylate, pentaerythritol oligoacrylate, ethoxylated neopentyl glycol di(meth)acrylate, propoxylated neopentyl glycol di(meth)acrylate, tripropylene glycol di(meth)acrylate, ethoxylated trimethylolpropane triacrylate, propoxylated trimethylolpropane triacrylate, and 2-(2-vinyloxyethoxy)ethyl acrylate; and silane coupling agents having functional groups such as acrylic groups, methacrylic groups, vinyl groups, epoxy groups, and thiol groups. These may be used alone or in combination. Among these, polyfunctional acrylic compounds are preferred, and polyethylene glycol diacrylate and polyethylene glycol dimethacrylate are even more preferred.

[0049] The content of the photocurable material in the sol-gel matrix is ​​not particularly limited, but is preferably 0.1% by mass or more and 50% by mass or less, more preferably 0.5% by mass or more and 25% by mass or less, and even more preferably 1% by mass or more and 10% by mass or less, relative to 100% by mass of the silane compound.

[0050] The sol-gel matrix preferably further contains a photopolymerization initiator. This allows the sol-gel matrix to be cured more efficiently. The photopolymerization initiator is not particularly limited, but examples include acylphosphine oxide compounds such as bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide, 2,4,6-trimethylbenzoyldiphenylphosphine oxide, benzoyldiphenylphosphine oxide, benzoyldiethoxyphosphine oxide, 2,4,6-trimethylbenzoyldiethoxyphenylphosphine oxide, and phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide. One or more of these compounds can be used. Among these, acylphosphine oxide compounds are preferred, and phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide is more preferred.

[0051] The content of the photopolymerization initiator in the sol-gel matrix is ​​not particularly limited, but is preferably 0.1% by mass or more and 10% by mass or less, more preferably 0.2% by mass or more and 5% by mass or less, and even more preferably 0.3% by mass or more and 3% by mass or less, relative to 100% by mass of the photocurable material.

[0052] <Electrode substrate> The electrode substrate of the enzyme electrode of the present invention is a conductive substrate that can be connected to an external circuit and can transfer electrons. The electrode substrate is not particularly limited in material or shape as long as it has the relevant properties. The material of the electrode substrate may be a conductive material, for example, carbon materials such as carbon cloth, carbon paper, graphite, glassy carbon, activated carbon, carbon black, and carbon nanotubes; metals or alloys such as gold, platinum, copper, palladium, titanium, aluminum, silver, and nickel; SnO ２ , ITO, In ２ O ３ , W.O. ３ , TiO ２Examples of the conductive oxide include those mentioned above. The electrode substrate may be composed of a single layer of one of these materials, or may be composed of a laminated structure of two or more layers. The conductive material is preferably a carbon material. Examples of the carbon material include glassy carbon, activated carbon, carbon black, and carbon nanotubes.

[0053] In the electrode substrate, for example, when two or more of the conductive materials are used, a binder such as a polymer may be used. The polymer is not particularly limited, but may be a fluorine-containing polymer such as polyvinylidene fluoride (PVDF) or polyvinyl fluoride (PVF), a copolymer thereof, or a copolymer of these monomers with ethylene or styrene. Further examples include polymers such as polystyrene, polyethylene, and polypropylene, as well as hydrophilic polymers such as polyacrylic acid, polylysine, and carboxymethyl cellulose, and conductive polymers such as polyaniline, polypyrrole, and their derivatives, polyaniline sulfonic acid.

[0054] The electrode substrate may have a flat surface or may have irregularities or pores on the surface, but preferably has pores. The size of the pores is not particularly limited, but is preferably 0.1 nm or more and 100 nm or less, and more preferably 1 nm or more and 50 nm or less.

[0055] Hereinafter, an embodiment of the enzyme electrode of the present invention will be described with reference to the drawings.

[0056] 1 is a schematic diagram of one embodiment of the enzyme electrode of the present invention. In the enzyme electrode 1, an oxidoreductase (a) 3a, an oxidoreductase (b) 3b, a conjugate 6a of an electron mediator (a) 5a and a silane coupling agent, and a conjugate 6b of an electron mediator (b) 5b and a silane coupling agent are immobilized on an electrode substrate 2. When the enzyme electrode 1 of the present invention is an anode, the electron mediator (a) 5a receives electrons generated by oxidation of a substrate 8 by the oxidoreductase (a) 3a and transfers the received electrons to the oxidoreductase (b) 3b, which transfers the electrons to the electron mediator (b) 5b, which transfers the electrons to the electrode substrate 2, thereby transferring the electrons extracted from the substrate 8 to the electrode substrate 2.

[0057] 2 is a schematic diagram showing the interaction between a silane coupling agent and an oxidoreductase in one embodiment of the enzyme electrode of the present invention. When the silane coupling agent has a hydrocarbon group with 4 or more carbon atoms in the linking group, the linking group forms a hydrophobic interaction with the hydrophobic portion of the oxidoreductase, thereby stabilizing the oxidoreductase. Furthermore, when the silane coupling agent has a positive or negative charge in the reactive functional group, it forms an electrostatic interaction with the negatively or positively charged portion on the surface of the oxidoreductase, thereby stabilizing the oxidoreductase.

[0058] [Method for producing enzyme electrode] The method for producing the enzyme electrode of the present invention is not particularly limited, and it is preferable to produce the enzyme electrode by immobilizing an oxidoreductase and a conjugate of a silane coupling agent and an electron mediator on an electrode substrate using a sol-gel matrix. That is, a method for producing an enzyme electrode including an immobilization step of immobilizing an oxidoreductase and a conjugate of a silane coupling agent and an electron mediator on an electrode substrate using a sol-gel matrix also constitutes one aspect of the present invention. Specific examples and preferred forms of the electrode substrate, oxidoreductase, conjugate of a silane coupling agent and an electron mediator, and sol-gel matrix are as described above.

[0059] The immobilization step is not particularly limited as long as it immobilizes the oxidoreductase, the conjugate of the silane coupling agent and the electron mediator to the electrode substrate using a sol-gel matrix, but can be carried out by, for example, applying a composition containing the oxidoreductase, the conjugate of the silane coupling agent and the electron mediator, and a sol-gel matrix material to the electrode substrate and drying it. The application method may be any commonly used method, such as spin coating, spraying, screen coating, dip coating, or blade coating.

[0060] The sol-gel matrix material used in the immobilization step is not particularly limited, but preferably contains a silane compound, specific examples and preferred forms of which are as described above.

[0061] The composition used in the immobilization step preferably contains a solvent and a buffer component. Examples of the solvent include aqueous solvents such as water and ethanol. Water is preferred. Examples of the buffer component include phosphates such as potassium phosphate and sodium phosphate, imidazole, carbonates, borates, tartrates, citrates, tris(hydroxymethyl)aminomethane (TRIS), 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES), and 3-morpholinopropanesulfonic acid (MOPS).

[0062] The content ratios of the oxidoreductase, the conjugate of the silane coupling agent and the electron mediator, and the sol-gel matrix material in the composition used in the immobilization step can be adjusted appropriately according to the ratios of these in the enzyme electrode.

[0063] The composition used in the fixing step preferably contains a photocurable material. The composition preferably further contains a photopolymerization initiator. Specific examples and preferred examples of the photocurable material and photopolymerization initiator are as described above. The preferred ranges of the amounts of the photocurable material and photopolymerization initiator used are the same as the content ratios in the sol-gel matrix described above.

[0064] When the photocurable material and photopolymerization initiator are used in the fixing step, it is preferable to apply the composition to an electrode substrate, dry it, and then perform a light irradiation step. The light irradiation method can be performed by a commonly used method. The light irradiation conditions vary depending on the energy ray used. For example, when curing is performed by ultraviolet irradiation, the ultraviolet irradiation dose is 10 mJ / cm. ２ Above, 3,000mJ / cm ２ Hereinafter, the irradiation time is preferably 1 second or more and 180 seconds or less.

[0065] [Biosensor] The present invention also relates to a biosensor equipped with the enzyme electrode of the present invention. The biosensor of the present invention preferably includes the enzyme electrode of the present invention as a working electrode and a counter electrode. Measurement using the biosensor is performed by bringing a measurement sample into contact with the biosensor, causing an oxidation-reduction reaction between the oxidoreductase contained in the enzyme electrode of the present invention and the substance to be measured, and detecting the resulting current. The presence or absence or concentration of a substrate in a sample can be determined based on the response current value.

[0066] Measurement methods using the biosensor of the present invention include commonly used methods such as chronoamperometry, which measures oxidation current or reduction current, or coulometry and cyclic voltammetry. [Biobattery] The present invention also relates to a biobattery equipped with the enzyme electrode of the present invention. In the biobattery, the enzyme electrode of the present invention is preferably an anode. The biobattery of the present invention is not particularly limited as long as it is equipped with the enzyme electrode of the present invention and the anode and cathode are connected by an external circuit, but it is preferable that the biobattery includes a diaphragm separating the anode and cathode.

[0067] In one embodiment, the biobattery of the present invention preferably comprises an anode made of the enzyme electrode of the present invention, a cathode, and a diaphragm separating the anode and the cathode.

[0068] The cathode in the biobattery of one embodiment of the present invention can be an enzyme catalyst such as a multi-copper enzyme, such as pyruvate oxidase, ascorbate oxidase, or laccase, or a metal catalyst, such as platinum. When an enzyme catalytic mechanism is used for the reaction on the cathode side, the enzyme can be preferably immobilized on an electrode substrate, or can be supplied to an appropriate electrode substrate as an enzyme solution without being immobilized. In this case, the electrode substrate can be the same as the electrode substrate described above for the enzyme electrode of the present invention.

[0069] The diaphragm may be made of any material or have any shape, so long as it has ionic conductivity that allows it to pass through protons and other ions, while preventing the passage of components on the negative electrode side and components on the positive electrode side other than ions such as protons. For example, a cellulose membrane or a solid electrolyte membrane may be used. Examples of solid electrolyte membranes include solid membranes with ion exchange functions, such as organic polymers having strong acid groups such as sulfo groups, phosphate groups, phosphonic groups, and phosphine groups, weak acid groups such as carboxy groups, and polar groups, but are not limited thereto. Specifically, cellulose membranes and perfluorocarbon sulfonic acid (PFS)-based resin membranes, such as Nafion®, a copolymer of tetrafluoroethylene and perfluoro[2-(fluorosulfonylethoxy)propylvinyl ether], can be used.

[0070] [Bioreactor] The enzyme electrode of the present invention can be used as a bioreactor. A bioreactor equipped with the enzyme electrode of the present invention also constitutes one aspect of the present invention. The bioreactor is not particularly limited as long as the enzyme electrode of the present invention acts as a reaction site with the reactant, but in one aspect, it is preferable that the enzyme electrode of the present invention is installed in a column reactor. In the above aspect, when a solution containing the reactant is passed through the column reactor and brought into contact with the enzyme electrode, a product is obtained from the reactant through an enzymatic reaction catalyzed by the oxidoreductase contained in the enzyme electrode of the present invention. The reactant applied to the bioreactor is not particularly limited, and may be a substrate that can be oxidized or reduced by the oxidoreductase. Specific examples include the substrates described above.

[0071] The present specification discloses the following:

[0072] <1> An enzyme-containing electrode, the electrode comprising: an electrode substrate; an oxidoreductase; a conjugate of a silane coupling agent and an electron mediator; and a sol-gel matrix, the oxidoreductase and the conjugate being fixed to the electrode substrate by the sol-gel matrix, and the silane coupling agent having a silicon atom, a reactive functional group, and a hydrolyzable group, the silicon atom and the reactive functional group being linked by a linking group having 4 or more carbon atoms.

[0073] <2> The enzyme electrode according to <1>, wherein the oxidoreductase includes an NAD(P)H- or NAD(P)-dependent oxidoreductase and diaphorase.

[0074] <3> The enzyme electrode according to <1> or <2>, wherein the electron mediator includes NAD(P)H and / or NAD(P) and an electron mediator other than these.

[0075] <4> The enzyme electrode according to any one of <1> to <3>, wherein the silane coupling agent has a positive or negative charge.

[0076] <5> The enzyme electrode according to any one of <1> to <4>, wherein the sol-gel matrix contains a silane compound.

[0077] <6> A method for producing an enzyme electrode, the method comprising: an immobilization step of immobilizing an oxidoreductase and a conjugate of a silane coupling agent and an electron mediator on an electrode substrate using a sol-gel matrix; the silane coupling agent has a silicon atom, a reactive functional group, and a hydrolyzable group, and the silicon atom and the reactive functional group are linked by a linking group having 4 or more carbon atoms.

[0078] <7> The method for producing an enzyme electrode according to <6>, wherein the immobilization step involves hardening the sol-gel matrix using a photo-hardening material.

[0079] <8> A biosensor comprising the enzyme electrode according to any one of <1> to <5>.

[0080] <9> A biobattery comprising the enzyme electrode according to any one of <1> to <5>.

[0081] <10> A bioreactor comprising the enzyme electrode according to any one of <1> to <5>.

[0082] EXAMPLES The present invention will be described in more detail below with reference to examples, although the present invention is not limited to these examples.

[0083] <Cyclic Voltammetry (CV) Measurement> CV measurements were performed under the following conditions. A 1280Z electrochemical measurement system (manufactured by Solartron Analytical) was used for the CV measurements. The solution was placed in an electrochemical measurement cell and kept at 37°C. A platinum counter electrode, an Ag / AgCl reference electrode, and a working electrode were immersed in the cell. Nitrogen gas was passed through the cell for 10 minutes to remove dissolved oxygen from the solution, and then the voltage was continuously changed between −0.6 V and +0.6 V vs. Ag / AgCl at a sweep rate of 5 mV / sec.

[0084] <Mechanical Strength Measurement Cross-Cut Peel Test> A cross-cut peel test was carried out by the micro-scratch method as follows: The composition solution was dropped onto a glass substrate, dried overnight at 4°C, and then scratched with 25 µmR and 10 mN, and the result was evaluated according to the following criteria: ◯: The base was not exposed even after micro-scratching ×: The base was exposed

[0085] <Production Example 1: Conjugate of NAD and GOS> 25 mg of NAD (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 48.6 mg of 8-glycidoxyoctyltrimethoxysilane (manufactured by Shin-Etsu Chemical Co., Ltd.; hereinafter, also referred to as GOS) were mixed with 400 μL of 0.1 M Tris-HCl buffer (pH 7.5) and stirred for 7 hours to obtain a conjugate of NAD and GOS (hereinafter, also referred to as NAD-GOS).

[0086] <Production Example 2: Conjugate of ANQ and GOS> 25 mg of aminonaphthoquinone (hereinafter also referred to as ANQ) and 48.6 mg of GOS were mixed in 400 μL of 0.1 M Tris-HCl buffer (pH 7.5) and stirred for 7 hours to obtain a conjugate of ANQ and GOS (hereinafter also referred to as ANQ-GOS).

[0087] Comparative Production Example 1 Conjugate of NAD and GPS 25 mg of NAD and 37.5 mg of 3-glycidoxypropylethoxysilane (manufactured by Shin-Etsu Chemical Co., Ltd., hereinafter also referred to as GPS) were mixed with 400 μL of 0.1 M Tris-HCl buffer (pH 7.5) and stirred for 7 hours to obtain a conjugate of NAD and GPS (hereinafter also referred to as NAD-GPS).

[0088] Comparative Preparation Example 2: Conjugate of ANQ and GPS 25 mg of ANQ and 37.5 mg of GPS were mixed in 400 μL of 0.1 M Tris-HCl buffer (pH 7.5) and stirred for 7 hours to obtain a conjugate of ANQ and GPS (hereinafter also referred to as ANQ-GPS).

[0089] <Production Example 3: Sol-gel matrix (I)> 0.18 g of tetraethoxysilane (manufactured by Tokyo Chemical Industry Co., Ltd., hereinafter also referred to as TEOS), 0.5 mL of water, and 0.625 mL of 0.01 M hydrochloric acid were mixed and stirred for 7 hours to obtain a sol-gel matrix (I) (hereinafter also referred to as Sol(I)).

[0090] Production Example 4 Sol-Gel Matrix (II) 0.16 g of TEOS, 0.01 g of methyltriethoxysilane (manufactured by Shin-Etsu Chemical Co., Ltd., hereinafter also referred to as MTES), 0.01 g of dimethyldiethoxysilane (manufactured by Shin-Etsu Chemical Co., Ltd., hereinafter also referred to as DMDES), 0.5 mL of water, and 0.625 mL of 0.01 M hydrochloric acid were mixed and stirred for 7 hours to obtain a sol-gel matrix (II) (hereinafter also referred to as Sol(II)).

[0091] <Production Example 5: 10% Polyethyleneimine Solution> Polyethyleneimine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., molecular weight 10,000, hereinafter also referred to as PEI) and pure water were mixed, and the pH was adjusted to 9 with hydrochloric acid to obtain a 10% polyethyleneimine solution (hereinafter also referred to as PEIaq).

[0092] Example 1 15 μL of 10 mg / mL glycerol dehydrogenase (manufactured by Toyobo Co., Ltd., hereinafter also referred to as GLDH), 10 μL of 5 mg / mL diaphorase (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter also referred to as DI), 10 μL of NAD-GOS, 20 μL of Sol(I), and 10 μL of PEI aq were added in this order to an Eppendorf tube, and after stirring, 10 μL of the resulting composition was immediately dropped onto a glassy carbon electrode (hereinafter also referred to as a GC electrode) and dried overnight at 4° C. to obtain an enzyme electrode 1.

[0093] Comparative Example 1 15 μL of 10 mg / mL GLDH, 10 μL of 5 mg / mL DI, 10 μL of NAD-GPS, 20 μL of Sol(I), and 10 μL of PEI aq were added to an Eppendorf tube in this order and stirred. 10 μL of the resulting composition was immediately dropped onto a GC electrode and dried overnight at 4° C. to obtain a comparative enzyme electrode 1.

[0094] The enzyme electrodes obtained in Example 1 and Comparative Example 1 were subjected to 0.1M NH ４ OH-NH ４Cyclic voltammetry (CV) measurements were performed using an electrochemical measurement cell 1 containing 10 mL of Cl buffer (pH 9.0), 500 μL of glycerol, and 10 mg of ANQ. The measurement results are shown in FIG. 3. In FIG. 3, Example 1 is shown by a solid line, and Comparative Example 1 is shown by a dashed line. The results in FIG. 3 show that Example 1 had a higher catalytic current value than Comparative Example 1. In Example 1, a silane coupling agent having a linking group with four or more carbon atoms was bonded to NAD, which is thought to have improved the mobility of NAD and increased the reaction rate between NAD and GLDH and DI.

[0095] Example 2 15 μL of 10 mg / mL GLDH, 10 μL of 5 mg / mL DI, 10 μL of NAD-GOS, 15 μL of ANQ-GOS, 20 μL of Sol(I), and 10 μL of PEI aq were added to an Eppendorf tube in this order and stirred. 10 μL of the resulting composition was immediately dropped onto a GC electrode and dried overnight at 4° C. to obtain an enzyme electrode 2.

[0096] Example 3 15 μL of 10 mg / mL GLDH, 10 μL of 5 mg / mL DI, 10 μL of NAD-GOS, 15 μL of ANQ-GOS, 20 μL of Sol(II), and 10 μL of PEI aq were added to an Eppendorf tube in this order and stirred. 10 μL of the resulting composition was immediately dropped onto a GC electrode and dried overnight at 4° C. to obtain an enzyme electrode 3.

[0097] The enzyme electrodes obtained in Examples 2 and 3 were treated with 0.1M NH ４ OH-NH ４ Cyclic voltammetry (CV) measurements were performed using an electrochemical measurement cell 2 containing 10 mL of Cl buffer (pH 9.0) and 500 μL of glycerol. Measurements were performed immediately after immersing the enzyme electrode in the electrochemical measurement cell 2 and 5 hours after immersion. The measurement results are shown in FIGS. 4 and 5. In FIGS. 4 and 5, the results immediately after immersion are shown by solid lines, and the results 5 hours after immersion are shown by dashed lines. As a result of the measurements, in Examples 2 and 3, the catalytic current values ​​immediately after immersing the enzyme electrode in the electrochemical measurement cell 2 were 2.1×10 －５ A, 3.3 x 10 －５ A. The catalytic current values ​​in Examples 2 and 3 after 5 hours of immersion were 1.2 × 10 －６A, 5.4 x 10 －６ In Example 3, the catalytic current value was improved both immediately after immersion and after 5 hours.

[0098] The compositions coated on the electrode surfaces of the enzyme electrodes obtained in Examples 2 and 3 were analyzed by small-angle X-ray scattering (SAXS) using a NANOPIX manufactured by Rigaku Corporation. The measurement results are shown in FIG. 6. In FIG. 6, Example 2 is shown by a dashed line and Example 3 is shown by a solid line. As a result of the measurement, the average pore size of the gel of Example 2, which contained only tetrafunctional alkoxysilane, was 10 nm (a network structure with a period of 10 nm was formed), while the average pore size of the gel of Example 3, which further contained difunctional alkoxysilane and trifunctional alkoxysilane, was 14 nm (a network structure with a period of 14 nm was formed), confirming that the molecular network in the sol-gel matrix of Example 3 was more expanded.

[0099] Example 4 Sol(I') was obtained by adding 0.1 g of polyethylene glycol dimethacrylate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter also referred to as PEGDMA) and 1 mg of phenylbis(2,3,4-trimethylbenzoyl)phosphine oxide (manufactured by IGM ResinS) to Sol(I) prepared in Production Example 3. 10 μL of a composition prepared in the same manner as in Example 1, except that the obtained Sol(I') was used instead of Sol(I), was dropped onto a glass substrate, dried, and then irradiated with ultraviolet light. A cross-cut peel test was performed on the obtained glass substrate, and the mechanical strength of the matrix was evaluated according to the above-mentioned evaluation criteria. The evaluation result was good. The use of a photocurable material in the sol-gel matrix improved the mechanical strength of the matrix, making it less likely to peel off from the electrode surface. This is thought to improve the durability of the electrode.

[0100] REFERENCE SIGNS LIST 1 enzyme electrode 2 electrode substrate 3a oxidoreductase (a) 3b oxidoreductase (b) 4 silane coupling agent 5a electron mediator (a) 5b electron mediator (b) 6a bond of silane coupling agent and electron mediator (a) 6b bond of silane coupling agent and electron mediator (b) 7 sol-gel matrix 8 substrate

Claims

1. An electrode comprising an enzyme, The electrode includes an electrode substrate, an oxidoreductase, a conjugate of a silane coupling agent and an electron mediator, and a sol-gel matrix; the oxidoreductase and the conjugate are immobilized on the electrode substrate by a sol-gel matrix; The silane coupling agent has a silicon atom, a reactive functional group, and a hydrolyzable group, and the silicon atom and the reactive functional group are linked by a linking group having 4 or more carbon atoms.

2. 2. The enzyme electrode according to claim 1, wherein the oxidoreductase comprises an NAD(P)H- or NAD(P)-dependent oxidoreductase and diaphorase.

3. 2. The enzyme electrode according to claim 1, wherein the electron mediator comprises NAD(P)H and / or NAD(P) and an electron mediator other than these.

4. The enzyme electrode according to claim 1 , wherein the silane coupling agent has a positive or negative charge.

5. The enzyme electrode according to claim 1 , wherein the sol-gel matrix contains a silane compound.

6. A method for producing an enzyme electrode, comprising: The production method includes an immobilization step of immobilizing an oxidoreductase and a conjugate of a silane coupling agent and an electron mediator on an electrode substrate using a sol-gel matrix; The silane coupling agent has a silicon atom, a reactive functional group, and a hydrolyzable group, and the silicon atom and the reactive functional group are linked by a linking group having 4 or more carbon atoms.

7. The method for producing an enzyme electrode according to claim 6 , wherein the immobilization step involves hardening the sol-gel matrix using a photo-hardening material.

8. A biosensor comprising the enzyme electrode according to any one of claims 1 to 5.

9. A biobattery comprising the enzyme electrode according to any one of claims 1 to 5.

10. A bioreactor comprising the enzyme electrode according to any one of claims 1 to 5.