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

Abstract

An enzyme electrode (1) includes an electrode base material (2), an oxidoreductase (3), a conjugate (6) of an electron mediator (5) and a silane coupling agent (4) having a silicon atom, a reactive functional group, and a hydrolyzable group, the oxidoreductase (3) and the conjugate (6) being immobilized on the electrode base material (2) via the sol-gel matrix (7). And a structure in which the silicon atom and the reactive functional group are linked via a linking group having 4 or more carbon atoms.

Description

Technical Field

[0001] This invention relates to enzyme electrodes, methods for manufacturing enzyme electrodes, biosensors, and biocells. Background Technology

[0002] In recent years, with increasing attention to energy issues, bio-batteries using biological substances such as sugars and alcohols as fuels have attracted attention. Bio-batteries use enzymes as electrode catalysts, combining the oxidation reaction of biofuels at the anode with the reduction reaction of oxygen at the cathode to generate electricity.

[0003] In addition, biosensors using enzyme electrodes are used in various fields such as medical and clinical testing to measure the target substance in biological samples. A redox reaction occurs between the target substance, the oxidoreductase contained in the enzyme electrode, and the target substance, and the resulting current can be detected to measure the substance.

[0004] In enzyme electrodes used in biocells and biosensors, methods utilizing substances called electron mediators are known to facilitate efficient electron transfer. Furthermore, to further improve the efficiency of this electron transfer, techniques are being developed to immobilize small molecules such as enzymes, electron mediators, and indicators onto the electrode. One known method for immobilizing enzymes is the sol-gel method, which involves encapsulating molecules within a matrix of metal oxides formed in a liquid to achieve comprehensive molecular immobilization.

[0005] Regarding enzyme immobilization methods, Patent Document 1 discloses an enzyme immobilization method characterized by immobilizing the enzyme in a structural unit having an inner diameter of more than 1.2 times the enzyme diameter and having structural stability, and then forming a network structure of gelled material obtained by the sol-gel method in the opening and / or internal voids of the above-mentioned structural unit to improve the stability of the immobilized enzyme.

[0006] Patent documents 2 and 3 disclose a technique for encapsulating small molecules such as electron mediators and indicators with enzymes for sensing purposes.

[0007] While the technologies in the aforementioned patent documents 1 to 3 can suppress enzyme leakage, there is a problem of small molecules such as electron mediators leaking from the gel matrix.

[0008] To address the aforementioned issues, Non-Patent Literature 1 discloses a method for preventing the leakage of electron mediators by covalently binding a small molecule electron mediator to a sol-gel matrix.

[0009] Non-patent document 2 discloses that the sol-gel matrix is ​​made hydrophobic in order to improve the stability of the immobilized enzyme.

[0010] Existing technical documents

[0011] Patent documents

[0012] Patent Document 1: Japanese Patent Application Publication No. 2001-178457

[0013] Patent Document 2: Japanese Patent Publication No. 2005-529309

[0014] Patent Document 3: Japanese Patent Publication No. 2006-512573

[0015] Non-patent literature

[0016] Non-patent literature 1: Z Wang et al., Biosens. Bioelectron., 2012, Vol. 32, pp. 111-117

[0017] Non-patent literature 2: D Weiser et al., Green Chem., 2017, Vol. 19, pp. 3927-3937 Summary of the Invention

[0018] As mentioned above, although various enzyme immobilization techniques have been disclosed in the past, enzyme electrodes using conventional immobilization methods are not sufficient in terms of electrode catalyst function and there is room for improvement.

[0019] The present invention was made in view of the above-mentioned situation, and its object is to provide an enzyme electrode with superior electrode catalyst function compared with conventional enzyme electrodes.

[0020] In order to solve the above-mentioned problems, the inventors conducted in-depth research and discovered that in enzyme electrodes using oxidoreductases and electron mediators, by using a sol-gel matrix to fix a complex formed by combining the electron mediator with a silane coupling agent having a structure in which silicon atoms and reactive functional groups are linked by linking groups having 4 or more carbon atoms, the electrode catalyst function is excellent. This invention was thus able to solve the above-mentioned problems and complete the present invention.

[0021] This invention provides an enzyme electrode, which is an electrode containing an enzyme. The electrode comprises an electrode substrate, an oxidoreductase, a combination of a silane coupling agent and an electron mediator, and a sol-gel matrix. The oxidoreductase and the combination are fixed to the electrode substrate by the sol-gel matrix. The silane coupling agent has silicon atoms, reactive functional groups, and hydrolyzable groups, and is a structure in which the silicon atoms and reactive functional groups are connected by linking groups with 4 or more carbon atoms.

[0022] The enzyme electrode of the present invention is formed by the above-described structure, thereby exhibiting excellent electrode catalyst function and thus being suitable for use in bio-batteries, biosensors, etc. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of one embodiment of the enzyme electrode of the present invention.

[0024] Figure 2 This is a schematic diagram illustrating the interaction between a silane coupling agent and an oxidoreductase in one embodiment of the enzyme electrode of the present invention.

[0025] Figure 3 This is a graph showing the voltammograms obtained by cyclic voltammetry (CV) measurements using the enzyme electrodes obtained in Example 1 and Comparative Example 1.

[0026] Figure 4 This is a graph showing the voltammogram obtained by cyclic voltammetry (CV) measurement using the enzyme electrode obtained in Example 2.

[0027] Figure 5 This is a graph showing the voltammogram obtained by cyclic voltammetry (CV) measurement using the enzyme electrode obtained in Example 3.

[0028] Figure 6 This is a graph showing the SAXS-based analytical results of the compositions coated on the electrode surfaces of the enzyme electrodes obtained in Examples 2 and 3. Detailed Implementation

[0029] The enzyme electrode, the method for manufacturing the enzyme electrode, the biosensor, and the biocell of the present invention will be described below.

[0030] However, the present invention is not limited to the following configuration, and can be appropriately modified and applied without changing the spirit of the invention. It should be noted that a configuration consisting of two or more of the preferred configurations of the invention described below is also part of the present invention.

[0031] [Enzyme Electrode]

[0032] The enzyme electrode of the present invention comprises an electrode substrate, an oxidoreductase, a combination of a silane coupling agent and an electron mediator, and a sol-gel matrix. The oxidoreductase and the combination are fixed to the electrode substrate by the sol-gel matrix. The silane coupling agent has silicon atoms, reactive functional groups, and hydrolyzable groups, and is a structure formed by connecting the silicon atoms and reactive functional groups through connecting groups with 4 or more carbon atoms.

[0033] For example, Non-Patent Document 1 discloses a technique for covalently linking electron mediators to a sol-gel matrix. While this technique can suppress leakage of the electron mediator matrix, its reactivity is poor due to the limited mobile area of ​​the electron mediator. In contrast, in this invention, by giving the aforementioned silane coupling agent the long-chain linking groups described above, even if the electron mediator is fixed by the sol-gel matrix, its mobile area is large and it is easy to move, thus enabling efficient electron transfer between enzymes and / or enzymes and electrodes. This increases the catalyst current, and when such an electrode is used in batteries, sensors, etc., the output is improved.

[0034] The ratio of the silane coupling agent to the electron mediator in the enzyme electrode is preferably 1,000 mol% to 10,000,000 mol% relative to 100 mol% of the oxidoreductase. More preferably, it is 10,000 mol% to 1,000,000 mol%, and even more preferably, it is 100,000 mol% to 1,000,000 mol%.

[0035]

[0036] There are no particular restrictions on the combination of the silane coupling agent and the electron mediator, as long as the silane coupling agent and the electron mediator are combined. However, it is preferred that the reactive functional groups of the silane coupling agent and the reactive functional groups of the electron mediator are combined through covalent bonds.

[0037] (Silane coupling agent)

[0038] There are no particular restrictions on the above-mentioned silane coupling agents as long as they have silicon atoms, reactive functional groups, and hydrolyzable groups, and the silicon atoms and reactive functional groups are connected by linking groups with 4 or more carbon atoms.

[0039] There are no particular limitations on the reactive functional groups of the aforementioned silane coupling agents, as long as they can bind to electron mediators. Examples of reactive functional groups that interact through intermolecular forces, hydrogen bonds, Coulomb forces, etc., or that can undergo covalent bonding are possible. However, from the viewpoint of bonding strength, reactive functional groups that can undergo covalent bonding are preferred. Reactive functional groups capable of covalent bonding include, specifically, groups such as amino, sulfonic acid, sulfate, phosphate, mercapto, carboxyl and their salts, epoxy, thiol, hydroxyl, polymerizable unsaturated groups, azide, azo, nitro, nitrile, cyano, propadienyl, isonitrile, urea, aldehyde, ketone, halogen, NHS ester, imide ester, maleimide, pyridyl dithiol, allyl azide, haloacetate, isocyanate, carbodiimide, allyl azide, diaziridine, hydrazide, psoralen, pyridine disulfide, vinyl sulfone, etc. Among these, amino and epoxy groups are preferred.

[0040] The silane coupling agent described above may have one or more of the aforementioned reactive functional groups.

[0041] The aforementioned silane coupling agent has a linking group having 4 or more carbon atoms that connect the silicon atom to the reactive functional group.

[0042] There are no particular restrictions on the above-mentioned linking groups as long as they are organic groups with 4 or more carbon atoms, but it is preferred that they are hydrocarbon groups with 4 to 30 carbon atoms that can have heteroatoms.

[0043] By structuring the linker group in such a way that it is hydrophobic and interacts hydrophobically with the hydrophobic portion of the enzyme, the enzyme becomes stable. Therefore, the enzyme electrode of this invention exhibits excellent durability when used as a battery or sensor.

[0044] Furthermore, when the linker group has a long-chain hydrophobic group with 4 or more carbon atoms, the hydrated water near the active site of the enzyme is eliminated, thus further increasing the rate of electron transfer reaction between the enzyme and the electron mediator.

[0045] There are no particular restrictions on the hydrocarbon group mentioned above, but it is preferred to be a group obtained by removing one or more hydrogen atoms from a straight-chain alkyl or branched-chain alkyl, alkenyl, alkynyl, aryl, etc.

[0046] Examples of straight-chain or branched alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecanyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecanyl, n-eicosyl, isopropyl, sec-butyl, isobutyl, tert-butyl, 1-methylbutyl, 1-ethylpropyl, and 2-methylbutyl. Butyl, isopentyl, neopentyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, tert-pentyl, 1,3-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl, 2-ethyl-2-methylpropyl, 1-methylheptyl, 2-ethylhexyl, 1,5-dimethylhexyl, tert-octyl, branched nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, stearyl, eicosyl, etc.

[0047] Examples of alkenyl groups include vinyl, allyl, 1-butenyl, 2-butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, dodecenyl, octadecenyl, and eicoseneyl.

[0048] Examples of the aforementioned alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, butynyl, pentynyl, hexynyl, heptynyl, octyynyl, nonynyl, decynyl, dodecynyl, octadecynyl, and icosynyl.

[0049] Examples of aryl groups include phenyl, naphthyl, benzyl, methylphenyl, ethylphenyl, propylphenyl, butylphenyl, butylmethylphenyl, dimethylphenyl, diethylphenyl, dibutylphenyl, biphenyl, methyl biphenyl, ethyl biphenyl, methyl naphthyl, ethyl naphthyl, cinnamyl (Ph-CH=CHCH2-yl), 1-benzocyclobutenyl, and other aryl groups.

[0050] The number of carbon atoms in the aforementioned hydrocarbon group is preferably 5 to 25, more preferably 6 to 20, even more preferably 7 to 18, and particularly preferably 8 to 15.

[0051] The aforementioned hydrocarbon groups may have heteroatoms such as nitrogen, sulfur, oxygen, phosphorus, and halogen atoms. In addition, they may have substituents containing heteroatoms such as hydroxyl, alkoxy, carboxyl, acyl, sulfonic acid, amino, and phosphate groups.

[0052] The aforementioned silane coupling agent preferably has a positive or negative charge. Thus, through electrostatic interactions with positively or negatively charged sites on the enzyme surface, the enzyme can be further stabilized, and its activity can be maintained for a longer period.

[0053] The aforementioned silane coupling agent preferably possesses functional groups with positive or negative charges, and these functional groups can be present in any part of the silane coupling agent. The aforementioned silane coupling agent preferably has positive or negative charges in the linking group and / or reactive functional groups. When the reactive functional groups have positive or negative charges, it is sufficient that they have positive or negative charges in the structure after binding with the electron mediator.

[0054] When the reactive functional group in the above-mentioned silane coupling agent has a positive or negative charge and has a hydrocarbon group with 4 or more carbon atoms as the linking group, the enzyme can be further stabilized through electrostatic and hydrophobic interactions.

[0055] Examples of functional groups with positive charges include primary to tertiary amino groups and quaternary ammonium groups.

[0056] Examples of functional groups with negative charges include epoxy groups, ether groups, carboxyl groups, sulfonic acid groups, sulfuric acid groups, phosphate groups, thiol groups, and halogen groups.

[0057] The aforementioned silane coupling agent preferably has a structure in which hydrolyzable groups such as alkoxy groups are bonded to silicon atoms, and more preferably has groups represented by the following formula (1).

[0058] -Si(R)1 ) 3－n (OR) 2 ) n (1)

[0059] (where R) 1 R 2 "Same" or "different" indicates hydrocarbon groups with 1 to 5 carbon atoms. n represents an integer from 1 to 3.

[0060] n is an integer from 1 to 3, preferably 2 or 3, and more preferably 3.

[0061] R is the R in the above equation (1) 1 R 2 There are no particular restrictions on the hydrocarbon group, but examples include alkyl, alkenyl, and aryl groups. Specific examples of these can be the hydrocarbon groups described in the linking groups above.

[0062] As R 1 R 2 The number of carbon atoms in the hydrocarbon group is preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 to 2.

[0063] As R 1 R 2 The hydrocarbon group is preferably an alkyl group, more preferably methyl, ethyl, n-propyl, or n-butyl, and even more preferably methyl or ethyl.

[0064] Examples of silane coupling agents 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)-γ-aminopropyltriethoxysilane. Silane coupling agents containing amino groups, including N-β-(aminoethyl)-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-epoxypropoxyoctyltrimethoxysilane... 8-Epoxypropoxyoctylmethyldimethoxysilane, 8-Epoxypropoxyoctylmethyldiethoxysilane, 8-Epoxypropoxyoctyltriethoxysilane, 7-Epoxypropoxyheptyltrimethoxysilane, 7-Epoxypropoxyheptylmethyldimethoxysilane, 7-Epoxypropoxyheptylmethyldiethoxysilane, 7-Epoxypropoxyheptyltriethoxysilane, 6-Epoxypropoxyhexyltrimethoxysilane, 6-Epoxypropoxyhexylmethyldimethoxysilane, 6-Epoxypropoxyhexylmethyldiethoxysilane, 6-Epoxypropoxyhexyltriethoxysilane, 5-Epoxypropoxypentyltriethoxysilane Methoxysilanes, 5-epoxypropoxypentylmethyldiethoxysilane, 5-epoxypropoxypentylmethyldiethoxysilane, 5-epoxypropoxypentyltriethoxysilane, 4-epoxypropoxybutyltrimethoxysilane, 4-epoxypropoxybutylmethyldimethoxysilane, 4-epoxypropoxybutylmethyldiethoxysilane, 4-epoxypropoxybutyltriethoxysilane, γ-epoxypropoxypropyl(ethyl)dimethoxysilane, β-3,4-epoxycyclohexylethyltrimethoxysilane, β-3,4-epoxycyclohexylethyltriethoxysilane, and other silane coupling agents containing epoxy groups.

[0065] (Electron mediator)

[0066] In the enzyme electrode of the present invention, there are no particular restrictions on the electron mediator that binds to the silane coupling agent, as long as it is capable of electron donation and acceptance with the oxidoreductase contained in the enzyme electrode of the present invention and can bind with the reactive functional group of the silane coupling agent. Preferably, it has functional groups such as amino, carboxyl, and aldehyde groups that can form bonds with the reactive functional group of the silane coupling agent.

[0067] Examples of electron mediators include coenzymes of oxidoreductases and electron transporters other than coenzymes.

[0068] As coenzymes for oxidoreductases, examples include vitamin coenzymes such as nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, and flavin mononucleotide; and quinone coenzymes such as pyrrolopyrrolinoquinone, 6-hydroxydopabenzoquinone, tryptophan-tryptophan quinone, lysine tyrosine quinone, and cysteine-tryptophan quinone.

[0069] 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. The oxidized form of nicotinamide adenine dinucleotide phosphate is also referred to as NADP, and its reduced form is also referred to as NADPH.

[0070] Additionally, NAD(P)H refers to NADH or NADPH, and NAD(P) refers to NAD or NADP. Furthermore, NAD refers to NAD+. ＋ NADP refers to NADP ＋ .

[0071] Other electron transporters besides the aforementioned coenzymes include metal complexes centered on metal elements such as Os, Fe, Ru, Co, Cu, Ni, V, Mo, Cr, Mn, Pt, and W, or their ions (ferrocene, potassium ferrocyanide, lithium ferrocyanide, sodium ferrocyanide, and other alkali metal ferrocyanides or their alkyl-substituted derivatives (methyl-substituted, ethyl-substituted, propyl-substituted, etc.), potassium octacyanotungsticate, etc.); quinones such as quinones, benzoquinones, anthraquinones, naphthoquinones, and aminonaphthoquinones; heterocyclic compounds such as toluidine blue, methylene blue, viologen, methyl viologen, benzyl viologen, methyl phenazine sulfate, ethyl phenazine sulfate, bipyridine, or their derivatives; and 2,6-dichlorophenolindophenol, methylene blue, and potassium β-naphthoquinone-4-sulfonate, etc.

[0072] As the aforementioned metal complex, metal complexes with iron as the central metal are preferred, and ferrocene compounds such as aminoferrocene and ferrocene formaldehyde are more preferred.

[0073] When the enzyme electrode of the present invention contains an oxidoreductase (A) of the substrate described later, it is preferable to use an electron mediator (a) as a coenzyme of the oxidoreductase (A).

[0074] When the coenzyme of the oxidoreductase (A) of the above-mentioned substrate 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 an electron mediator (a).

[0075] In cases where the enzyme electrode of the present invention further contains an enzyme (B) that accepts electrons from an electron mediator (a) such as a coenzyme and transfers the accepted electrons to the electrode substrate or transfers electrons accepted from the electrode substrate to the electron mediator (a), it is preferable to use an electron mediator (b) that coordinates the electron transfer between the enzyme (B) and the electrode substrate.

[0076] One of the preferred embodiments of the present invention is that the electron mediator includes NAD(P)H and / or NAD(P) as an electron mediator (a), or includes electron mediators other than NAD(P)H and NAD(P) as an electron mediator (b).

[0077] There are no particular limitations on the electron mediator (b) mentioned above, but heterocyclic compounds with a phenothiazine skeleton, such as quinones, ferrocene compounds, and toluidine blue, are preferred. Quinones such as aminonaphthoquinone are more preferred.

[0078] The preferred embodiment of the present invention comprises a combination of a silane coupling agent and an electron mediator such as a coenzyme (hereinafter also referred to as a combination (α)) and a combination of a silane coupling agent and an electron mediator (b) (hereinafter also referred to as a combination (β)).

[0079] <Oxidoreductase>

[0080] The aforementioned oxidoreductases are not particularly limited as long as they can accept and accept electrons with an electron mediator. However, the enzyme electrode of the present invention preferably contains an enzyme (A) that oxidizes and / or reduces the substrate described below and accepts and accepts electrons with an electron mediator (hereinafter also referred to as oxidoreductase (A) or simply enzyme (A)). As enzyme (A), substrate oxidase is more preferred. More preferred is NAD(P)-dependent oxidase that accepts and accepts electrons with NAD(P)H or NAD(P) (hereinafter also referred to as NAD(P)-dependent oxidase).

[0081] Preferred oxidases include glycerol dehydrogenase, glucose dehydrogenase, a series of enzymes in the electron transport chain, ATP synthase, and enzymes related to carbohydrate metabolism (e.g., hexokinase, glucose-phosphogluconate isomerase, fructose-phosphofructokinase, fructose-2-phosphate aldolase, triose-phosphogluconate isomerase, glyceraldehyde-2-phosphate dehydrogenase, glycerol-2-phosphate mutase, pyruvate hydratase, pyruvate kinase, L-lactate dehydrogenase, D-lactate dehydrogenase, pyruvate dehydrogenase, citrate synthase, cis-aconitase, isocitrate dehydrogenase, 2-ketoglutarate dehydrogenase, succinyl-CoA synthase, succinate dehydrogenase, fumarate, malate dehydrogenase, etc.). One or more of these may be used.

[0082] Among them, glycerol dehydrogenase and glucose dehydrogenase are preferred.

[0083] The enzyme electrode of the present invention preferably further contains an enzyme (B) that accepts electrons from the above-mentioned electron mediator (a) and transfers the accepted electrons to the electrode substrate or transfers electrons accepted from the electrode substrate to the electron mediator (a) (hereinafter also referred to as oxidoreductase (B) or simply enzyme (B)).

[0084] The enzyme electrode of the present invention preferably contains myocardial flavin as enzyme (B).

[0085] Myocardial flavin is an enzyme that can catalyze the redox reaction of NAD(P) redox pair and conduct electron donation and acceptance with electron mediators (b) or electrode surfaces.

[0086] When the enzyme electrode of the present invention contains NAD(P)-dependent oxidase and myocardial flavin, NAD(P) accepts electrons generated by the oxidation of the substrate, and NAD(P) becomes NAD(P)H. The NAD(P)H transfers electrons to the myocardial flavin, the myocardial flavin transfers electrons to the aforementioned electron mediator (b), and then the electron mediator (b) transfers electrons to the electrode substrate, thereby enabling the transfer of electrons taken from the substrate to the electrode substrate.

[0087] When the above-mentioned oxidoreductase contains oxidoreductase (A) and oxidoreductase (B), the content ratio of oxidoreductase (B) relative to 100 mol% of oxidoreductase (A) is preferably 10 mol% to 1000 mol%. More preferably, it is 20 mol% to 500 mol%, and even more preferably, it is 30 mol% to 100 mol%.

[0088] The enzyme electrode described above preferably comprises a conjugate (α), a conjugate (β), an enzyme (A), and an enzyme (B).

[0089] In this case, the proportion of the complex (α) is not particularly limited, but it is preferably 1,000 mol% to 10,000,000 mol% relative to 100 mol% of enzyme (A). More preferably, it is 10,000 mol% to 1,000,000 mol%, and even more preferably, it is 100,000 mol% to 1,000,000 mol%.

[0090] Furthermore, there is no particular limitation on the content of the conjugate (β), but it is preferably 1,000 mol% to 10,000,000 mol% relative to 100 mol% of enzyme (B). More preferably, it is 10,000 mol% to 1,000,000 mol%, and even more preferably, it is 100,000 mol% to 1,000,000 mol%.

[0091] The embodiment of the present invention in which the enzyme electrode comprises conjugate (α), conjugate (β), NAD-dependent oxidase and myocardial flavin is also one of the preferred embodiments.

[0092] <sol-gel matrix>

[0093] The enzyme electrode of the present invention contains a sol-gel matrix.

[0094] There are no particular limitations on the sol-gel matrix as long as it can fix the combination of oxidoreductase and silane coupling agent with electron mediator to the electrode substrate, and it is preferred to contain silane compound.

[0095] As the above-mentioned silane compound, it is preferable to have a structure in which hydrolyzable groups such as alkoxy groups are bonded to silicon atoms, and more preferably to be a silane compound with alkoxy groups as shown in the following formula (2):

[0096] Si(R) 3 ) 4－m (OR) 4 ) m (2)

[0097] (where R) 3 R 4 "Same" or "different" indicates hydrocarbon groups with 1 to 5 carbon atoms. m represents an integer from 1 to 4.

[0098] R is the value of equation (2) above. 3 R 4 The hydrocarbon group is not particularly limited, but examples include alkyl, alkenyl, aryl, etc. Specific examples can be the hydrocarbon groups described in the linking groups above.

[0099] As R 3 R 4 The hydrocarbon group preferably has 1 to 4 carbon atoms, more preferably 1 to 3, and even more preferably 1 to 2.

[0100] As R 3 R 4 The hydrocarbon group is preferably an alkyl group, more preferably methyl, ethyl, n-propyl, or n-butyl, and even more preferably methyl or ethyl.

[0101] Specifically, examples of compounds represented by formula (2) above include tetramethoxysilanes, tetraethoxysilanes, tetraisopropoxysilanes, tetrabutoxysilanes, dimethoxydiethoxysilanes, etc., which are tetrafunctional alkoxysilanes having four alkoxy groups; trifunctional alkoxysilanes, methyltrimethoxysilanes, methyltriethoxysilanes, ethyltrimethoxysilanes, ethyltriethoxysilanes, etc., which are trifunctional alkoxysilanes having three alkoxy groups; difunctional alkoxysilanes, dimethyldiethoxysilanes, diethyldimethoxysilanes, diethyldiethoxysilanes, diethyldiethoxysilanes, etc., which are difunctional alkoxysilanes having two alkoxy groups; and monofunctional alkoxysilanes, trimethylmethoxysilanes, trimethylethoxysilanes, triethylmethoxysilanes, triethylethoxysilanes, etc., which are monofunctional alkoxysilanes having one alkoxy group.

[0102] You can use one or more of these.

[0103] The molecular structure of silane compounds can be determined by taking a gel coated on the electrode surface, dissolving the gel with deuterated sodium hydroxide, and then performing NMR (Si, H) determination.

[0104] To promote the hydrolysis and condensation reaction of the silane compounds, the sol-gel matrix preferably contains a curing catalyst.

[0105] There are no particular limitations on what can be used as a curing catalyst; examples include alkaline catalysts and acidic catalysts.

[0106] Examples of alkaline catalysts include polyethyleneimine, N,N-diethylethanolamine, N,N-dimethylethanolamine, triethanolamine, triethylamine, and 3-morpholinopropylamine.

[0107] 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, formic acid, acetic acid, and other carboxylic acids.

[0108] The preferred curing catalyst is an alkaline catalyst, more preferably an amine, and even more preferably polyethyleneimine.

[0109] The aforementioned sol-gel matrix preferably further contains a photocurable material. This allows the sol-gel matrix to cure more fully.

[0110] There are no particular limitations on photocurable materials, but examples include polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, tripropylene glycol triacrylate, dipentaerythritol hexaacrylate, ethylene glycol diacrylate, 1,3-butanediol 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, cyclohexanedimethyl diacrylate, tricyclodecanedimethyl diacrylate, bisphenol A polyethoxylated diacrylate, bisphenol F polyethoxylated diacrylate, and pentaerythritol. Tetraacrylate, propoxylated (2) neopentyl glycol diacrylate, trimethylolpropane triacrylate, tri(2-hydroxyethyl) isocyanurate triacrylate, pentaerythritol triacrylate, ethoxylated (3) trimethylolpropane triacrylate, propoxylated (3) triglyceride triacrylate, pentaerythritol tetraacrylate, bis(trimethylolpropane) tetraacrylate, ethoxylated (4) pentaerythritol tetraacrylate, dipentaerythritol pentaacrylate, 2-(2-ethoxyethoxy)ethyl acrylate, hexanediol diacrylate, 1,6-hexanediol diacrylate, ethoxylated 1,6-hexanediol diacrylate, polypropylene glycol diacrylate, 1,4-butanediol diacrylate Diacrylates of alcohols, 1,9-nonanediol diacrylate, tetraethylene glycol diacrylate, 2-n-butyl-2-ethyl-1,3-propanediol diacrylate, neopentyl hydroxypentanoic acid neopentyl glycol diacrylate, trimethylolpropane triacrylate of hydroxypentanoic acid, ethoxylated phosphate triacrylate, ethoxylated tripropylene glycol diacrylate, neopentyl glycol modified trimethylolpropane diacrylate, stearic acid modified pentaerythritol diacrylate, tetramethylolpropane triacrylate, tetramethylolmethane triacrylate, caprolactone modified trimethylolpropane triacrylate, propoxylated glycerol triacrylate, tetramethylolmethane tetraacrylate, ethoxylated pentaerythritol tetraacrylate Multifunctional acrylic compounds, including dipentaerythritol hexaacrylate, caprolactone-modified dipentaerythritol hexaacrylate, dipentaerythritol hydroxypentaacrylate, neopentyl glycol low-polyacrylate, trimethylolpropane low-polyacrylate, pentaerythritol low-polyacrylate, 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 propenyl, methpropenyl, vinyl, epoxy, and thiol groups, etc., may be used. One or more of these may be used. Multifunctional acrylic compounds are preferred, and polyethylene glycol diacrylate and polyethylene glycol dimethacrylate are more preferred.

[0111] There is no particular limitation on the proportion of photocurable material in the above-mentioned sol-gel matrix, but it is preferably 0.1% to 50% by mass relative to 100% by mass of the silane compound. More preferably, it is 0.5% to 25% by mass, and even more preferably, it is 1% to 10% by mass.

[0112] The sol-gel matrix preferably further contains a photopolymerization initiator. This allows the sol-gel matrix to solidify more efficiently.

[0113] There are no particular limitations on the photopolymerization initiator, but examples include bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide, 2,4,6-trimethylbenzoyl diphenylphosphine oxide, benzoyl diphenylphosphine oxide, benzoyl diethoxyphosphine oxide, 2,4,6-trimethylbenzoyl diethoxyphenylphosphine oxide, phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide, and other acylphosphine oxide compounds. One or more of these can be used. Among them, acylphosphine oxide compounds are preferred, and phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide is more preferred.

[0114] The proportion of photopolymerization initiator in the above-mentioned sol-gel matrix is ​​not particularly limited, but is preferably 0.1% to 10% by mass relative to 100% by mass of the photocurable material. More preferably, it is 0.2% to 5% by mass, and even more preferably, it is 0.3% to 3% by mass.

[0115] <Electrode substrate>

[0116] The enzyme electrode of the present invention has an electrode substrate that is conductive and capable of connecting to an external circuit and transferring electrons. As long as the electrode substrate possesses this property, there are no particular limitations on its material, shape, etc.

[0117] The electrode substrate can be any conductive material, such as carbon materials like carbon cloth, carbon paper, graphite, glassy carbon, activated carbon, carbon black, and carbon nanotubes; metals or alloys like gold, platinum, copper, palladium, titanium, aluminum, silver, and nickel; and conductive oxides like SnO2, ITO, In2O3, WO3, and TiO2.

[0118] The electrode substrates described above can be composed of one of these as a single layer, or they can be composed of a stacked structure of two or more layers.

[0119] Carbon materials are preferred as the aforementioned conductive materials.

[0120] Glassy carbon, activated carbon, carbon black, and carbon nanotubes are preferred as the aforementioned carbon materials.

[0121] In the electrode substrate described above, for example, when using two or more of the above-mentioned conductive materials, a polymer or other adhesive can be used.

[0122] There are no particular restrictions on the polymers mentioned above, but polymers containing fluorine atoms, such as polyvinylidene fluoride (PVDF) and polyvinyl fluoride (PVF), as well as their copolymers and copolymers of their monomers with ethylene, styrene, etc., can be used. In addition to polymers such as polystyrene, polyethylene, and polypropylene, hydrophilic polymers such as polyacrylic acid, polylysine, and carboxymethyl cellulose, as well as conductive polymers such as polyaniline, polypyrrole, and their derivatives, such as polyaniline sulfonic acid, can also be used.

[0123] The surface of the electrode substrate can be flat or it can have irregularities or fine pores, preferably an electrode substrate with fine pores.

[0124] There is no particular limitation on the size of the aforementioned pores, but it is preferably 0.1 nm to 100 nm. More preferably, it is 1 nm to 50 nm.

[0125] The following is a reference to the appendix. Figure 1 The embodiments of the enzyme electrode of the present invention will be described below.

[0126] Figure 1 This is a schematic diagram of one embodiment of the enzyme electrode of the present invention. For the enzyme electrode 1, oxidoreductase (a) 3a, oxidoreductase (b) 3b, electron mediator (a) 5a combined with silane coupling agent 6a, and electron mediator (b) 5b combined with silane coupling agent 6b are fixed to the electrode substrate 2.

[0127] When the enzyme electrode 1 of the present invention is the anode, the electron mediator (a) 5a accepts electrons generated by the oxidation of the substrate 8 of the oxidoreductase (a) 3a and transfers the accepted electrons to the oxidoreductase (b) 3b. The oxidoreductase (b) 3b transfers electrons to the electron mediator (b) 5b, and the electron mediator (b) 5b transfers electrons to the electrode substrate 2. Electrons taken from the substrate 8 are transferred to the electrode substrate 2.

[0128] Figure 2 This is a schematic diagram illustrating the interaction between a silane coupling agent and an oxidoreductase in one embodiment of the enzyme electrode of the present invention.

[0129] Silane coupling agents, when the linking group has 4 or more carbon atoms in the hydrocarbon group, can form a hydrophobic interaction with the hydrophobic portion of the oxidoreductase, thereby stabilizing the oxidoreductase.

[0130] In addition, silane coupling agents, when having positive or negative charges in their reactive functional groups, can form electrostatic interactions with negatively or positively charged portions on the surface of oxidoreductases, thereby stabilizing the oxidoreductases.

[0131] [Manufacturing method of enzyme electrode]

[0132] The method for manufacturing the enzyme electrode of the present invention is not particularly limited, but it is preferable to manufacture it by immobilizing the oxidoreductase and the combination of the silane coupling agent and the electron mediator onto the electrode substrate using a sol-gel matrix. That is, the method for manufacturing the enzyme electrode, which includes an immobilization step of immobilizing the oxidoreductase and the combination of the silane coupling agent and the electron mediator onto the electrode substrate using a sol-gel matrix, is also one aspect of the present invention.

[0133] Specific examples and preferred embodiments of the electrode substrate, oxidoreductase, silane coupling agent and electron mediator combination, and sol-gel matrix are described above.

[0134] The aforementioned immobilization process is not particularly limited as long as the oxidoreductase and the silane coupling agent-electron mediator combination are fixed to the electrode substrate using a sol-gel matrix. It can be performed by coating the electrode substrate with a composition containing the oxidoreductase, the silane coupling agent-electron mediator combination, and the sol-gel matrix material, followed by drying. Commonly used methods can be employed, such as spin coating, spray coating, screen coating, dip coating, and doctor blade coating.

[0135] There are no particular limitations on the sol-gel matrix material used in the above-described immobilization process, but it is preferable to contain a silane compound. Specific examples and preferred embodiments of the silane compound are described above.

[0136] The composition used in the above immobilization process preferably contains a solvent and a buffering component.

[0137] Examples of solvents include water and ethanol. Water is preferred.

[0138] Examples of buffer components 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).

[0139] The proportions of the oxidoreductase, the silane coupling agent and electron mediator combination, and the sol-gel matrix material in the above composition used in the above immobilization process can be appropriately adjusted according to their proportions in the enzyme electrode.

[0140] The composition used in the above-described immobilization process preferably contains a photocurable material.

[0141] In addition, the above composition preferably further contains a photopolymerization initiator.

[0142] Specific and preferred examples of the aforementioned photocurable materials and photopolymerization initiators are as described above.

[0143] Furthermore, the preferred range of the amount of photocurable material and photopolymerization initiator used is the same as the content ratio in the sol-gel matrix mentioned above.

[0144] In the above immobilization process, when using the above-mentioned photocurable material and photopolymerization initiator, it is preferable to coat the above-mentioned composition onto the electrode substrate and dry it before performing the light irradiation process.

[0145] The above-described irradiation method can be performed using commonly used methods. The irradiation conditions vary depending on the energy of the radiation used; for example, in the case of curing using ultraviolet (UV) irradiation, a UV irradiation dose of 10 mJ / cm² is preferred. 2 ~3000mJ / cm 2 The irradiation time is 1 second to 180 seconds.

[0146] [Biosensors]

[0147] The present invention also provides a biosensor incorporating the enzyme electrode of the present invention.

[0148] The biosensor of the present invention is preferably configured to include the enzyme electrode of the present invention as the active electrode and its counter electrode.

[0149] The above-described biosensor measurement is performed by contacting the sample with the biosensor, thereby generating a redox reaction between the oxidoreductase contained in the enzyme electrode of the present invention and the analyte, and detecting the resulting current. The presence or concentration of the substrate in the sample can be determined using the aforementioned response current value.

[0150] Examples of measurement methods using the biosensor of the present invention include commonly used methods such as chronoamperometry, coulometric method, and cyclic voltammetry for measuring oxidation current or reduction current.

[0151] [Bio-battery]

[0152] The present invention also provides a bio-battery incorporating the enzyme electrode of the present invention.

[0153] The enzyme electrode of the present invention in the above-mentioned bio-battery is preferably the anode.

[0154] There are no particular limitations on the bio-battery of the present invention as long as it has the enzyme electrode of the present invention and the anode and cathode are connected by an external circuit, but it is preferred to be configured to include a membrane that isolates the anode and cathode.

[0155] In one embodiment, the bio-battery of the present invention is preferably configured to include an anode, a cathode, and a separator that isolates the anode and cathode, both of which are made of the enzyme electrode of the present invention.

[0156] In one embodiment of the present invention, the cathode of the bio-battery can be an enzyme catalyst such as pyruvate oxidase, ascorbate oxidase, laccase, or other copper-based enzymes, or a metal catalyst such as platinum. When the reaction on the cathode side utilizes an enzyme catalyst mechanism, it is preferable that the enzyme is immobilized on the electrode substrate or, without immobilization, is supplied as an enzyme solution to a suitable electrode substrate. In this case, the electrode substrate described in the enzyme electrode of the present invention can be used similarly.

[0157] The aforementioned membrane is not limited in its raw materials or shape, as long as it possesses ionic conductivity that allows protons to pass through while simultaneously preventing the passage of components other than protons and ions on both the negative and positive sides. For example, cellulose membranes can be used, as well as solid electrolyte membranes. Examples of solid electrolyte membranes include, but are not limited to, solid membranes with ion-exchange functions made from organic polymers containing strong acid groups such as sulfonyl, phosphate, phosphonic acid, and phosphine, weak acid groups such as carboxyl, and polar groups. Specifically, cellulose membranes and perfluorocarbon sulfonic acid (PFS) based resin membranes, such as Nafion (registered trademark), which is a copolymer of tetrafluoroethylene and perfluoro[2-(fluorosulfonylethoxy)propylvinyl ether], can be used.

[0158] [Bioreactor]

[0159] The enzyme electrode of the present invention can be used as a bioreactor. A bioreactor equipped with the enzyme electrode of the present invention is also part of the present invention.

[0160] There are no particular limitations as long as the above-mentioned bioreactor functions as the reaction site between the enzyme electrode of the present invention and the reactants. However, in one embodiment, it is preferred that the enzyme electrode of the present invention is provided in a column reactor.

[0161] In one of the above methods, if a solution containing reactants is fed into a column reactor and brought into contact with an enzyme electrode, a product is obtained from the reactants through an enzymatic reaction utilizing the oxidoreductase contained in the enzyme electrode of the present invention.

[0162] There are no particular restrictions on the reactants used in the above-mentioned bioreactors, as long as they are substrates that are oxidized or reduced by oxidoreductases. Specifically, the above-mentioned substrates can be cited as examples.

[0163] The following information is disclosed in this specification.

[0164] <1>

[0165] An enzyme electrode is an electrode containing an enzyme.

[0166] The electrode comprises an electrode substrate, an oxidoreductase, a combination of a silane coupling agent and an electron mediator, and a sol-gel matrix.

[0167] The oxidoreductase and its conjugate are immobilized on the electrode substrate using a sol-gel matrix.

[0168] This silane coupling agent has silicon atoms, reactive functional groups, and hydrolyzable groups. It is a structure in which silicon atoms and reactive functional groups are linked by linking groups with 4 or more carbon atoms.

[0169] <2>

[0170] According to the enzyme electrode described in <1>, the above-mentioned oxidoreductase includes NAD(P)H or NAD(P)-dependent oxidoreductase and myocardial flavin.

[0171] <3>

[0172] According to the enzyme electrode described in <1> or <2>, the electron mediators include NAD(P)H and / or NAD(P) and other electron mediators.

[0173] <4>

[0174] The enzyme electrode according to any one of <1> to <3>, wherein the silane coupling agent has a positive or negative charge.

[0175] <5>

[0176] The enzyme electrode according to any one of <1> to <4>, wherein the sol-gel matrix contains a silane compound.

[0177] <6>

[0178] A method for manufacturing an enzyme electrode, which is a method for manufacturing an enzyme electrode.

[0179] The manufacturing method includes an immobilization step, in which a conjugate of oxidoreductase and silane coupling agent with an electron mediator is immobilized on an electrode substrate using a sol-gel matrix.

[0180] This silane coupling agent has silicon atoms, reactive functional groups, and hydrolyzable groups. It is a structure in which silicon atoms and reactive functional groups are linked by linking groups with 4 or more carbon atoms.

[0181] <7>

[0182] According to the method for manufacturing the enzyme electrode described in <6>, the immobilization step involves using a photocurable material to solidify the sol-gel matrix.

[0183] <8>

[0184] A biosensor comprising any one of <1> to <5>.

[0185] <9>

[0186] A bio-battery comprising any one of <1> to <5>.

[0187] <10>

[0188] A bioreactor comprising any one of <1> to <5>.

[0189] Example

[0190] The following describes embodiments that illustrate the invention in more detail. It should be noted that the invention is not limited to these embodiments.

[0191] <Cyclic Voltammetry (CV) Determination>

[0192] CV measurements were performed under the following conditions.

[0193] The CV measurements were performed using a 1280Z electrochemical measurement system (Solartron Analytical). A solution was added to the electrochemical measurement cell and maintained at 37°C. Platinum (counter electrode), Ag / AgCl (reference electrode), and the working electrode were immersed in the cell. Nitrogen gas was purged for 10 minutes to remove dissolved oxygen from the solution. Then, the voltage was continuously varied relative to Ag / AgCl at a scan rate of 5 mV / sec between -0.6 V and +0.6 V.

[0194] <Mechanical Strength Determination: Checkerboard Peel Test>

[0195] The checkerboard peeling test was performed using the micro-scratching method.

[0196] After adding the constituent droplets to the glass substrate and drying it overnight at 4°C, the substrate was scratched with 25 μmR and 10 mN, and the results were judged according to the following criteria.

[0197] 〇: Even after micro-scratching, the base layer was not exposed.

[0198] ×: Exposed base

[0199] <Manufacturing Example 1: A combination of NAD and GOS>

[0200] 25 mg of NAD (manufactured by Fujifilm and Kohden Chemical Co., Ltd.) and 48.6 mg of 8-epoxypropoxyoctyltrimethoxysilane (manufactured by Shin-Etsu Chemical Co., Ltd., hereinafter also referred to as GOS) were mixed in 400 μL of 0.1 M Tris-HCl buffer (pH 7.5) and stirred for 7 hours to obtain the NAD-GOS conjugate (hereinafter also referred to as NAD-GOS).

[0201] <Manufacturing Example 2: A combination of ANQ and GOS>

[0202] 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 the ANQ-GOS conjugate (hereinafter also referred to as ANQ-GOS).

[0203] <Comparative Manufacturing Example 1: A Combination of NAD and GPS>

[0204] 25 mg of NAD and 37.5 mg of 3-epoxypropoxypropylethoxysilane (manufactured by Shin-Etsu Chemical Industry Co., Ltd., hereinafter also referred to as GPS) were mixed in 400 μL of 0.1 M Tris-HCl buffer (pH 7.5) and stirred for 7 hours to obtain the NAD-GPS conjugate (hereinafter also referred to as NAD-GPS).

[0205] <Comparative Manufacturing Example 2: The Combination of ANQ and GPS>

[0206] ANQ 25 mg and GPS 37.5 mg were mixed in 400 μL of 0.1 M Tris-HCl buffer (pH 7.5) and stirred for 7 hours to obtain the ANQ-GPS conjugate (hereinafter also referred to as ANQ-GPS).

[0207] <Manufacturing Example 3: Sol-Gel Matrix (I)>

[0208] 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 sol-gel matrix (I) (hereinafter also referred to as Sol (I)).

[0209] <Manufacturing Example 4: Sol-Gel Matrix (II)>

[0210] 0.16 g of TEOS, 0.01 g of methyltriethoxysilane (manufactured by Shin-Etsu Chemical Industry Co., Ltd., hereinafter also referred to as MTES), 0.01 g of dimethyldiethoxysilane (manufactured by Shin-Etsu Chemical Industry 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 sol-gel matrix (II) (hereinafter also referred to as Sol(II)).

[0211] <Manufacturing Example 5: 10% Polyethyleneimine Solution>

[0212] Polyethyleneimine (manufactured by Fujifilm and Koh Genuine Chemicals Co., Ltd., molecular weight 10,000, hereinafter also referred to as PEI) was mixed with pure water, and the pH was adjusted to 9 with hydrochloric acid to obtain a 10% polyethyleneimine solution (hereinafter also referred to as PEIaq).

[0213] <Example 1>

[0214] 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 myocardial flavonoid (manufactured by Fujifilm and Koko Pure Chemicals Co., Ltd., hereinafter also referred to as DI), 10 μL of NAD-GOS, 20 μL of Sol(I) and 10 μL of PEIaq were added sequentially to an Eppendorf tube and stirred. Then, 10 μL of the resulting composition was immediately added dropwise to a glassy carbon electrode (hereinafter also referred to as GC electrode) and dried overnight at 4°C to obtain enzyme electrode 1.

[0215] <Comparative Example 1>

[0216] 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 PEIaq were added sequentially to an Eppendorf tube and stirred. Then, 10 μL of the resulting composition was immediately added dropwise to the GC electrode and dried overnight at 4 °C to obtain the comparative enzyme electrode 1.

[0217] For the enzyme electrodes obtained in Example 1 and Comparative Example 1, cyclic voltammetry (CV) was performed using electrochemical assay cell 1 containing 10 mL of 0.1 M NH4OH-NH4Cl buffer (pH 9.0) + 500 μL of glycerol + 10 mg of ANQ. The results are shown below. Figure 3 .exist Figure 3 In the text, solid lines represent Example 1, and dashed lines represent Comparative Example 1.

[0218] according to Figure 3As a result, the catalyst current value was increased in Example 1 compared to Comparative Example 1. It is believed that in Example 1, the mobility of NAD was improved by combining a silane coupling agent having a linking group having 4 or more carbon atoms with NAD, thereby increasing the reaction rate of NAD with GLDH and DI.

[0219] <Example 2>

[0220] 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 PEIaq were added sequentially to an Eppendorf tube and stirred. Then, 10 μL of the resulting composition was immediately added dropwise to the GC electrode and dried overnight at 4 °C to obtain enzyme electrode 2.

[0221] <Example 3>

[0222] 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 PEIaq were added sequentially to an Eppendorf tube and stirred. Then, 10 μL of the resulting composition was immediately added dropwise to the GC electrode and dried overnight at 4 °C to obtain enzyme electrode 3.

[0223] For the enzyme electrodes obtained in Examples 2 and 3, cyclic voltammetry (CV) was performed using electrochemical assay cell 2 containing 10 mL of 0.1 M NH4OH-NH4Cl buffer (pH 9.0) + 500 μL of glycerol. Measurements were performed immediately after immersion of the enzyme electrode in electrochemical assay cell 2 and 5 hours after immersion. The results are shown below. Figure 4 and Figure 5 .exist Figure 4 and Figure 5 In the diagram, solid lines represent the results immediately after immersion, and dashed lines represent the results 5 hours after immersion.

[0224] Regarding the measurement results, in Examples 2 and 3, the catalyst current values ​​immediately after immersing the enzyme electrode in the electrochemical measurement cell 2 were 2.1 × 10⁻⁶. -5 A, 3.3 × 10 -5 A. Additionally, the catalyst current values ​​in Examples 2 and 3, 5 hours after impregnation, were 1.2 × 10⁻⁶. -6 A, 5.4 × 10 -6 A. In Example 3, the catalyst current value increased both immediately after impregnation and 5 hours later.

[0225] The compositions coated on the electrode surfaces of the enzyme electrodes obtained in Examples 2 and 3 were analyzed using small-angle X-ray scattering (SAXS) with Rigaku NANOPIX. The results are shown below. Figure 6 .exist Figure 6 In the diagram, Example 2 is represented by a dashed line, and Example 3 by a solid line. The results show that the gel of Example 2, containing only a 4-functional alkoxysilane, had an average pore size of 10 nm (forming a mesh structure with a 10 nm period). In contrast, the gel of Example 3, which further contained 2-functional and 3-functional alkoxysilanes, had an average pore size of 14 nm (forming a mesh structure with a 14 nm period), confirming the expansion of the molecular network in the sol-gel matrix of Example 3.

[0226] <Example 4>

[0227] In the Sol(I) prepared in Manufacturing Example 3, 0.1 g of polyethylene glycol dimethacrylate (manufactured by Fujifilm and Kojun Chemical Co., Ltd., hereinafter also referred to as PEGDMA) and 1 mg of phenylbis(2,3,4-trimethylbenzoyl)phosphine oxide (manufactured by IGMRESINS) were added to obtain Sol(I'). Using the obtained Sol(I') instead of Sol(I), 10 μL of the prepared composition was dropped onto a glass substrate in the same manner as in the examples, and after drying, it was irradiated with ultraviolet light. A checkerboard peel test was performed on the obtained glass substrate, and the mechanical strength of the matrix was evaluated according to the above evaluation criteria. The evaluation result was 0.

[0228] By using photocurable materials in the sol-gel matrix, the mechanical strength of the matrix is ​​improved, making it less prone to peeling off from the electrode surface. Therefore, the electrode durability is considered to be improved.

[0229] Symbol Explanation

[0230] 1. Enzyme electrode

[0231] 2 Electrode substrate

[0232] 3a Oxidoreductase (a)

[0233] 3b Oxidoreductase (b)

[0234] 4. Silane coupling agent

[0235] 5a Electron mediator (a)

[0236] 5b Electron mediator (b)

[0237] 6a A combination of silane coupling agent and electron mediator (a)

[0238] 6b The combination of silane coupling agent and electron mediator (b)

[0239] 7 Sol-gel matrix

[0240] 8. Substrate

Claims

1. An enzyme electrode, which is an electrode containing an enzyme. The electrode comprises an electrode substrate, an oxidoreductase, a combination of a silane coupling agent and an electron mediator, and a sol-gel matrix. The oxidoreductase and its conjugate are immobilized on the electrode substrate using a sol-gel matrix. This silane coupling agent has silicon atoms, reactive functional groups, and hydrolyzable groups. It is a structure in which silicon atoms and reactive functional groups are linked by linking groups with 4 or more carbon atoms.

2. The enzyme electrode according to claim 1, wherein, The oxidoreductases include NAD(P)H or NAD(P)-dependent oxidoreductases and myocardial flavins.

3. The enzyme electrode according to claim 1 or 2, wherein, The electron mediators include NAD(P)H and / or NAD(P) as well as other electron mediators.

4. The enzyme electrode according to any one of claims 1 to 3, wherein, The silane coupling agent has a positive or negative charge.

5. The enzyme electrode according to any one of claims 1 to 4, wherein, The sol-gel matrix contains silane compounds.

6. A method for manufacturing an enzyme electrode, which is a method for manufacturing an enzyme electrode. The manufacturing method includes an immobilization process in which oxidoreductases and complexes of silane coupling agents and electron mediators are immobilized on an electrode substrate using a sol-gel matrix. This silane coupling agent has silicon atoms, reactive functional groups, and hydrolyzable groups. It is a structure in which silicon atoms and reactive functional groups are linked by linking groups with 4 or more carbon atoms.

7. The method for manufacturing an enzyme electrode according to claim 6, wherein, The immobilization process uses a photocurable material to solidify the sol-gel matrix.

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

9. A bio-battery 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.

Images