Enzyme electrode, method for manufacturing same, and enzyme sensor

Abstract

Disclosed is a method for manufacturing an enzyme electrode. This method for manufacturing an enzyme electrode comprises: a step for preparing a laminate including an electrode and a mediator layer provided on the surface of the electrode and including a mediator crosslinked by a crosslinking agent; and a step for forming a reaction layer by adding a solution containing an enzyme to the mediator layer of the laminate.

Description

Enzyme electrode, its manufacturing method, and enzyme sensor

[0001] The present disclosure relates to an enzyme electrode, a method for manufacturing the same, and an enzyme sensor.

[0002] An enzyme electrode is an electrode on whose surface an enzyme, which is a catalyst for promoting chemical reactions (metabolism) in the body, is immobilized. By using an enzyme electrode, only a specific reaction proceeds selectively on the electrode due to the enzyme's substrate specificity, and the change in substance caused by the reaction can be converted into an electrical signal by the electrode. Enzyme electrodes are used as electrodes for biobatteries, biosensors, etc. Enzyme electrodes usually have a reaction layer containing an enzyme, a mediator, etc.

[0003] The mediator is a compound with redox ability that mediates electron transfer between the enzyme and the electrode. Therefore, if the mediator is desorbed from the reaction layer of the enzyme electrode, problems such as a decrease in the output of the enzyme electrode may occur.

[0004] One known method for suppressing the detachment of a mediator is to use a redox polymer as a mediator, in which a hydrophilic polymer arm containing a mediator is bound to a hydrophobic polymer main chain (see, for example, Patent Document 1).

[0005] Japanese Patent Application Laid-Open No. 2006-078468

[0006] However, in conventional enzyme electrodes, the polymer design required to suppress mediator desorption is complicated, and a simpler method for suppressing mediator desorption is desired.

[0007] Therefore, a main object of the present disclosure is to provide a method for producing an enzyme electrode that can suppress detachment of a mediator from a reaction layer with a simpler design than conventional methods.

[0008] The present inventors conducted extensive research to solve the above-mentioned problems and discovered that crosslinking the mediator with a crosslinking agent suppresses detachment from the reaction layer, thereby completing the presently disclosed invention. Conventional redox polymers are designed to facilitate the mediator's entry into the active center of the enzyme by placing a linker (crosslinking group) of a certain length between the hydrophobic polymer and the mediator. This is because, in order for the mediator to receive and transfer electrons from the enzyme, the coenzyme and the mediator must be close to each other. Therefore, when the mediator itself is polymerized, the polymer of the mediator may have difficulty entering the active center of the enzyme, making it difficult to obtain a sufficient electrical signal. The present inventors' research confirmed that an electrode containing a mediator crosslinked with a crosslinking agent can obtain a sufficient electrical signal. From this, it can be inferred that the mediator can adequately enter the active center of the enzyme when a mediator crosslinked with a crosslinking agent is used.

[0009] The present disclosure provides a method for producing an enzyme electrode according to [1] to [4], an enzyme electrode according to [5] to [7], and an enzyme sensor according to [8]. [1] A method for producing an enzyme electrode, comprising the steps of: preparing a laminate including an electrode and a mediator layer provided on the surface of the electrode, the mediator layer including a mediator cross-linked by a cross-linking agent; and adding a solution containing an enzyme to the mediator layer of the laminate to form a reaction layer. [2] The method for producing an enzyme electrode according to [1], wherein the laminate is produced by arranging a mediator cross-linked by the cross-linking agent, which has been synthesized in advance, on the surface of the electrode. [3] The method for producing an enzyme electrode according to [1] or [2], wherein the mediator is a compound having two or more amino groups in one molecule. [4] The method for producing an enzyme electrode according to [3], wherein the cross-linking agent is a compound having two or more reactive groups in one molecule that are reactive with the amino groups. [5] An enzyme electrode comprising: an electrode; and a reaction layer provided on a surface of the electrode, wherein the reaction layer contains an enzyme and a mediator crosslinked by a crosslinking agent. [6] The enzyme electrode according to [5], wherein the mediator is a compound having two or more amino groups per molecule. [7] The enzyme electrode according to [6], wherein the crosslinking agent is a compound having two or more reactive groups per molecule that are reactive with the amino groups. [8] An enzyme sensor having the enzyme electrode according to any one of [5] to [7] as a working electrode.

[0010] The present disclosure provides an enzyme electrode that can suppress detachment of a mediator from a reaction layer with a simpler design than conventional methods, a method for manufacturing the same, and an enzyme sensor having such an enzyme electrode as a working electrode.

[0011] Fig. 1 is a graph showing the results of chronoamperometry measurement of the GDH electrode of Example 1. Fig. 2 shows the sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

[0012] Hereinafter, embodiments of the present disclosure will be described in detail, but the present disclosure is not limited to the following embodiments.

[0013] In this specification, a numerical range indicated using "to" indicates a range that includes the numerical values ​​before and after "to" as the minimum and maximum values, respectively. In the numerical ranges described in stages in this specification, the upper or lower limit of a numerical range in a certain stage may be replaced with the upper or lower limit of a numerical range in another stage. Furthermore, in the numerical ranges described in this specification, the upper or lower limit of the numerical range may be replaced with a value shown in the examples.

[0014] In this specification, unless otherwise specified, the materials exemplified below may be used alone or in combination of two or more within the range that meets the conditions. When multiple substances corresponding to each component are present, the content of each component means the total amount of the multiple substances unless otherwise specified.

[0015] [Enzyme Electrode] The enzyme electrode of this embodiment includes an electrode and a reaction layer provided on the surface of the electrode. The enzyme electrode may further include a protective film layer provided so as to cover the reaction layer.

[0016] <Electrode (Working Electrode)> The electrode may be a known electrode used in the relevant field. Examples of the electrode include carbon electrodes; metal electrodes such as Pt, Au, Ag, Ni, and Pd. These electrodes may further contain metal fine particles containing at least one element selected from the group consisting of Co, Pd, Rh, Ir, Ru, Os, Re, Ni, Cr, Fe, Mo, Ti, Al, Cu, V, Nb, Zr, Sn, In, Ga, Mg, Pb, Au, Pt, and Ag as a conductive material. These metal fine particles may be alloys or plated. The electrode may be, for example, a carbon electrode. Examples of materials constituting the carbon electrode include pyrolytic graphite carbon (PG), glassy carbon (GC), carbon paste, and plastic-formed carbon (PFC).

[0017] <Insulating Substrate> The enzyme electrode may further include an insulating substrate, and the electrode may be formed on the insulating substrate. Examples of materials for the insulating substrate include thermoplastic resins such as polyetherimide (PEI), polyethylene terephthalate (PET), and polyethylene (PE); resins (plastics) such as polyimide resin and epoxy resin; and insulating materials such as glass, ceramic, and paper.

[0018] <Reaction Layer> The reaction layer contains an enzyme and a mediator cross-linked by a cross-linking agent (hereinafter, may be referred to as a "cross-linked mediator"). The reaction layer may further contain, for example, a polymer electrolyte.

[0019] (Enzymes) The enzyme is not particularly limited as long as it has substrate specificity for the analyte substance. Examples of enzymes include various oxidoreductases classified into EC group 1. More specific examples include dehydrogenases, oxidases, reductases, etc. Examples of dehydrogenases include glucose dehydrogenase, alcohol dehydrogenase, glutamate dehydrogenase, cholesterol dehydrogenase, aldehyde dehydrogenase, fructose dehydrogenase, sorbitol dehydrogenase, D- or L-lactate dehydrogenase, malate dehydrogenase, glycerol dehydrogenase, D- or L-amino acid dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, 3-hydroxysteroid dehydrogenase, pyranose dehydrogenase, formate dehydrogenase, phosphate dehydrogenase, and diaphorase. Examples of oxidases include glucose oxidase, galactose oxidase, pyruvate oxidase, D- or L-amino acid oxidase, amine oxidase, cholesterol oxidase, choline oxidase, bilirubin oxidase, xanthine oxidase, sarcosine oxidase, D- or L-lactate oxidase, ascorbic acid oxidase, cytochrome oxidase, NADH (nicotinamide adenine dinucleotide) oxidase, pyranose oxidase, polyamine oxidase, amadoriase (also called fructosyl peptide oxidase or fructosyl amino acid oxidase), peroxidase, laccase, etc. Examples of reductases include catalase, glutathione reductase, cytochrome b5 reductase, adrenoxin reductase, nitrate reductase, etc.

[0020] The enzyme may be, for example, at least one selected from the group consisting of dehydrogenase, oxidase, and reductase, or at least one selected from the group consisting of dehydrogenase and oxidase. In one embodiment, the enzyme may be a dehydrogenase, such as glucose dehydrogenase (GDH) or lactate dehydrogenase (LDH). In one embodiment, the enzyme may be an oxidase, such as lactate oxidase (LOX) or NADH oxidase (NOX).

[0021] Examples of coenzymes for the above enzymes include nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), pyrroloquinoline quinone (PQQ), etc. For example, glucose dehydrogenase (GDH) having a coenzyme includes FAD-dependent GDH, NAD-dependent GDH, NADP-dependent GDH, PQQ-dependent GDH, etc. The coenzyme may be, for example, flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN), and the enzyme may be, for example, a flavin enzyme having these coenzymes.

[0022] The activity of these oxidoreductases can be measured using various substrates, for example, by the method described in Methods in Enzymology (vols. 1-602).

[0023] The enzyme content in the reaction layer is calculated based on the total amount of the reaction layer, and is expressed as the amount of the enzyme per 1 cm of the area of ​​the electrode (working electrode). ２ The effective area of ​​the electrode (working electrode) may be 0.001 U or more, 0.01 U or more, 0.1 U or more, 1 U or more, or 10 U or more, and may be 10 kU or less, 1 kU or less, or 500 U or less. These values ​​are calculated based on the effective area of ​​the electrode (working electrode) per cm. ２ When using carbon nanotubes, graphene, or the like with a large specific surface area, the effective area may increase even if the geometric area remains the same. Hereinafter, the area of ​​the electrode (working electrode) is 1 cm ２ The content per unit is similar.

[0024] (Crosslinked Mediator) A crosslinked mediator is a mediator crosslinked by a crosslinking agent. A crosslinked mediator can also be considered a reaction product between a mediator and a crosslinking agent. It is presumed that crosslinking the mediator with a crosslinking agent reduces its solubility in an aqueous solution, improves its hydrophobicity, and strengthens the hydrophobic interaction with the electrode, thereby making it possible to suppress detachment of the mediator from the reaction layer.

[0025] - Mediator The mediator can be any compound that has redox ability to mediate electron transfer between the enzyme and the electrode and has two or more functional groups per molecule that can react with the reactive group of the crosslinker described below to form a crosslinked structure. The mediator may be a neutral molecule that does not have ionicity, or may be a salt composed of a cation and an anion. These may be acid addition salts or solvates. The salt is not particularly limited, but examples include Na salts, K salts, Cl salts, and Br salts. The acid addition salt is not particularly limited, but examples include acetates, hydrochlorides, sulfates, sulfites, and nitrates.

[0026] Examples of functional groups include amino groups and thiol groups. Among these, the functional group may be an amino group. The number of functional groups per molecule may be two or more, or may be 10 or less, 8 or less, 6 or less, or 4 or less, or may be 2. The mediator may be, for example, a compound having two functional groups per molecule.

[0027] The mediator may be, for example, a compound having two or more amino groups per molecule. The number of amino groups per molecule may be 10 or less, 8 or less, 6 or less, or 4 or less, or may be 2. The mediator may be, for example, a compound having two amino groups per molecule.

[0028] The compound having two or more amino groups per molecule may be a compound in which the amino groups do not contribute to a redox reaction. For example, it may be a compound in which, when the amino groups of the compound are substituted with hydrogen atoms, the hypothetical compound exhibits a redox potential. The crosslinked mediator forms a crosslinked structure by reaction between the amino group and the reactive group. Therefore, by using such a compound as a mediator, the resulting crosslinked mediator can exhibit sufficient redox ability.

[0029] The molecular weight of the mediator may be, for example, 100 or more, 150 or more, or 200 or more, and may be 1000 or less, 700 or less, or 500 or less.

[0030] The content of the mediator in the reaction layer is calculated based on the mediator monomolecules before crosslinking, and is calculated based on the total mass of the mediator per 1 cm of the electrode (working electrode) area. ２ The content of the mediator in the reaction layer may be 0.1 pmol or more, 0.2 pmol or more, 0.3 pmol or more, 0.4 pmol or more, 0.5 pmol or more, or 1 pmol or more, and may be 10 mmol or less, 5 mmol or less, 1 mmol or less, 800 μmol or less, 600 μmol or less, 500 μmol or less, 400 μmol or less, 300 μmol or less, 200 μmol or less, 100 μmol or less, or 50 μmol or less, per 1 cm of the area of ​​the electrode (working electrode). ２ For example, the amount may be 0.1 pmol to 10 mmol, 0.1 pmol to 5 mmol, 0.2 pmol to 1 mmol, 0.3 pmol to 800 μmol, 0.4 pmol to 600 μmol, 0.5 pmol to 500 μmol, 0.6 pmol to 400 μmol, 0.7 pmol to 300 μmol, 0.8 pmol to 200 μmol, 0.9 pmol to 100 μmol, or 1 pmol to 50 μmol.

[0031] The mediator may be, for example, a compound represented by formula (1): H ２ N-X-NH ２ In formula (1), X represents a divalent group.

[0032] The divalent group represented by X may be, for example, a divalent fused ring group containing a heteroatom as a constituent atom of the ring. The divalent fused ring group refers to a group obtained by removing two hydrogen atoms directly bonded to carbon atoms on the fused ring from a fused ring compound containing a heteroatom as a constituent atom of the ring. Examples of heteroatoms include a nitrogen atom, an oxygen atom, and a sulfur atom. The fused ring group may be an aromatic fused ring group having aromaticity. When the fused ring group has aromaticity, the effect of π-π interaction can be expected, making it possible to further suppress detachment of the mediator. The fused ring group may be tricyclic or more, and may be octacyclic or less, hexacyclic or less, or tetracyclic or less. The fused ring group may be, for example, tricyclic.

[0033] The divalent group represented by X may be, for example, a group that, when a compound (H-X-H) in which the amino group in compound (1) is substituted with a hydrogen atom is assumed, the assumed compound is a compound that exhibits a redox potential.

[0034] From the viewpoint of suppressing detachment from the reaction layer, the mediator is preferably a highly hydrophobic compound. The divalent group represented by X may be, for example, a group that, when a compound (H-X-H) in which the amino group in compound (1) is substituted with a hydrogen atom is assumed, the Log P calculated for the assumed compound is 1.5 or more. Note that Log P refers to the partition coefficient between water and octanol (1-octanol). A larger Log P indicates lower polarity and higher hydrophobicity. Log P may be, for example, 2.0 or more, 2.5 or more, or 3.0 or more.

[0035] In this specification, LogP means the value at 25°C calculated for the compound using the software ChemDraw (Prime 6.01, manufactured by PerkinElmer).

[0036] Specific examples of the mediator include a compound represented by the following formula (1-1) (thionine salt), a compound represented by the following formula (1-2) (acriflavine), and a compound represented by the following formula (1-3) (2,3-diaminophenazine). Log P values ​​calculated for compounds in which the amino group is substituted with a hydrogen atom are also shown below. The mediator may be, for example, one selected from the group consisting of a compound represented by the formula (1-1) (thionine salt), a compound represented by the formula (1-2) (acriflavine), and a compound represented by the formula (1-3) (2,3-diaminophenazine).

[0037]

[0038] Crosslinking Agent The crosslinking agent is not particularly limited as long as it is a compound having a reactive group capable of reacting with a functional group (for example, an amino group or a thiol group) of the mediator to form a crosslinked structure.

[0039] The crosslinking agent may be, for example, a compound having two or more reactive groups reactive with functional groups per molecule, or may be a compound having two or more reactive groups reactive with amino groups per molecule. Examples of reactive groups include groups containing cyclic ether groups (epoxy groups, glycidyl groups, glycidyl ether groups, etc.); formyl groups; carboxyl groups; azide groups; and active ester groups. Here, the term "active ester group" refers to a group obtained by esterifying a carboxyl group with a condensing agent such as N-hydroxysuccinimide (NHS) or a triazine-based condensing agent (DMT-MM). The reactive group may be, for example, at least one selected from the group consisting of groups containing cyclic ether groups, formyl groups, and active ester groups. The number of reactive groups per molecule may be 10 or less, 8 or less, 6 or less, or 4 or less, or may be 2. The crosslinking agent may be, for example, a compound having two reactive groups reactive with functional groups (amino groups) per molecule.

[0040] The molecular weight or number average molecular weight (Mn) of the crosslinking agent may be, for example, 25 or more, 50 or more, 100 or more, or 150 or more, and may be 100,000 or less, 50,000 or less, or 10,000 or less. Here, the number average molecular weight (Mn) is a converted value obtained by gel permeation chromatography (GPC) using a calibration curve of polyethylene glycol (PEG) / polyethylene oxide (PEO) (standard substance).

[0041] The crosslinking agent may be, for example, a compound represented by formula (2): Z-Y-Z. In formula (2), Y represents a divalent group having an alkylene group or a polyoxyalkylene chain, and Z represents a reactive group. Multiple Zs may be the same or different.

[0042] The alkylene group may be, for example, a linear or branched alkylene group having 1 to 50 carbon atoms. The number of carbon atoms in the alkylene group may be 2 to 30 or 3 to 20. The alkylene group may be a linear alkylene group.

[0043] The divalent group having a polyoxyalkylene chain is, for example, a group represented by formula (3): —O—(Y １ -O) ｎ１ In formula (3), Y may be a group represented by １ represents an alkylene group having 1 to 3 carbon atoms, and n1 represents an integer of 1 or more. n1 may be, for example, 2 or more, and may be 1000 or less, or 500 or less.

[0044] Crosslinked Mediators Crosslinked mediators can be obtained, for example, by reacting amino groups of a mediator with reactive groups of a crosslinking agent in the presence of a solvent. In the reaction between the mediator and the crosslinking agent, the ratio of the total number of moles of reactive groups of the crosslinking agent to the total number of moles of amino groups of the mediator (total number of moles of reactive groups of the crosslinking agent / total number of moles of amino groups of the mediator) may be, for example, 0.1 or more, 0.2 or more, 0.5 or more, 1.0 or more, 3.0 or more, 5.0 or more, 10 or more, or 15 or more, and may be 50 or less, or 30 or less.

[0045] The solvent is not particularly limited as long as it dissolves the mediator and crosslinking agent. Examples of the solvent include water; alcohols such as methanol, ethanol, and 2-propanol; aromatic hydrocarbons such as toluene and xylene; aliphatic hydrocarbons such as hexane, heptane, and octane; alicyclic hydrocarbons such as cyclohexane and methylcyclohexane; ethers such as diethyl ether, di-n-propyl ether, diisopropyl ether, di-n-butyl ether, tert-butyl methyl ether, and tetrahydrofuran; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, and cyclohexanone; esters such as ethyl acetate, n-propyl acetate, isopropyl acetate, and n-butyl acetate; and mixtures thereof.

[0046] The solvent may contain other components such as a buffer for adjusting the pH of the reaction solution, such as potassium phosphate, MES (2-morpholinoethanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), acetic acid, and sodium acetate.

[0047] The reaction temperature when reacting the mediator with the crosslinking agent may be, for example, 20 to 200°C, and may be 30°C or higher, 40°C or higher, 50°C or higher, or 60°C or higher, and may be 150°C or lower, 130°C or lower, 110°C or lower, or 100°C or lower. The reaction time when reacting the mediator with the crosslinking agent may be, for example, 0.1 hours or longer, 0.5 hours or longer, or 1 hour or longer. The upper limit of the reaction time is not particularly limited, but may be, for example, 72 hours or shorter.

[0048] In the crosslinked mediator, the ratio of the total number of moles of structural units derived from the crosslinking agent (e.g., a compound having two reactive groups per molecule that are reactive with functional groups) to the total number of moles of structural units derived from the mediator (e.g., a compound having two functional groups per molecule) (total number of moles of structural units derived from the crosslinking agent / total number of moles of structural units derived from the mediator) may be, for example, 0.80 or more, 0.90 or more, or 0.95 or more, and may be 1.20 or less, 1.10 or less, or 1.05 or less.

[0049] The number average molecular weight (Mn) of the crosslinking mediator may be, for example, 250 or more, 500 or more, 1000 or more, 2000 or more, 3000 or more, 4000 or more, 5000 or more, 6000 or more, 7000 or more, 8000 or more, 9000 or more, 10000 or more, 15000 or more, 20000 or more, 30000 or more, 50000 or more, 100000 or more, 200000 or more, 500000 or more, or 1000000 or more. The number average molecular weight (Mn) of the crosslinking mediator may be, for example, 10,000,000 or less or 5,000,000 or less. Here, the number average molecular weight (Mn) is a converted value obtained by gel permeation chromatography (GPC) using a calibration curve of polyethylene glycol (PEG) / polyethylene oxide (PEO) (standard substance).

[0050] In the crosslinking mediator, the average number of structural units (or structural units derived from the mediator) derived from one mediator (e.g., a compound having two functional groups per molecule) and one crosslinking agent (e.g., a compound having two reactive groups per molecule that are reactive with functional groups) may be, for example, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, 100 or more, 1000 or more, 10,000 or more, or 100,000 or more. The average number of units may be, for example, 1,000 or less or 500 or less. Here, the average number of units can be calculated from the following formula (X): Average number of units = [number average molecular weight (Mn) of crosslinking mediator] / [molecular weight of structural units derived from one mediator and one crosslinking agent] (X)

[0051] In the crosslinked mediator, the maximum number of structural units (or structural units derived from the mediator) derived from one mediator (e.g., a compound having two functional groups per molecule) and one crosslinking agent (e.g., a compound having two reactive groups per molecule that are reactive with functional groups) may be, for example, 12 units or more, 16 units or more, 20 units or more, 40 units or more, 60 units or more, 80 units or more, 200 units or more, 400 units or more, 4000 units or more, 40,000 units or more, or 400,000 units or more. The maximum number of units may be, for example, 4,000 units or less or 2,000 units or less. Here, the maximum number of units can be calculated from the following formula (Y). The maximum molecular weight can be calculated using an approximation formula (third order) obtained from the retention time of the peak start and a standard substance. Maximum number of units = [maximum molecular weight of crosslinked mediator] / [molecular weight of structural units derived from one mediator and one crosslinking agent] (Y)

[0052] An example of the crosslinking mediator is a polymer having a structural unit represented by the following formula (A) (a condensation polymer of benzoquinone, formaldehyde, and piperazine hydrochloride). It is preferable that the crosslinking mediator does not include a polymer having a structural unit represented by formula (A).

[0053]

[0054] (Polyelectrolyte) The polyelectrolyte may be a component capable of forming a polyion complex encapsulating the enzyme. The polyelectrolyte may be, for example, a combination of a cationic polymer and an anionic polymer. By forming a polyion complex encapsulating the enzyme in the reaction layer, it becomes possible to suppress the detachment of the enzyme. The polyelectrolyte may have a charge opposite to the surface charge of the enzyme.

[0055] Cationic polymers refer to hydrophilic polymers having cationic groups in their constituent units. Examples of cationic polymers include polylysine, chitosan, polyethyleneimine, polyarginine, polyornithine, polyallylamine, polyvinylamine, and salts thereof. Anionic polymers refer to hydrophilic polymers having anionic groups in their constituent units. Examples of anionic polymers include polystyrene sulfonate, polyglutamic acid, polyacrylic acid, polyaspartic acid, hyaluronic acid, polyglucuronic acid, carboxymethylcellulose, and salts thereof.

[0056] Other examples of polyelectrolytes include, for example, polymers selected from the group consisting of polypyrrole (PPY), polythiophene (PT), polyaniline (PANI), poly(p-phenylene sulfide) (PPS), poly(acetylene) (PAC), poly(p-phenylene vinylene) (PPV), poly(3,4-ethylenedioxythiophene) (PEDOT), poly(fluorene), polyphenylene, propylene, polyazulene, polynaphthalene, polycarbazole, polyindole, polyazepine, or functionalized derivatives, precursors, or salts thereof, and combinations thereof. Polyelectrolytes can be natural, non-natural, modified, or synthetic.

[0057] (Other Components) The reaction layer may contain other components. Examples of the other components include enzyme stabilizers (e.g., organic acids such as malic acid, citric acid, and gluconic acid; amino acids such as lysine, arginine, glutamic acid, and glycine; sugars such as trehalose, xylitol, sucrose, dextrin, and dextran; water-soluble polymers such as polylysine, polyethyleneimine, and polyacrylic acid; and proteins such as serum albumin), dispersants (e.g., phosphate buffer, citrate buffer, and Good's buffer), crosslinkers (e.g., aldehyde group-containing compounds such as glutaraldehyde and formaldehyde; carbodiimide group-containing compounds such as hexamethylene diisocyanate and hydrogenated xylylene diisocyanate; m-maleimidobenzoyl-N-hydroxybenzoyl Examples of suitable additives include succinimide esters, sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate and other imide group-containing compounds, 2,2'-bis-(2-oxazoline), 2,2'-methylene-bis-(2-oxazoline) and other oxazoline group-containing compounds, rubitol polyglycidyl ether, polyglycerol polyglycidyl ether and other epoxy group-containing compounds), thickeners (Natrosol™, DEAE-dextran hydrochloride, etc.), film-forming aids (acetonitrile plasma-polymerized membranes, hydrophilic polymers such as carboxymethyl cellulose, methyl cellulose, amphiphilic polymers such as polyvinylpyrrolidone), curing agents, antifoaming agents, preservatives, pH adjusters, surfactants (Triton X-100, sodium dodecyl sulfate, perfluorooctanesulfonic acid, sodium stearate, etc.), and the like.

[0058] <Protective Film Layer> The protective film layer suppresses the detachment of enzymes, crosslinking mediators, and the like contained in the reaction layer. Furthermore, since the analyte substance present outside the protective film layer must penetrate into the protective film layer where the reaction layer is present, the protective film layer has pores that allow the analyte substance to pass through. The material forming the protective film may be a biocompatible material that does not or is difficult to adsorb proteins, cells, and the like, and may also be a biocompatible polymer. Examples of biocompatible polymers include poly-4-vinylpyridine, a copolymer of methyl methacrylate and hydroxyethyl methacrylate, a copolymer of butyl methacrylate and hydroxyethyl methacrylate, poly(2-methacryloyloxyethylphosphorylcholine-co-n-butyl methacrylate), polyurethane, and zwitterionic polymers (see, for example, JP 2019-84519 A and JP 2020-38059 A).

[0059] [Method for manufacturing enzyme electrode] The method for manufacturing an enzyme electrode of this embodiment includes a step (step (A)) of preparing a laminate including an electrode and a mediator layer containing a crosslinked mediator provided on the surface of the electrode, and a step (step (B)) of adding a solution containing an enzyme to the mediator layer of the laminate to form a reaction layer. The method for manufacturing an enzyme electrode may further include a step (step (C)) of forming a protective film layer so as to cover the formed reaction layer.

[0060] <Step (A)> In step (A), a laminate including a predetermined electrode and a mediator layer is prepared. The mediator layer is a layer that can become a reactive layer by containing an enzyme. The laminate may be prepared by disposing a pre-synthesized cross-linked mediator on the surface of the electrode, or by synthesizing the cross-linked mediator by reacting a cross-linking agent with a mediator on the surface of the electrode. The laminate may be prepared by disposing a pre-synthesized cross-linked mediator on the surface of the electrode. The synthesis conditions (reaction conditions) of the cross-linked mediator may be the same as those described above.

[0061] A method for disposing a cross-linked mediator on an electrode may include, for example, the steps of preparing a solution (or dispersion) A containing the cross-linked mediator and a solvent, applying the prepared solution A, and removing at least a portion of the solvent from the applied solution A. Examples of the solvent include the same solvents as those used in synthesizing the cross-linked mediator.

[0062] There are no particular limitations on the method for applying Solution A, and any conventionally known method can be used. The amount of the crosslinking mediator to be applied is appropriately adjusted so as to fall within a desired range, taking into consideration the concentration of Solution A and the like.

[0063] The method for removing at least a portion of the solvent from Solution A is not particularly limited, and may be, for example, a method of drying at 4 to 100°C, preferably 4 to 60°C, for 1 minute to 48 hours, preferably 5 minutes to 24 hours.

[0064] In this way, a laminate can be obtained that includes an electrode and a mediator layer containing a cross-linked mediator provided on the surface of the electrode. The electrode provided with the mediator layer may be washed with water, if necessary. By washing the electrode provided with the mediator layer with water, it is possible to remove insufficiently cross-linked low-molecular-weight mediators from the mediator layer.

[0065] <Step (B)> In step (B), a solution containing an enzyme is added to the mediator layer of the laminate to form a reaction layer containing the enzyme and a crosslinked mediator. The enzyme typically permeates the mediator layer and can be immobilized on the electrode via the crosslinked mediator. The method for adding the enzyme solution to the mediator layer may, for example, be a method comprising the steps of preparing a solution (or dispersion) B containing the enzyme and a solvent and applying the prepared solution B, and may further comprise the step of removing at least a portion of the solvent from the applied solution B, as necessary. Examples of the solvent include those similar to those used in synthesizing the crosslinked mediator.

[0066] Solution B may contain components other than the enzyme and the solvent. Examples of the components other than the enzyme and the solvent include the other components in the reaction layer described above.

[0067] There are no particular limitations on the method for applying Solution B, and any conventionally known method can be used. The amount of enzyme to be applied is appropriately adjusted so as to fall within a desired range, taking into consideration the concentration of Solution B and the like.

[0068] The method for removing at least a portion of the solvent from Solution B is not particularly limited, and may be, for example, a method of drying at 4 to 60° C. for 5 minutes to 24 hours.

[0069] After adding a solution containing an enzyme to the mediator layer of the laminate, if necessary, a solution containing a polymer electrolyte may be added to the mediator layer, and the polymer electrolyte and the crosslinked mediator may be mixed to form a polyion complex, thereby entrapping and immobilizing the enzyme. The method for adding the solution containing a polymer electrolyte to the mediator layer may, for example, be a method comprising the steps of preparing a solution (or dispersion) C containing a polymer electrolyte and a solvent, and applying the prepared solution C, and, if necessary, may further comprise the step of removing at least a portion of the solvent from the applied solution C. Examples of the solvent include those similar to those used in synthesizing the crosslinked mediator.

[0070] There are no particular limitations on the method for applying Solution C, and any conventionally known method can be used. The amount of polymer electrolyte to be applied is appropriately adjusted to fall within a desired range, taking into account the concentration of Solution C and the like.

[0071] The method for removing at least a portion of the solvent from Solution C is not particularly limited, and may be, for example, a method of drying at 4 to 60° C. for 1 minute to 24 hours.

[0072] In this way, an enzyme electrode can be produced which comprises an electrode and a reaction layer provided on the surface of the electrode, the reaction layer containing an enzyme and a mediator cross-linked by a cross-linking agent.

[0073] <Step (C)> In step (C), a protective film layer is formed so as to cover the formed reaction layer. The method for forming the protective film layer is not particularly limited as long as it covers the reaction layer, and may be, for example, a method including a step of preparing a solution containing a material for forming the protective film and a solvent, and a step of immersing the formed reaction layer in the prepared solution.

[0074] [Enzyme Sensor] The enzyme sensor of this embodiment has an enzyme electrode as a working electrode. Examples of analytes for the enzyme sensor include glucose, glucose derivatives, lactic acid, pyruvic acid, cholesterol, theophylline, alcohol, glutamic acid, 3-hydroxybutyric acid, ketones, ketone bodies, glutamine, creatinine, acetylcholine, cortisol, hydrogen peroxide, choline, glycerol, glycine, gluconic acid, sucrose, maltose, maltooligosaccharides, lactose, xylose, cellobiose, alditol, acetic acid, L-ascorbic acid, malic acid, lysine, ammonia, tyramine, histamine, arginine, alanine, phenylalanine, histidine, hyboxanthine, inosine, tryptophan, threonine, alanylglutamine, kynurenine, glycated amino acids, glycated peptides, and glycated proteins.

[0075] The present disclosure will be specifically described below using examples, but the present disclosure is not limited to these examples.

[0076] [Fabrication of Enzyme Electrode (GDH Electrode)] Example 1 First, in order to crosslink thionine molecules, 37 μL of thionine acetate solution (1.2 mg / mL) and 2 μL of glutaraldehyde solution (15% (w / v)), each prepared using 10 mM potassium phosphate buffer (pH 6.5), were mixed and reacted at 60° C. for 1 hour. 3 μL of the reaction solution was applied to the working electrode of a printed electrode and dried. The printed electrode was a working electrode (12.6 mm) made of carbon. ２SCREEN-PRINTED ELECTRODES (DRP-C110, manufactured by Metrohm) were used, on which a reference electrode made of silver was printed. Subsequently, 2 μL of glucose dehydrogenase (GDH, manufactured by Kikkoman Biochemifa Corporation) solution (10 mg / mL) and 0.2 μL of glutaraldehyde solution (1% (w / v)), each prepared using 10 mM potassium phosphate buffer (pH 6.5), were mixed, applied to the working electrode, and dried. After drying, the mixture was washed with ultrapure water to remove insufficiently crosslinked thionin. Subsequently, the printed electrode was immersed in poly-4-vinylpyridine dissolved in ethanol, pulled out, and then immersed again after 10 minutes and pulled out to form a protective film layer. This produced the GDH electrode of Example 1, which included a working electrode, a reaction layer, and a protective film layer.

[0077] Comparative Example 1-1 A GDH electrode of Comparative Example 1-1 was prepared in the same manner as in Example 1, except that the thionine molecules were not crosslinked and 3 μL of a thionine acetate solution (1.2 mg / mL) was applied to the working electrode of the printed electrode and dried.

[0078] Comparative Example 1-2 36 μL of thionine acetate solution (2.5 mg / mL), 5 μL of GDH solution (20 mg / mL), and 2 μL of glutaraldehyde solution (15% (w / v)) were mixed, and the thionine and GDH were crosslinked with a crosslinker. 7 μL of the mixture was applied to the working electrode of the printed electrode and dried. After drying for 20 hours, the electrode was washed with ultrapure water, and a protective film layer was formed in the same manner as in Example 1, thereby producing the GDH electrode of Comparative Example 1-2.

[0079] Evaluation of the Enzyme Electrode (GDH Electrode) Evaluation was performed using the GDH electrodes of Example 1 and Comparative Example 1-1. To perform chronoamperometry using a printed electrode, the printed electrode was connected to an ALS Electrochemical Analyzer 814D (manufactured by BAS) using a dedicated connector (DRP-CAC, manufactured by Drop Sense). Measurements were performed using a three-electrode system with the printed electrode as the working electrode, a platinum counter electrode, and a silver-silver chloride electrode as the reference electrode. 10 mL of PBS (phosphate-buffered saline) was used as the buffer solution. The applied voltage was +100 mV (vs. Ag / AgCl). Specifically, starting 60 seconds after the start of measurement, glucose was added every 60 seconds to a concentration of 2 to 20 mM, and the current response value was continuously measured. After measurement, the printed electrode was immersed in PBS and stored at 37°C. After one week, the printed electrode was removed and the amount of thionine contained in the PBS was measured. The amount of thionine was quantified by measuring absorbance at 600 nm using a spectrophotometer. 0.2 nmol of thionine was detected from the GDH electrode of Example 1, whereas 3.9 nmol of thionine was detected from the GDH electrode of Comparative Example 1-1. This indicates that the amount of thionine eluted is significantly reduced by cross-linking thionine molecules.

[0080] FIG. 1 is a graph showing the results of chronoamperometry measurements of the GDH electrode of Example 1. The current value at each glucose concentration is the current value after background correction, in which the current value at a glucose concentration of 0 mM is subtracted. In FIG. 1, a good linear relationship was confirmed between the glucose concentration and the current value in the glucose concentration range of 0 to 20 mM. Furthermore, the ratio of the current value at a glucose concentration of 20 mM was 104% before and after storage at 37°C for one week, indicating that the GDH electrode of Example 1 exhibited high durability (storage stability). This confirmed that the enzyme electrode of the present disclosure is capable of suppressing desorption of the mediator from the reaction layer.

[0081] Chronoamperometry measurements were also performed on the GDH electrode of Comparative Example 1-2. As a result, the current value reached saturation at a glucose concentration of 6 mM or higher. Therefore, when quantification is performed under these conditions, it is believed that the glucose concentration range in which the glucose concentration and the measured value show a valid linear relationship is 6 mM or lower. However, when considering the needs of practical glucose measurement, for example, when glucose measurement during cell culture is assumed, RPMI 1640 medium, which is commonly used as a culture medium, contains 2000 mg / dL (11.11 mM) of glucose. Taking this into consideration, measurements using the GDH electrode of Comparative Example 1-2 exceeded the measurable glucose concentration range from the start, confirming that the quantitative glucose concentration range is insufficient for practical use.

[0082] Furthermore, after the above measurements, the printed electrode of the GDH electrode of Comparative Example 1-2 was immersed in PBS and stored at 37 ° C. After one day had passed, the printed electrode was removed from the PBS and chronoamperometry measurements were performed again in the same manner as the previous day. The ratio of the current value at a glucose concentration of 6 mM before and after storage at 37 ° C for one day was significantly reduced. Specifically, if the measured value before storage at 37 ° C for one day was taken as 100%, the current value after storage for one day was reduced to only 2.3%. This confirmed that the GDH electrode of Comparative Example 1-2 had insufficient durability (storage stability).

[0083] [Preparation of Enzyme Electrodes (LDH Electrodes) Using Various Mediators] Example 2-1 The 1503-bp gene (including the stop codon TAA) shown in SEQ ID NO: 2, which encodes the amino acid sequence shown in SEQ ID NO: 1, was inserted into the multicloning site of the plasmid pKK223-3 by a standard method to obtain recombinant plasmid pKK223-3-LDH. The sequences of SEQ ID NO: 1 and SEQ ID NO: 2 are shown in Figure 2. Hereinafter, when simply referred to as "LDH," this refers to the protein shown in SEQ ID NO: 1.

[0084] The E. coli BL21 strain was used as the LDH-producing bacterium. First, E. coli BL21 transformed with the recombinant plasmid pKK223-3-LDH was cultured on an LB plate (containing 100 μg / mL ampicillin (Amp)). A colony of the strain was picked with a toothpick and cultured overnight at 30°C and 180 rpm in a small test tube containing 2.5 mL of LB medium (containing 100 μg / mL Amp). This culture was cultured in a Sakaguchi flask containing 250 mL of LB medium (containing 100 μg / mL Amp, 0.1 mM isopropyl-β-thiogalactopyranoside (hereinafter referred to as "IPTG")) with shaking at 30°C and 130 rpm for 24 hours. After completion of the culture, the culture was centrifuged at 8500 rpm at 4°C for 5 minutes, the supernatant was removed, and the bacterial cells were collected. The resulting bacterial cells were then suspended in 30 mL of 150 mM potassium phosphate buffer (pH 6.5). The above bacterial cell suspension was sonicated using an ultrasonic homogenizer US-150E (manufactured by Nippon Seiki Seisakusho) until the suspension became translucent, and then heated at 50°C for 20 minutes to denature contaminating proteins. This treated solution was centrifuged at 8,500 rpm and 4°C for 5 minutes to recover the supernatant. The resulting supernatant was purified using an AKTA avant 25 (manufactured by Cytiva). First, it was applied to 5 mL of Q Sepharose Fast Flow resin (manufactured by Cytiva) equilibrated with 20 mM potassium phosphate buffer (pH 7.0) and adsorbed to the resin. Next, proteins not adsorbed to the resin were eluted with 20 mM potassium phosphate buffer containing 200 mM sodium chloride. Subsequently, the adsorbed LDH was eluted using 20 mM potassium phosphate buffer (pH 7.0) containing 350 mM sodium chloride. The resulting LDH was recovered, and the enzyme solution was replaced with 20 mM potassium phosphate buffer (pH 7.0) containing 200 mM sodium chloride, after which it was re-adsorbed onto 5 mL of Q Sepharose Fast Flow resin equilibrated with the same buffer. Next, the LDH adsorbed to the resin was eluted and recovered by gradually increasing the NaCl concentration using a gradient up to 350 mM NaCl / 20 mM potassium phosphate buffer (pH 7.0). The resulting fraction was replaced with 10 mM potassium phosphate buffer (pH 6.5).This LDH solution was analyzed by SDS-PAGE to confirm that it was purified to a degree that it contained no other contaminating proteins, and was used as a purified LDH preparation (purified LDH).

[0085] To crosslink thionine molecules, glutaraldehyde (final concentration: 20 mM) and thionine acetate (final concentration: 20 mM) were reacted in 10 mM potassium phosphate buffer (pH 6.5) at 45°C for 1 hour. The reaction solution was diluted 2-fold with 10 mM potassium phosphate buffer (pH 6.5), and 4 μL of the diluted solution was applied to the working electrode of SCREEN-PRINTED ELECTRODES (DRP-C110, manufactured by Metrohm) and dried. After drying, the solution was washed with ultrapure water to remove thionine that was not immobilized on the working electrode. Next, purified LDH at a final concentration of 3.8 mg / mL, polylysine at a final concentration of 0.1% (w / v), and polyethylene glycol diglycidyl ether (PEGDGE, Mn: 6000) at a final concentration of 1% (w / v) were mixed in 10 mM potassium phosphate buffer (pH 6.5), and 10 μL of this mixture was applied to the working electrode for immobilization. Next, 10 μL of a 0.1% (w / v) sodium polyacrylate solution was applied to the working electrode and dried. Subsequently, the printed electrode was immersed in poly-4-vinylpyridine dissolved in ethanol, pulled out, dried, and then immersed again and pulled out to form a protective film layer. This produced the LDH electrode of Example 2-1, which included a working electrode, a reaction layer, and a protective film layer.

[0086] Example 2-2 An LDH electrode of Example 2-2 was prepared in the same manner as in Example 2-1, except that thionine was changed to acriflavine as the mediator.

[0087] Example 2-3 An LDH electrode of Example 2-3 was prepared in the same manner as in Example 2-1, except that thionine was changed to 2,3-diaminophenazine as the mediator.

[0088] Comparative Example 2-1 An LDH electrode of Comparative Example 2-1 was prepared in the same manner as in Example 2-1, except that the thionine molecules were not crosslinked and 4 μL of a thionine acetate solution (10 mM) was applied to the working electrode of the printed electrode and dried.

[0089] Comparative Example 2-2 An LDH electrode of Comparative Example 2-2 was prepared in the same manner as in Example 2-2, except that the acriflavine molecules were not crosslinked and 4 μL of an acriflavine solution (10 mM) was applied to the working electrode of the printed electrode and dried.

[0090] Comparative Example 2-3 An LDH electrode of Comparative Example 2-3 was prepared in the same manner as in Example 2-3, except that the 2,3-diaminophenazine molecules were not crosslinked and 4 μL of a 2,3-diaminophenazine solution (10 mM) was applied to the working electrode of the printed electrode and dried.

[0091] [Evaluation of enzyme electrodes (LDH electrodes) using various mediators] Using the LDH electrodes of Examples 2-1 to 2-3 and Comparative Examples 2-1 to 2-3, the effect of inhibiting detachment from the electrode surface was evaluated by electrochemical measurement. Specifically, chronoamperometry was performed using the LDH electrodes, and the coefficient of determination (R ２ The sodium lactate concentration was 0, 2, 4, 6, 8, 10, 12, 14, and 16 mM. When a sufficient amount of mediator is present on the working electrode, the response current is proportional to the lactic acid concentration. ２ On the other hand, if the mediator is desorbed and not present on the working electrode in sufficient quantity, the current value will saturate at a certain lactic acid concentration, and R ２ The values ​​were far from 1. After storing the LDH electrodes of Examples 2-1 to 2-3 and Comparative Examples 2-1 to 2-3 in a PBS solution at 37° C. for 4 days, the R ２ The value was calculated and R ２The effect of inhibiting detachment from the electrode surface was evaluated by comparing the values. For chronoamperometry measurements, the printed electrode was connected to a high-sensitivity potentiostat analyzer ECstat-400 (EC Frontier) using a dedicated connector (DRP-CAC, manufactured by Drop Sense). Measurements were performed using a three-electrode system with the printed electrode as the working electrode, a platinum counter electrode, and a silver-silver chloride electrode as the reference electrode. A 100 mM potassium phosphate buffer solution (pH 7.5) was used as the buffer solution. The applied voltage was +100 mV (vs. Ag / AgCl). Specifically, sodium lactate was added every 60 seconds from 120 seconds after the start of measurement to a concentration of 0 to 16 mM, and the current response value was continuously measured. The results are shown in Table 1.

[0092]

[0093] Comparing the mediator and the cross-linked mediator, the LDH electrode of the example using the cross-linked mediator has a higher R than the LDH electrode of the comparative example not using the cross-linked mediator. ２ The value was close to 1. This confirmed that crosslinking the mediator suppressed the detachment of the mediator. It was also found that the effect of suppressing detachment from the electrode surface does not tend to be largely dependent on the type of mediator in the crosslinked mediator.

[0094] [Preparation of Enzyme Electrodes (LDH Electrodes) Using Various Crosslinking Agents] Example 3-1 An LDH electrode of Example 3-1 comprising a working electrode, a reaction layer, and a protective film layer was prepared in the same manner as in Example 2-1, except that glutaraldehyde at a final concentration of 20 mM was replaced with ethylene glycol diglycidyl ether at a final concentration of 20 mM.

[0095] Example 3-2 An LDH electrode of Example 3-2 comprising a working electrode, a reaction layer, and a protective film layer was prepared in the same manner as in Example 2-1, except that glutaraldehyde at a final concentration of 20 mM was replaced with suberic acid bis(3-sulfo-N-hydroxysuccinimide ester) sodium salt (BS3) at a final concentration of 20 mM.

[0096] [Evaluation of enzyme electrodes (LDH electrodes) using various crosslinking agents] The LDH electrodes of Examples 3-1 and 3-2 were used to evaluate the effect of inhibiting detachment from the electrode surface by electrochemical measurement (chronoamperometry measurement). The chronoamperometry measurement was performed in the same manner as in Example 2-1, except that the LDH electrodes of Examples 3-1 and 3-2 were used instead of the LDH electrode of Example 2-1. The results are shown in Table 2. Table 2 also shows the results of Example 2-1.

[0097]

[0098] The LDH electrode of Example 3-1 using ethylene glycol diglycidyl ether as a crosslinking agent and the LDH electrode of Example 3-2 using BS3 were similar to the LDH electrode of Example 2-1 using glutaraldehyde. ２ The values ​​were similar. This confirmed that the detachment of the mediator from the LDH electrodes of Examples 3-1 and 3-2 was suppressed. It was also found that the effect of suppressing detachment from the electrode surface does not tend to be largely dependent on the type of cross-linking agent in the cross-linked mediator.

[0099] [Preparation of Enzyme Electrodes (LDH Electrodes) Using Various Crosslinkers with Different Molecular Lengths] Example 4-1 An LDH electrode of Example 4-1 including a working electrode, a reaction layer, and a protective film layer was prepared in the same manner as in Example 2-1, except that glutaraldehyde at a final concentration of 20 mM was replaced with ethylene glycol diglycidyl ether (molecular weight: 218) at a final concentration of 20 mM. The LDH electrode of Example 4-1 is the same electrode as the LDH electrode of Example 3-1.

[0100] Example 4-2 An LDH electrode of Example 4-2 including a working electrode, a reaction layer, and a protective film layer was prepared in the same manner as in Example 2-1, except that glutaraldehyde at a final concentration of 20 mM was replaced with polyethylene glycol diglycidyl ether (PEGDGE (Mn: 500)) at a final concentration of 20 mM.

[0101] Example 4-3 An LDH electrode of Example 4-3 including a working electrode, a reaction layer, and a protective film layer was prepared in the same manner as in Example 2-1, except that glutaraldehyde at a final concentration of 20 mM was replaced with polyethylene glycol diglycidyl ether (PEGDGE (Mn: 6000)) at a final concentration of 20 mM.

[0102] The LDH electrode of Comparative Example 4-1 was prepared in the same manner as in Example 2-1, except that 4 μL of a thionine acetate solution (10 mM) was applied to the working electrode of the printed electrode and dried without crosslinking the thionine molecules. The LDH electrode of Comparative Example 4-1 is the same as the LDH electrode of Comparative Example 2-1.

[0103] [Evaluation of Enzyme Electrodes (LDH Electrodes) Using Various Crosslinkers of Different Molecular Lengths] Using the LDH electrodes of Examples 4-1 to 4-3 and Comparative Example 2-1, the effect of inhibiting detachment from the electrode surface was evaluated by electrochemical measurement (chronoamperometry measurement). The chronoamperometry measurement was performed in the same manner as in Example 2-1, except that instead of the LDH electrode of Example 2-1, Examples 4-1 to 4-3 and Comparative Example 2-1 were used on the day of preparation without storing in PBS solution at 37°C for 4 days. The results are shown in Table 3.

[0104]

[0105] The LDH electrodes of the examples using crosslinking mediators of various crosslinking agents with different molecular lengths had a higher R than the LDH electrodes of the comparative examples that did not use crosslinking mediators. ２ The value was close to 1. This confirmed that crosslinking the mediator suppressed the detachment of the mediator. It was also found that the effect of suppressing detachment from the electrode surface does not tend to be largely dependent on the molecular length of the crosslinking agent in the crosslinked mediator.

[0106] [Synthesis of Crosslinking Mediator] Synthesis Example 1 In order to crosslink thionine molecules, glutaraldehyde at a final concentration of 20 mM and thionine acetate at a final concentration of 20 mM were reacted in 10 mM potassium phosphate buffer (pH 6.5) at 45°C for 1 hour to prepare a reaction solution containing the crosslinking mediator of Synthesis Example 1.

[0107] Synthesis Example 2 A reaction solution containing the crosslinking mediator of Synthesis Example 2 was prepared in the same manner as in Synthesis Example 1, except that glutaraldehyde and thionine acetate at final concentrations of 20 mM and 20 mM were changed to glutaraldehyde and thionine acetate at final concentrations of 77 mM and 6 mM, respectively, and the heating temperature was changed to 60°C.

[0108] Synthesis Example 3 A reaction solution containing the crosslinking mediator of Synthesis Example 3 was prepared in the same manner as in Synthesis Example 1, except that glutaraldehyde and thionine acetate at final concentrations of 20 mM and 20 mM were changed to glutaraldehyde and thionine acetate at final concentrations of 160 mM and 20 mM, respectively, and methanol was added to make the final concentration 20% by mass.

[0109] [Maximum Molecular Weight of Crosslinked Mediator] The molecular weights (number average molecular weights) of the reaction solutions containing the crosslinked mediators of Synthesis Examples 1, 2, and 3 were measured. The number average molecular weights were measured using gel permeation chromatography (GPC) under the following conditions. The molecular weights (number average molecular weights) were calculated in terms of standard polyethylene glycol (PEG) (standard substance) using a calibration curve. The maximum molecular weight was calculated using an approximate equation (third order) determined from the retention time of the peak start and the standard substance. The results are shown in Table 4. Apparatus: HLC-8420GPC Column: TSKgel SuperAWM-H (6.0 mm ID x 15 cm) (two columns connected) (manufactured by Tosoh Corporation) Detector: UV detector (λ = 280 nm) Eluent: DMSO (dimethyl sulfoxide, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) + 10 mM LiBr (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) Flow rate: 0.5 mL / min Concentration: 1 mg / mL Injection volume: 20 μL Column temperature: 40°C

[0110]

[0111] [Preparation of Enzyme Electrodes (GDH Electrodes) Using Crosslinked Mediators with Different Preparation Conditions] Example 5-1 3 μL of the reaction solution containing the crosslinked mediator prepared in Synthesis Example 1 was applied to the working electrode of SCREEN-PRINTED ELECTRODES (Metrohm, DRP-C110) and dried. Subsequently, the working electrode was washed with ultrapure water to remove insufficiently crosslinked thionin. Next, 40 μL of 10 mg / mL GDH-AD / 10 mM potassium phosphate buffer (pH 6.5) and 4 μL of 1% by mass glutaraldehyde were mixed, and 2.2 μL of the mixture was applied to the working electrode and dried. Subsequently, the working electrode was immersed in 7% by mass poly-4-vinylpyridine / ethanol, removed, and dried twice to prepare the GDH electrode of Example 5-1.

[0112] Example 5-2 A GDH electrode of Example 5-2 was prepared in the same manner as in Example 5-1, except that the reaction solution containing the crosslinking mediator prepared in Synthesis Example 1 was changed to the reaction solution containing the crosslinking mediator prepared in Synthesis Example 2.

[0113] Example 5-3 A GDH electrode of Example 5-3 was prepared in the same manner as in Example 5-1, except that the reaction solution containing the crosslinking mediator prepared in Synthesis Example 1 was changed to the reaction solution containing the crosslinking mediator prepared in Synthesis Example 3.

[0114] Comparative Example 5-1 A GDH electrode of Comparative Example 5-1 was prepared in the same manner as in Example 5-1, except that the reaction solution containing the crosslinking mediator prepared in Synthesis Example 1 was not applied, i.e., the crosslinking mediator of Synthesis Example 1 was not used.

[0115] Comparative Example 5-2 A GDH electrode of Comparative Example 5-2 was prepared in the same manner as in Example 5-1, except that the reaction solution containing the crosslinking mediator prepared in Synthesis Example 1 was changed to a solution containing 20 mM thionine acetate.

[0116] [Evaluation of Enzyme Electrode (GDH Electrode)] The effect of inhibiting detachment from the electrode surface was evaluated by electrochemical measurement. Specifically, chronoamperometry measurements were performed using the GDH electrodes of Example 5-1, Example 5-2, Example 5-3, Comparative Example 5-1, and Comparative Example 5-2, and the response current values ​​when glucose was added to a final concentration of 20 mM were compared. For chronoamperometry measurements, the printed electrode was connected to a high-sensitivity potentiostat analyzer ECstat-400 (manufactured by EC FRONTIER) using a dedicated connector (DRP-CAC, manufactured by Drop Sense). Measurements were performed using a three-electrode system with the printed electrode as the working electrode, a platinum counter electrode, and a silver-silver chloride electrode as the reference electrode, and 100 mM potassium phosphate buffer (pH 7.5) was used as the buffer solution. The applied voltage was +100 mV (vs. Ag / AgCl). Specifically, glucose was added to a concentration of 20 mM 120 seconds after the start of measurement, and the response current value was measured 720 seconds after the start of measurement. The response current value 120 seconds after the start of measurement was used as the reference, and the increase in the response current value 720 seconds after the start of measurement was compared. The results are shown in Table 5. The relative values ​​in Table 5 are relative values ​​when the result of Example 5-1 is set to 100%.

[0117]

[0118] The GDH electrodes of the examples using the crosslinked mediators prepared under different conditions had higher relative values ​​than the GDH electrodes of the comparative examples that did not use the crosslinked mediator. This confirmed that mediator detachment was suppressed by crosslinking the mediator. Note that the GDH electrode of comparative example 1 did not reach equilibrium 120 seconds after the start of measurement, and the response current value did not increase at all even when glucose was added.

[0119] [Preparation of LOX Electrode Using Lactate Oxidase (LOX)] Example 6-1 2 μL of the reaction solution containing the crosslinking mediator prepared in Synthesis Example 1 was applied to the working electrode of SCREEN-PRINTED ELECTRODES (DRP-C110, manufactured by Metrohm), dried, and then washed with ultrapure water. A mixture of 15 μL of 20 mg / mL LOX (lactate oxidase, manufactured by Asahi Kasei Pharma Corporation) in 10 mM potassium phosphate buffer (pH 7.5) and 3 μL of BIOSURFINE-AWP-MRH (manufactured by Toyo Gosei Co., Ltd.) was prepared, and 3 μL of this mixture was applied to the working electrode and dried. Furthermore, the mixture was irradiated with 365 nm ultraviolet light for 10 minutes to crosslink the enzymes, thereby producing the LOX electrode of Example 6-1.

[0120] Comparative Example 6-1 A LOX electrode of Comparative Example 6-1 was prepared in the same manner as in Example 6-1, except that the reaction solution containing the crosslinking mediator prepared in Synthesis Example 1 was not applied, i.e., the crosslinking mediator of Synthesis Example 1 was not used.

[0121] Comparative Example 6-2 A LOX electrode of Comparative Example 6-2 was prepared in the same manner as in Example 6-1, except that the reaction solution containing the crosslinking mediator prepared in Synthesis Example 1 was changed to a solution containing 20 mM thionine acetate.

[0122] [Evaluation of Enzyme Electrode (LOX Electrode)] The effect of inhibiting detachment from the electrode surface was evaluated by electrochemical measurement. Specifically, chronoamperometry measurements were performed using the LOX electrodes of Example 6-1, Comparative Example 6-1, and Comparative Example 6-2, and the response current values ​​were compared when sodium lactate was added to a final concentration of 1.6 mM. The chronoamperometry measurements were performed in the same manner as in the evaluation of the enzyme electrode (GDH electrode) described above. Specifically, sodium lactate was added 120 seconds after the start of measurement to a final concentration of 1.6 mM, and the response current value was measured 360 seconds after the start of measurement. The response current value 120 seconds after the start of measurement was used as the reference, and the increase in the response current value 360 ​​seconds after the start of measurement was compared. The results are shown in Table 6. Note that the relative values ​​in Table 6 are relative values ​​when the result of Example 6-1 is set to 100%.

[0123]

[0124] The LOX electrode of the example had a larger relative value than the LOX electrode of the comparative example, which did not use a cross-linked mediator. This confirmed that cross-linking the mediator suppressed the detachment of the mediator.

[0125] [Preparation of NOX electrode using NADH oxidase (NOX)] Example 7-1 An NOX electrode of Example 7-1 was prepared in the same manner as in Example 6-1, except that the mixture of 15 μL of 20 mg / mL LOX (manufactured by Asahi Kasei Pharma Corporation) / 10 mM potassium phosphate buffer (pH 7.5) and 3 μL of BIOSURFINE-AWP-MRH (manufactured by Toyo Gosei Co., Ltd.) was replaced with a mixture of 50 μL of 15 U / mL NOX (NADH oxidase, Sigma-Aldrich) and 5 μL of 1 mM FAD (flavin adenine dinucleotide) solution.

[0126] Comparative Example 7-1 An NO electrode of Comparative Example 7-1 was prepared in the same manner as in Example 7-1, except that the reaction solution containing the crosslinking mediator prepared in Synthesis Example 1 was not applied, i.e., the crosslinking mediator of Synthesis Example 1 was not used.

[0127] Comparative Example 7-2 An NOx electrode of Comparative Example 7-2 was prepared in the same manner as in Example 7-1, except that the reaction solution containing the crosslinked mediator prepared in Synthesis Example 1 was changed to a solution containing 20 mM thionine acetate.

[0128] [Evaluation of Enzyme Electrode (NOX Electrode)] The effect of inhibiting detachment from the electrode surface was evaluated by electrochemical measurement. Specifically, chronoamperometry measurements were performed using the NOX electrodes of Example 7-1, Comparative Example 7-1, and Comparative Example 7-2, and the response current values ​​when NADH (nicotinamide adenine dinucleotide) was added to a final concentration of 0.24 mM were compared. The chronoamperometry measurements were performed in the same manner as in the evaluation of the enzyme electrode (GDH electrode) described above. Specifically, NADH was added to a final concentration of 0.24 mM 120 seconds after the start of measurement, and the response current value 360 ​​seconds after the start of measurement was measured. The response current value 120 seconds after the start of measurement was used as the reference, and the increase in the response current value 360 ​​seconds after the start of measurement was compared. The results are shown in Table 7. Note that the relative values ​​in Table 7 are relative values ​​when the result of Example 7-1 is set to 100%.

[0129]

[0130] The NO electrode of the Example had a larger relative value than the NO electrode of the Comparative Example, which did not use a cross-linked mediator. This confirmed that cross-linking the mediator suppressed desorption of the mediator.

Claims

1. A step of preparing a laminate comprising an electrode and a mediator layer provided on the surface of the electrode, the mediator layer containing a mediator crosslinked with a crosslinking agent, The process involves adding an enzyme-containing solution to the mediator layer of the laminate to form a reaction layer, Equipped with, A method for manufacturing enzyme electrodes.

2. The laminate is a laminate prepared by arranging mediators, which have been crosslinked with a pre-synthesized crosslinking agent, on the surface of the electrode. A method for producing an enzyme electrode according to claim 1.

3. The mediator is a compound having two or more amino groups in one molecule. A method for producing an enzyme electrode according to claim 1 or 2.

4. The crosslinking agent is a compound having two or more reactive groups in one molecule that are reactive with the amino group. A method for producing an enzyme electrode according to claim 3.

5. The molecular weight of the mediator is 100 or more and 1000 or less. A method for producing an enzyme electrode according to claim 1 or 2.

6. The mediator is a compound represented by formula (1): H₂N-X-NH₂, where X is a divalent fused ring group containing a heteroatom as a constituent atom of the ring. A method for producing an enzyme electrode according to claim 1 or 2.

7. Electrodes and, A reaction layer provided on the surface of the electrode, Equipped with, The reaction layer comprises an enzyme and a mediator crosslinked with a crosslinking agent. Enzyme electrode.

8. The mediator is a compound having two or more amino groups in one molecule. The enzyme electrode according to claim 7.

9. The crosslinking agent is a compound having two or more reactive groups in one molecule that are reactive with the amino group. The enzyme electrode according to claim 8.

10. The molecular weight of the mediator is 100 or more and 1000 or less. The enzyme electrode according to claim 7.

11. The mediator is a compound represented by formula (1): H₂N-X-NH₂, where X is a divalent fused ring group containing a heteroatom as a constituent atom of the ring. The enzyme electrode according to claim 7.

12. The enzyme electrode described in any one of claims 7 to 11 is used as the working electrode. Enzyme sensor.