JP2023128506A - Enzyme crosslinked product, bioelectrode material, bioelectrode, and electrochemical device - Google Patents

Abstract

To provide an enzyme crosslinked product, bioelectrode material, bioelectrode, and electrochemical device by which electrochemical devices that can maintain high current density can be obtained.SOLUTION: Provided is an enzyme crosslinked product having a structure represented by the following formula (1). In formula (1), R represents an enzyme, R' represents an organic group, and n is a number greater than or equal to 2.SELECTED DRAWING: Figure 4

Description

The present invention relates to an enzyme crosslinked product, a bioelectrode material, a bioelectrode, and an electrochemical device.

Research and development are underway on electrochemical devices such as biofuel cells that use enzymes as electrode catalysts and generate electricity using sugars, alcohol, and other biomass resources as fuel.
Electrochemical devices that utilize enzyme catalysis generally include a negative electrode (anode) containing an enzyme that promotes fuel oxidation and a positive electrode (cathode) containing an enzyme that promotes oxygen reduction. Electrons removed by fuel oxidation (for example, glucose to gluconolactone) at the negative electrode are transferred to the positive electrode, where they are used to reduce oxygen and produce water (H ₂ O). The movement of electrons that occurs during this process is used for things such as power generation.

In electrochemical devices that utilize the catalytic action of enzymes, in order to increase current density, enzyme molecules may be bonded (crosslinked) to each other to suppress detachment of the enzyme from the electrode.
For example, Non-Patent Document 1 describes an electrochemical device using glutaraldehyde as a crosslinking agent for bilirubin oxidase.

G.C. Sedenho et al. Journal of Power Sources 482 (2021) 229035.

In addition to high current density, the ability to maintain high current density is also important for electrochemical devices.
In view of the above-mentioned circumstances, an object of the present invention is to provide an enzyme crosslinked product, a bioelectrode material, a bioelectrode, and an electrochemical device from which an electrochemical device capable of maintaining a high current density can be obtained.

Specific means for solving the above problems include the following embodiments.
<1> An enzyme crosslinked product having a structure represented by the following formula (1).

In formula (1), R represents an enzyme, R' represents an organic group, and n is a number of 2 or more.
<2> The enzyme crosslinked product according to <1>, which is a reaction product of an enzyme and a multi-epoxy compound.
<3> The enzyme crosslinked product according to <2>, wherein the multi-epoxy compound is water-soluble.
<4> The enzyme crosslinked product according to any one of <1> to <3>, wherein the enzyme promotes oxygen reduction or fuel oxidation.
<5> A bioelectrode material comprising the enzyme crosslinked product according to any one of <1> to <4>.
<6> A bioelectrode comprising the bioelectrode material according to <5> and a conductive base material.
<7> An electrochemical device comprising the bioelectrode according to <6>.
<8> Including an anode and a cathode,
The anode includes a copolymer and an enzyme, and the copolymer includes a structural unit having a structure bonded to the enzyme via an amide group, a structural unit having a hydrophilic group, and a structural unit having a mediator molecule. A bioelectrode including
The electrochemical device, wherein the cathode is the bioelectrode according to <6>.

According to the present invention, an enzyme crosslinked product, a bioelectrode material, a bioelectrode, and an electrochemical device are provided, which provide an electrochemical device that can maintain a high current density.

FIG. 2 is a diagram showing a disassembled state of the measurement system used in Examples. FIG. 2 is a cross-sectional view of a measurement system used in Examples. It is a graph showing the results of electrochemical measurements carried out in Examples. It is a graph showing the results of electrochemical measurements carried out in Examples. It is a graph showing the results of electrochemical measurements carried out in Examples. It is a figure which shows the measurement result of ^1HNMR spectrum carried out in the Example. It is a figure showing the measurement result of GPC carried out in an example. FIG. 3 is a diagram showing the results of electrochemical measurements carried out in Examples.

EMBODIMENT OF THE INVENTION Hereinafter, the form for implementing this invention is demonstrated in detail. However, the present invention is not limited to the following embodiments. In the following embodiments, the constituent elements (including elemental steps and the like) are not essential unless otherwise specified. The same applies to numerical values and their ranges, and they do not limit the present invention.
In the present disclosure, numerical ranges indicated using "~" include the numerical values written before and after "~" as minimum and maximum values, respectively.
In this disclosure, (meth)acrylic, (meth)acryloyl, and (meth)acrylate mean acrylic or methacrylic, acryloyl or methacryloyl, and acrylate or methacrylate, respectively.

<Enzyme crosslinked product>
The enzyme crosslinked product of the present disclosure is an enzyme crosslinked product having a structure represented by the following formula (1).

In the formula, R represents an enzyme, R' represents an organic group, and n is a number of 2 or more.

As a result of studies by the present inventors, an electrochemical device using the above-mentioned enzyme crosslinked product as an electrode material has an electrochemical device in which an enzyme crosslinked with a compound that does not form the structure represented by formula (1), such as glutaraldehyde, is used as an electrode material. It was found that the current density increased compared to the electrochemical device used as the material. Furthermore, an electrochemical device using the above-mentioned enzyme crosslinked product as an electrode material has a higher cost than an electrochemical device using an enzyme crosslinked with a compound that does not form the structure represented by formula (1) as an electrode material. It was found that the current density was maintained over a long period of time and exhibited excellent durability.

In formula (1), R represents an enzyme, R' represents an organic group, and n is a number of 2 or more.
Each of the enzymes represented by R may have only one or two or more structures represented by formula (1).

In formula (1), n means the number of binding sites with the enzyme that the organic group represented by R' has. From the viewpoint of increasing the current density and its durability, n is preferably 3 or more, more preferably 4 or more. The upper limit of n is not particularly limited. For example, n may be 10 or less, or may be 5 or less.

In formula (1), the organic group represented by R' may have a structure corresponding to the skeleton of a compound having n epoxy groups.

The structure represented by formula (1) in the enzyme crosslinked product is, for example, a compound having two or more epoxy groups in one molecule (hereinafter also referred to as a multi-epoxy compound) and an amino group (-NH ₂ ) is formed by reaction with That is, the enzyme crosslinked product may be a reaction product of an enzyme and a multi-epoxy compound.

By reacting the epoxy group of the multi-epoxy compound with the amino group of the enzyme, a crosslinked enzyme having the structure represented by formula (1) can be obtained.
Whether or not the enzyme has reacted with the multi-epoxy compound to form an enzyme cross-linked product can be determined by, for example, mixing the enzyme and the multi-epoxy compound in water, reacting, drying, and adding water again. This can be determined by its solubility in water. If the water solubility when water is added after the reaction is lower than the water solubility of the enzyme before the reaction, it is determined that at least a portion of the enzyme has reacted with the multi-epoxy compound to form an enzyme crosslinked product. can.

The type of multi-epoxy compound is not particularly limited, and can be selected depending on the use of the enzyme crosslinked product obtained using the multi-epoxy compound. When using the enzyme crosslinked product with an aqueous medium, the multi-epoxy compound is preferably water-soluble.
In the present disclosure, a water-soluble multi-epoxy compound means a multi-epoxy compound whose dissolution rate is 30% or more when the multi-epoxy compound (10 parts by mass) is dissolved in pure water (90 parts by mass) at 25°C. .

Specifically, water-soluble multi-epoxy compounds include multi-epoxy compounds having two epoxy groups in one molecule such as ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, and glycerol polyglycidyl ether. , multi-epoxy compounds having three epoxy groups in one molecule such as diglycerol polyglycidyl ether, multi-epoxy compounds having four epoxy groups in one molecule such as sorbitol polyglycidyl ether, and polyglycerol polyglycidyl ether. Examples include multi-epoxy compounds having four or more epoxy groups in one molecule.
The number of multi-epoxy compounds reacted with the enzyme may be one or two or more.

From the viewpoint of improving current density, sorbitol polyglycidyl ether (SPGE) is preferred among multi-epoxy compounds.
SPGE has a molecular structure in which epoxy groups are distributed in four directions. Therefore, a state where the enzyme is crosslinked with the multi-epoxy compound (a state where two or more epoxy groups in one molecule react with the amino group of the enzyme) is more likely to be formed than with other multi-epoxy compounds.

The type of enzyme to be reacted with the multi-epoxy compound is not particularly limited, and can be selected depending on the type of fuel to be supplied to the electrochemical device using the enzyme crosslinked product.
When an enzyme crosslinker is used as a cathode material, the enzyme crosslinker contains an enzyme that promotes the reduction of oxygen.
Specific examples of enzymes that promote oxygen reduction include bilirubin oxidase, laccase, polyphenol oxidase, and ascorbate oxidase.

When the enzyme crosslinker is used as an anode material, the enzyme crosslinker contains an enzyme that promotes the oxidation of the fuel. The enzyme that promotes oxidation of fuel may be a combination of an enzyme that renders fuel oxidizable by hydrolysis or the like, and an enzyme that promotes oxidation.

Enzymes when the fuel is glucose include glucose oxidase, glucose dehydrogenase, pyranose oxidase, and cellobiose dehydrogenase.
Enzymes when the fuel is fructose include fructose oxidase and fructose dehydrogenase.
When the fuel is sucrose, the enzyme includes a combination of invertase and glucose dehydrogenase.
When the fuel is starch, the enzyme includes a combination of amylase and glucose dehydrogenase.
Enzymes when the fuel is lactic acid include lactate oxidase and lactate dehydrogenase.
Enzymes when the fuel is alcohol include alcohol oxidase and alcohol dehydrogenase.
Enzymes when the fuel is an aldehyde include aldehyde oxidase and aldehyde dehydrogenase.
Enzymes when the fuel is pyruvate include pyruvate oxidase and pyruvate dehydrogenase.

The enzyme crosslinked product may contain only one type of enzyme or two or more types of enzymes.

When the enzyme crosslinked product is a reaction product of an enzyme and a multi-epoxy compound, the amount of the multi-epoxy compound reacted with the enzyme is not particularly limited.
For example, the amount of the multi-epoxy compound to be reacted with 100 parts by mass of the enzyme may be selected from 1 part by mass to 1000 parts by mass, may be selected from 10 parts by mass to 100 parts by mass, and may be selected from 10 parts by mass to 50 parts by mass. You may also select from parts by mass.

When the enzyme crosslinked product is used as a material for a bioelectrode, the enzyme crosslinked product may be incorporated into a polymer in order to protect the enzyme crosslinked product, suppress its detachment from the electrode, and the like.
Although the type of polymer is not particularly limited, it is preferable to use a polymer that does not hinder the movement of electrons or ions (electrolyte) accompanying the oxidation of fuel or reduction of oxygen by enzymes. For example, it is preferable to use a cation-permeable polymer such as Nafion (registered trademark, hereinafter the same), an anion-permeable polymer such as polyamine, polyvinyl alcohol, or the like.

<Bioelectrode material>
The bioelectrode material of the present disclosure is a bioelectrode material containing the enzyme crosslinked product described above.
By using the bioelectrode material of the present disclosure, the current density of an electrochemical device can be increased. Furthermore, high current density can be maintained for a long period of time.

(Other ingredients)
The bioelectrode material may contain components other than the enzyme crosslinked product.
For example, it may contain a binder that binds the enzyme crosslinked product. Examples of the binder include cation-permeable polymers such as polyvinylidene fluoride (PVDF), styrene-butadiene rubber (SBR), polytetrafluoroethylene (PTFE), polyvinyl alcohol (PVA), and Nafion.

<Bioelectrode>
The bioelectrode of the present disclosure includes the bioelectrode material described above and a conductive base material.
The bioelectrode may be in a state in which the above-mentioned bioelectrode material is placed on a conductive base material.

The material of the conductive base material is not particularly limited, and can be selected from conductive materials such as carbon materials and metals.
Examples of carbon materials include graphite, carbon black (Ketjenblack, acetylene black, etc.), MgO-templated carbon particles, carbon nanomaterials (carbon nanotubes, graphene, carbon nanohorns, carbon nanowires, etc.). Only one type of carbon material may be used or two or more types may be used in combination.

From the perspective of increasing the area where the enzyme contained in the bioelectrode material comes into contact with fuel or oxygen and improving power generation efficiency, the part of the conductive base material where the bioelectrode material is placed contains particulate conductive material. It is preferable. Examples of the particulate conductive material include porous carbon particles produced by a molding method such as MgO molded carbon.

The form of the bioelectrode is not particularly limited. For example, the bioelectrode may be a gas diffusion electrode.
Gas diffusion electrodes incorporate a gas diffusion layer with strong hydrophobicity, and receive oxygen from the atmosphere, protons from the electrolyte, and a catalyst that receives electrons supplied from the electrode surface to perform an oxygen reduction reaction on the electrode surface. This electrode has a three-phase structure.
Gas diffusion electrodes are generally composed of a porous substrate and a catalyst so that a large number of three-phase interfaces are formed where gas, electrolyte, and catalyst come into contact at the same time.

The entire electrically conductive material may be electrically conductive, or a portion thereof may be electrically conductive.
The bioelectrode may include a support for supporting the conductive substrate.

<Electrochemical device>
The electrochemical device of the present disclosure includes the bioelectrode described above. The electrochemical device may include the above-described bioelectrode as an anode, a cathode, or both an anode and a cathode.
Specific examples of electrochemical devices include biofuel cells, bioreactors, biosensors, and the like.

The configuration of a biofuel cell will be described below as an example of an electrochemical device.
A biofuel cell includes at least a cathode and an anode.
When a biofuel cell is supplied with fuel, electrons generated by oxidation of the fuel at the anode are transferred to the cathode and used to reduce oxygen at the cathode. The movement of electrons that occurs during this process is used to generate electricity.

The type of fuel used in the biofuel cell is not particularly limited as long as it is a substance whose oxidation is promoted by the enzyme contained in the anode. Specific examples of the fuel include sugars, alcohols, aldehydes, amino acids, amines, lactic acid, pyruvic acid, uric acid, hydrogen, and the like.

The number of fuels used in a biofuel cell may be one or two or more. The fuel may be one that can be oxidized as is by an enzyme contained in the anode, or one that can be oxidized by hydrolysis or the like (for example, starch that becomes glucose by hydrolysis).

The electrochemical device of the present disclosure may include the bioelectrode of the present disclosure described above as a cathode, and may include the bioelectrode described below as an anode.
That is, one embodiment of the electrochemical device of the present disclosure includes an anode and a cathode,
The anode includes a copolymer and an enzyme, and the copolymer includes a structural unit having a structure bonded to the enzyme via an amide group, a structural unit having a hydrophilic group, and a structural unit having a mediator molecule. A bioelectrode including
The cathode is an electrochemical device, which is the bioelectrode of the present disclosure described above.

(copolymer)
The copolymer contained in the anode includes a structural unit having a structure bonded to an enzyme via an amide group, a structural unit having a hydrophilic group, and a structural unit having a mediator molecule.

An electrochemical device using the above copolymer as an anode material has a significantly higher current density than an electrochemical device using a mediator molecule not bonded to the copolymer as an anode material. This is thought to be because the mediator molecule, which is fixed in the vicinity of the enzyme by bonding to the copolymer via the amide group, efficiently mediates electron transfer between the electrode and the enzyme.

Furthermore, the above-mentioned copolymer contains a structural unit having a hydrophilic group. It is thought that this hydrophilic group increases the copolymer's affinity for the enzyme, and that the mediator molecules contained in the copolymer efficiently mediate electron transfer between the electrode and the enzyme.

(Functional group that reacts with a primary amine to form an amide group)
A structural unit having a structure bonded to an enzyme in a copolymer via an amide group is, for example, an enzyme having a copolymer containing a structural unit having a functional group that reacts with a primary amine to form an amide group. It can be formed by reacting with a primary amine (-NH ₂ ).
The type of functional group that reacts with the primary amine contained in the copolymer to form an amide group is not particularly limited.
From the viewpoint of reactivity with primary amines, NHS (N-hydroxysuccinimide) ester groups are preferred. In the reaction between the HNS ester group and the primary amino group, an amide bond is formed and N-hydroxysuccinimide (NHS) is liberated.
The number of functional groups that react with the primary amine contained in the copolymer to form an amide group may be one or two or more.

(Hydrophilic group)
The type of hydrophilic group contained in the copolymer is not particularly limited.
The hydrophilic group may contain a nitrogen atom. For example, when the surface of an enzyme is negatively charged with a carboxy group or the like, the copolymer's affinity for the enzyme can be further enhanced by electrostatic interaction with a hydrophilic group containing a nitrogen atom.
Examples of the hydrophilic group containing a nitrogen atom include a secondary amino group, a tertiary amino group, and an ammonium group. Among these, a tertiary amino group and an ammonium group are preferred, and a dimethylamino group and an ammonium group are more preferred.
The hydrophilic group may be an anionic hydrophilic group. For example, if the surface of the enzyme is positively charged with amino groups or the like, the copolymer's affinity for the enzyme can be further enhanced by electrostatic interaction with the hydrophilic group, which is an anionic group.
Examples of anionic hydrophilic groups include carboxy groups, sulfonic acid groups, and phosphoric acid groups.
The number of hydrophilic groups contained in the copolymer may be one or two or more. The hydrophilic groups contained in the copolymer may form a salt.

(mediator molecule)
The mediator molecule contained in the copolymer is not particularly limited as long as it mediates electron transfer between the enzyme and the electrode.
Specifically, the mediator molecules include AzureA (3-amino-7-(dimethylamino)phenothiazin-5-ium chloride), thionine (3,7-diaminophenothiazine), and toluidine blue (3-amino-2-methyl- 7-(dimethylamino)phenothiazin-5-ium chloride), aminoferrocene, neutral red, aminoanthraquinone, aminonaphthoquinone, aminobenzoquinone, a ligand having an amino group (aminopyridine, diaminobipyridine, etc.) as a ligand, and iron , metal complexes having a transition metal such as osmium, ruthenium, nickel, copper, etc. as a central metal.

From the viewpoint of ease of synthesizing the copolymer, it is preferable that the mediator molecule has a primary amino group. If the mediator molecule has a primary amino group, the mediator molecule can be introduced into the copolymer by reacting the primary amine contained in the copolymer with a functional group that forms an amide group. . That is, the mediator molecule contained in the copolymer may be bonded to the main chain via an amide group.
The number of mediator molecules contained in the copolymer may be one or two or more.

The proportion of the structural units having a functional group that reacts with a primary amine to form an amide group in the total structural units in the copolymer is not particularly limited.
For example, the ratio of structural units having functional groups that react with primary amines to form amide groups in the copolymer to all structural units may be selected from the range of 1 mol% to 90 mol%; It may be selected from the range of mol% to 50 mol%, or may be selected from the range of 3 mol% to 10 mol%.

The proportion of the structural units having mediator molecules in the total structural units in the copolymer is not particularly limited.
For example, the ratio of the structural units having mediator molecules to the total structural units in the copolymer may be selected from the range of 1 mol% to 20 mol%, or may be selected from the range of 2 mol% to 10 mol%. It may be selected from the range of 3 mol % to 5 mol %.

The proportion of the structural units having a hydrophilic group in the total structural units in the copolymer is not particularly limited.
For example, the proportion of the structural units having a hydrophilic group in the total structural units in the copolymer may be selected from the range of 60 mol% to 98 mol%, or may be selected from the range of 70 mol% to 95 mol%. It may be selected from the range of 80 mol% to 90 mol%.

If necessary, the copolymer may further include a structural unit other than a structural unit having a structure bonded to an enzyme via an amide group, a structural unit having a hydrophilic group, and a structural unit having a mediator molecule. For example, it may contain a structural unit containing a substituent such as an alkyl group, an aryl group, or an alkyleneoxy group.
When the copolymer contains structural units other than structural units having a structure bonded to an enzyme via an amide group, structural units having a hydrophilic group, and structural units having a mediator molecule, the proportion thereof is 15 mol of the total structural units. % or less, more preferably 10 mol% or less, even more preferably 5 mol% or less.

The molecular weight of the copolymer is not particularly limited. For example, the weight average molecular weight of the copolymer as measured by gel permeation chromatography (GPC) may be in the range of 10,000 to 500,000. Further, the number average molecular weight of the copolymer measured by gel permeation chromatography (GPC) may be within the range of 1,000 to 200,000.

The copolymer may be poly(meth)acrylate.
The copolymer may be a random copolymer or a block copolymer.

(Method of synthesizing copolymer)
The method for synthesizing the copolymer is not particularly limited, and any known method can be employed.
The copolymer may be synthesized in a one-step reaction or in two or more steps.
A method for synthesizing a copolymer through a one-step reaction involves using a polymerizable compound containing a functional group that reacts with a primary amino group to form an amide group and a polymerizable group, and a hydrophilic group and a polymerizable group. A method of reacting a polymerizable compound containing a mediator molecule and a polymerizable group with a polymerizable compound containing a mediator molecule and a polymerizable group.

As a method for synthesizing a copolymer through a two-step reaction, a polymerizable compound containing a functional group and a polymerizable group that reacts with a primary amino group to form an amide group, and a hydrophilic group and a polymerizable group are used. and a functional group that reacts with the primary amino group in the base polymer to form an amide group, and a mediator molecule containing the primary amino group. One example is a method of reacting with and. At this time, by reacting a part of the functional group that reacts with the primary amino group to form an amide group with the mediator molecule, the unreacted functional group can be used for bonding with the enzyme.

The enzyme and other components contained in the anode containing the copolymer are not particularly limited, and may be selected from the components contained in the above-mentioned bioelectrode.

Hereinafter, the present disclosure will be described in more detail based on Examples, but the present disclosure is not limited to these Examples.

(1) Preparation of composition A 0.1M phosphate buffer (pH 7.0) solution of bilirubin oxidase (BOD), a 0.1M phosphate buffer (pH 7.0) solution of Nafion 117, and a 0.0% crosslinking agent (CL) solution. A composition was prepared by mixing a 1M phosphate buffer (pH 7.0) solution such that the amounts (solid content) of BOD, CL, and Nafion were in the combinations shown in Table 1.

Examples of crosslinking agents include sorbitol polyglycidyl ether (SPGE), which has an epoxy group as a functional group that reacts with the amino group of the enzyme, glutaraldehyde (GA), which has an aldehyde group as a functional group that reacts with the amino group of the enzyme, and amino acid of the enzyme. Bis(NHS)PEG5 and bis(NHS)PEG9 having an NHS ester group as a functional group that reacts with the group were used, respectively.

(2) Preparation of gas diffusion electrode One side of a carbon cloth was uniformly coated with a 40% polytetrafluoroethylene (PTFE) dispersion and dried at room temperature for 30 minutes. Thereafter, it was heated in an oven at 370°C for 15 minutes and cooled to room temperature. Then, a further 40% PTFE dispersion was evenly coated on the dried PTFE layer and dried for 30 minutes. Thereafter, it was heated in an oven at 370°C for 15 minutes and cooled to room temperature. Through this water repellent treatment, a gas diffusion layer made of PTFE was formed on the carbon cloth.
A mixture was obtained by mixing MgO template carbon particles (average pore diameter: 100 nm), PTFE as a binder, and isopropanol as a solvent. This mixture (MgOC ink) was evenly applied to the entire surface of the gas diffusion carbon cloth opposite to the water-repellent surface, and dried at 60°C for 16 hours to form an MgOC layer containing porous carbon particles and the carbon cloth. A gas diffusion electrode was manufactured in which a gas diffusion layer and a gas diffusion layer were laminated in this order.
The composition prepared in (1) was applied to the MgOC layer of the fabricated gas diffusion electrode, and the composition was dried at 4°C overnight to react with BOD and SPGE to form an enzyme crosslinked product, which was then used for electrochemical measurements. A sample of a gas diffusion electrode was fabricated.

For comparison, a composition containing only BOD (3.0 mg) (BOD only) and a composition containing only BOD (3.0 mg) and Nafion (0.5 mg) (BOD + Nafion) were used in the MgOC layer of the gas diffusion electrode. A sample of a gas diffusion electrode for electrochemical measurement was prepared by coating the sample on the surface of the substrate and drying it at 4° C. overnight.

(3) Electrochemical measurements Electrochemical measurements were performed on the prepared gas diffusion electrode using a Pt electrode as a counter electrode, an Ag/AgCl electrode as a reference electrode, and a phosphate buffer (1M, pH 7.0).
For electrochemical measurements, chronoamperometry measurements (CA) were performed using a measurement system with the configuration shown in Figures 1 and 2 under conditions of 0.2V and 300 seconds, and the current was measured 250 seconds after the start of the measurement. Density values were measured. The results are shown in Figure 3.

Figure 1 shows an exploded state of the measurement system used in electrochemical measurements, and Figure 2 shows a cross-sectional view of the measurement system.
As shown in FIG. 1, a sample 1 of the gas diffusion electrode is sandwiched between blocks A and B together with a titanium mesh sheet 2 as a current collector and a spacer 3 made of silicone rubber.

As shown in FIG. 2, block A has a ventilation hole 4 in the center. Sample 1 is arranged such that the PTFE layer faces vent hole 4 .
Block B has a space 7 in which a counter electrode 5 and a reference electrode 6 are placed. Electrochemical measurements are performed with the space 7 filled with phosphate buffer.

As shown in FIG. 3, the sample containing the enzyme crosslinked product using SPGE showed a higher current density than the other samples.

(4) Long-term continuous measurement experiment A 0.1M phosphate buffer (pH 7.0) solution of BOD, a 0.1M phosphate buffer (pH 7.0) solution of Nafion 117, and a 0.1M crosslinking agent (SPGE or GA). A composition was prepared by mixing a phosphate buffer (pH 7.0) solution so that the BOD was 3.0 mg, the crosslinking agent was 0.5 mg, and Nafion was 0.5 mg (all solid content). ) A sample of a gas diffusion electrode for electrochemical measurements was prepared.

Using the same measurement system as in (3) above, the current density at 0.2 V was measured for the prepared sample using a potentiostat. Measurements were carried out for three consecutive days. The results are shown in Figure 4.

As shown in Figure 4, the sample containing the enzyme cross-linked product using SPGE showed a higher current density than the sample containing the enzyme cross-linked product using GA, and the high current density was maintained over a long period of time. Ta.

(5) Electrochemical measurement (using glucose dehydrogenase)
A composition for forming an electrode was prepared by mixing a phosphate buffer solution of glucose dehydrogenase (25 mg/mL) and a phosphate buffer solution of a crosslinking agent (12.5 mg/mL) in respective amounts of 7 μL and 8 μL. . This composition (2 μL) was placed on the end of a glassy carbon disk electrode (3 mm diameter) and dried at 26° C. for 30 hours to form a layer containing the enzyme crosslinked product.
As the crosslinking agent, SPGE as a multi-epoxy compound and GA and bis(NHS) PEG5 as comparative crosslinking agents were used.
The obtained electrode was washed with a phosphate buffer (pH 7.0) and cyclically washed with a phosphate buffer containing 0.1M glucose and a phosphate buffer containing 0.1mM 1,4-naphthoquinone. Electrochemical measurements were performed using voltammetry. In the measurement, a Pt electrode was used as a counter electrode, and an Ag/AgCl electrode was used as a reference electrode. The temperature of the solution was 25°C, the sweep rate was 10 mVs ^-1 , and the measurement range was -0.4V to 0.5V. The results are shown in Figure 5.

<Electrode material for anode>
(1) Synthesis of base polymer A base polymer was synthesized according to the reaction scheme below.

In 8.56 mL of toluene, 187.3 mg (50 mM) of N-succinimidyl methacrylate (NHSMA), 0.2 mL of tetralin (internal standard), and 1.55 mL of 2-(dimethylamino)ethyl methacrylate (DMAEMA). (450mM) and nitrogen bubbling was performed for 5 minutes. Thereafter, 10.22 mL of a toluene solution (100 mM) of 2,2'-azobis(isobutyronitrile) (AIBN) was added (final concentration 50 mM), and the mixture was reacted at 65° C. for 6 hours with stirring. Thereafter, the reaction was stopped by briefly contacting the container with liquid nitrogen.
The resulting reaction solution was dialyzed against acetone (molecular weight cut off: 3500) for 24 hours. Thereafter, dialysis against water was performed for 24 hours. The reaction solution after dialysis was collected and the polymer was taken out by freeze-drying.

FIG. 6 shows ¹ H NMR spectra before and after the reaction (the upper side is before the reaction, the lower side is after the reaction). Bruker's AVANCE-600 NMR Spectrometer was used as a measuring device, and CDCl ₃ was used as a measuring solvent.
As shown in FIG. 6, the monomer was consumed by the reaction, and a new peak representing the polymer appeared. When the monomer consumption was determined from the integral value of the peak, the structural ratio of the purified polymer was 10.1 mol% for the NHSMA site and 89.9 mol% for the DMAEMA site.
The molecular weight of the obtained polymer was measured by GPC. The equipment was JASCO Corporation's UV-4075 (UV/VisDetector), AS-4050 (HPLC Autosampler), and PU-4180 (RHPLC Pump), and the filter was Tosoh Corporation's TSKgel α-M 7.8 mm I.D. D. x 30 cm, DMF containing LiBr (10 mM) was used as the solvent, the measurement time was 50 minutes, the injection volume was 20 μL, and the flow rate was 0.6 mL/min.
As a result of GPC, the Mn of the polymer was 5574, the Mw was 20797, and the Mw/Mn was 3.7309.

(2) Introduction of mediator molecule A mediator molecule (AzureA) was introduced into the base polymer according to the reaction scheme below.

Polymer (10 mg/mL) and Azure A (1.72 mg/mL) were dissolved in distilled water, and reacted with stirring at room temperature for 24 hours.
The concentration of AzureA was determined such that a portion of the NHSMA sites within the base polymer reacted with the amine sites of AzureA, and the remaining NHSMA sites remained unreacted.

FIG. 7 shows the results of GPC after the reaction. The same device as above was used as the measurement device, DMF containing LiBr (10mM) was used as the solvent, the measurement time was 50 minutes, the injection volume was 20μL, the flow rate was 0.6mL/min, and UV-Vis measurement was performed. The wavelength was 600 nm (absorption wavelength of Azure A).
As shown in FIG. 7, absorption at 600 nm was observed on the high molecular weight side, indicating that AzureA was bonded to the base polymer.

(3) Preparation of electrode 20 μL of an aqueous solution of flavin adenine dinucleotide-dependent glucose dehydrogenase (50,000 Units) (FAD-GDH), 5 μL of an aqueous solution of PEGDGE (10.0 mg/mL), and an aqueous solution of Bis-AEE (3. 0mg/mL) were mixed to obtain a mixed solution. 14 μL of this mixed solution was mixed with 10 μL of a solution of the polymer into which Azure A was introduced.

2.4 μL of the obtained liquid mixture was dropped onto the end of a glassy carbon disk electrode (diameter 3.0 mm, BAS) that had been previously subjected to plasma treatment (hydrophilic treatment) for 10 minutes. The dropping was performed in two portions of 1.2 μL each. After dropping, it was dried at room temperature overnight. The electrode thus produced was designated as sample A.

For comparison, an electrode was produced in the same manner as Sample A except that the same amount (1.72 mg/mL) of an aqueous solution of Azure A was used instead of the solution of the polymer introduced with Azure A. The electrode thus produced was designated as sample B.

(4) Electrochemical measurements Electrochemical measurements were carried out by cyclic voltammetry using the prepared electrodes.
A 0.1M phosphate buffer (pH 7.0) containing 0.5M glucose was used as the measurement solution, a sample A or sample B electrode was used as the working electrode, a Pt electrode was used as the counter electrode, and an Ag/AgCl electrode was used as the reference electrode. did. Palmsens' MultiEmStat was used as the measurement device, and the measurement range was -0.6 to 0.4V vs. Ag/AgCl was used, and the sweep rate was 10 mV/s.

The results of the second cycle of electrochemical measurements are shown in FIG. Sample A, which contained AzureA bound to a polymer, exhibited a current density approximately 10 times higher than Sample B, which contained AzureA not bound to a polymer.
The average value of the current density at a potential of 0.1 V when electrochemical measurements were performed four times was 258 μA/cm ² for sample A, which was larger than 19 μA/cm ² for sample B.

Claims (8)

An enzyme crosslinked product having a structure represented by the following formula (1).

In formula (1), R represents an enzyme, R' represents an organic group, and n is a number of 2 or more. The enzyme crosslinked product according to claim 1, which is a reaction product of an enzyme and a multi-epoxy compound. The enzyme crosslinked product according to claim 2, wherein the multi-epoxy compound is water-soluble. The enzyme crosslinked product according to any one of claims 1 to 3, wherein the enzyme promotes oxygen reduction or fuel oxidation. A bioelectrode material comprising the enzyme crosslinked product according to any one of claims 1 to 4. A bioelectrode comprising the bioelectrode material according to claim 5 and a conductive base material. An electrochemical device comprising the bioelectrode according to claim 6. including an anode and a cathode;
The anode includes a copolymer and an enzyme, and the copolymer includes a structural unit having a structure bonded to the enzyme via an amide group, a structural unit having a hydrophilic group, and a structural unit having a mediator molecule. A bioelectrode including
An electrochemical device, wherein the cathode is a bioelectrode according to claim 6.