JP2013254724A - Enzyme ink, enzyme fuel cell and electronic apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an enzyme ink which allows for production of an enzyme electrode that can use a hydrophilic enzyme and a promoter molecule while mixing them uniformly, and can obtain a high current density by direct electron transfer reaction, while suppressing elution of the promoter molecule, and to provide a production method of an enzyme electrode using the enzyme ink, and an enzyme fuel cell using an enzyme electrode produced using the enzyme ink.SOLUTION: The enzyme ink contains carbon particles, a hydrophilic binder, an oxidoreductase, a promoter molecule promoting electron transition between the carbon particles and oxidoreductase, and a solvent containing at least water. An enzyme electrode 13 is produced by applying or printing the enzyme ink 12 onto a substrate 11. The enzyme electrode 13 is used as a positive electrode or a negative electrode of an enzyme fuel cell.

Description

  The present disclosure relates to enzyme inks, enzyme fuel cells, and electronic devices. More specifically, the present disclosure relates to, for example, an enzyme ink suitable for use in manufacturing an enzyme electrode, an enzyme fuel cell having an enzyme electrode manufactured using the enzyme ink, and various types using the enzyme fuel cell as a power source. It is suitable for application to electronic equipment.

In recent years, enzyme fuel cells using enzymes (also referred to as biofuel cells) have attracted attention (see, for example, Patent Documents 1 to 12). In this enzyme fuel cell, fuel is decomposed by an enzyme and separated into protons (H ⁺ ) and electrons. As fuel, alcohols such as methanol and ethanol, monosaccharides such as glucose, or polysaccharides such as starch are used. What has been developed.

  Enzyme fuel cells are classified into a mediator type and a direct electron transfer type depending on whether or not a mediator is used for an electron transfer reaction between an enzyme and an electrode. In the mediator type enzyme fuel cell, a high current density can be expected, but the crossover between the positive electrode (cathode) and the negative electrode (anode) due to the elution of the mediator, and the output decrease due to it, become problems. Although the direct electron transfer type enzyme fuel cell has a low current density, it does not easily cause crossover. Therefore, it is desirable to obtain a high current density with a direct electron transfer type enzyme fuel cell.

  Many direct electron transfer reactions of multi-copper oxidase that perform four-electron reduction of oxygen have been reported so far. However, both of these have the problem that the current density is low.

  CueO (copper efflux oxidase) (a type of multi-copper oxidase) has been reported as an enzyme that can obtain a high current value by direct electron transfer reaction (see Patent Document 13). However, since the redox potential of CueO is low, there is a problem that the potential loss is large. For this reason, it is desirable to obtain a high current value using bilirubin oxidase or laccase having a high redox potential.

  In the direct electron transfer reaction of bilirubin oxidase, pyrroloquinoline quinone (PQQ) is known to be effective as a promoter molecule that promotes the direct electron transfer reaction of bilirubin oxidase (see Non-Patent Document 1). However, since PQQ itself may reversibly react with the electrode in the same manner as the mediator molecule, elution of PQQ causes a crossover between the positive electrode and the negative electrode, which may cause a problem in that the output decreases.

  Anthracene derivatives are known to be effective as laccase promoter molecules (see Non-Patent Documents 2 to 6 and Patent Document 14). This molecule shows no redox reaction with the electrode, so there is no crossover problem.

JP 2000-133297 A JP 2003-282124 A JP 2004-71559 A JP 2005-13210 A JP 2005-310613 A JP 2006-24555 A JP 2006-49215 A JP 2006-93090 A JP 2006-127957 A JP 2006-156354 A JP 2007-12281 A JP 2007-35437 A JP 2008-64514 A International Publication No. 08/062216 JP 2008-273816 A

Electrochem.Commun., 10,2008,1691-1694 Chem.Commun., 2007,1710-1712 Faraday Discuss., 2008,140,319-335 Phys.Chem.Chem.Phys., 2010,12,10018-10026 J. Phys. Chem. Lett. 2010, 1,2251-2254 ACS Catal. 2011,1,1683-1690

  However, since anthracene derivatives are highly hydrophobic, they are insoluble in water. For this reason, the hydrophilic enzyme and the anthracene derivative cannot be mixed uniformly and used.

  Therefore, the problem to be solved by the present disclosure is that a hydrophilic enzyme and a promoter molecule can be mixed uniformly and used, and a high current density can be obtained by direct electron transfer reaction. An enzyme ink capable of producing an enzyme electrode capable of suppressing elution is provided.

  Another problem to be solved by the present disclosure is to provide an enzyme fuel cell using an enzyme electrode capable of obtaining a high current density by direct electron transfer reaction and suppressing elution of promoter molecules.

  Still another problem to be solved by the present disclosure is to provide a high-performance electronic device having the enzyme fuel cell described above.

  The above and other problems will become apparent from the following description of the present specification with reference to the accompanying drawings.

In order to solve the above problems, the present disclosure provides:
Carbon particles,
A hydrophilic binder,
Oxidoreductase,
A promoter molecule that promotes electron transfer between the carbon particles and the redox enzyme;
An enzyme ink containing at least a solvent containing water.

In addition, this disclosure
An enzyme ink containing carbon particles, a hydrophilic binder, an oxidoreductase, a promoter molecule that promotes electron transfer between the carbon particles and the oxidoreductase, and a solvent containing at least water is placed on the substrate. It is the manufacturing method of the enzyme electrode which has the process of apply | coating or printing on.

In addition, this disclosure
An enzyme ink containing carbon particles, a hydrophilic binder, an oxidoreductase, a promoter molecule that promotes electron transfer between the carbon particles and the oxidoreductase, and a solvent containing at least water is placed on the substrate. It is a manufacturing method of the enzyme reaction utilization apparatus which has the process of apply | coating or printing on.

  Here, the enzyme reaction utilization apparatus is, for example, an enzyme fuel cell, a biosensor, a bioreactor, or the like.

In addition, this disclosure
An enzyme ink containing carbon particles, a hydrophilic binder, an oxidoreductase, a promoter molecule that promotes electron transfer between the carbon particles and the oxidoreductase, and a solvent containing at least water is placed on the substrate. It is an enzyme reaction utilization apparatus which has the enzyme electrode manufactured by apply | coating or printing on.

In addition, this disclosure
An enzyme ink containing carbon particles, a hydrophilic binder, an oxidoreductase, a promoter molecule that promotes electron transfer between the carbon particles and the oxidoreductase, and a solvent containing at least water is placed on the substrate. It is the electronic device which has the enzyme fuel cell which has the enzyme electrode manufactured by apply | coating or printing on.

In addition, this disclosure
A positive electrode;
A negative electrode,
A proton conductor provided between the positive electrode and the negative electrode;
The positive electrode is
Carbon particles,
A hydrophilic binder,
Oxidoreductase,
It is an enzyme fuel cell having a promoter molecule that promotes electron transfer between the carbon particles and the oxidoreductase.

In addition, this disclosure
Having at least one enzyme fuel cell;
The enzyme fuel cell is
A positive electrode;
A negative electrode,
A proton conductor provided between the positive electrode and the negative electrode;
The positive electrode is
Carbon particles,
A hydrophilic binder,
Oxidoreductase,
An electronic device having a promoter molecule that promotes electron transfer between the carbon particles and the oxidoreductase.

  In the present disclosure, the promoter molecule typically has a site that binds to carbon particles and a site that binds to oxidoreductase. The bond between the promoter molecule and the carbon particle or oxidoreductase may be basically any bond as long as electron transfer is possible through this bond, for example, electrostatic interaction, In addition to van der Waals interaction, hydrogen bond interaction, etc., a covalent bond may be used. Promoter molecules are typically organic acids, preferably organic acids that are sparingly soluble in water, but may be water soluble. As the organic acid, for example, an organic acid having a ππ bond with a carbon particle (for example, a ππ bond between a π electron of an aromatic ring and a π electron on the surface of the carbon particle) can be used. A compound having a hydrophobic site such as a chain alkyl can be used. The organic acid is most preferably a carboxylic acid. The carboxylic acid is at least one selected from the group consisting of a compound having at least one benzene ring, a compound having at least one heterocyclic ring, a fatty acid and citric acid. The carboxylic acid is, for example, an anthracene derivative, a pyrene derivative or a naphthalene derivative. As the promoter molecule, 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) may be used.

  As the oxidoreductase, various conventionally known enzymes can be used, and are appropriately selected depending on whether the enzyme electrode is used as a positive electrode or a negative electrode.

  As a solvent containing at least water contained in the enzyme ink, a solvent mainly containing water is preferably used, and water is typically used. As a solvent other than water, for example, an organic or inorganic solvent can be used.

  From the viewpoint of improving the heat resistance and current density of the enzyme electrode, particularly the positive electrode, if necessary, the enzyme ink may contain at least one saccharide. Examples of the saccharide include saccharides such as sucrose, trehalose, maltose, lactose, cellobiose, raffinose, melezitose, maltotriose, stachyose, maltohexaose, cyclodextrin, dextrin, arabitol, sorbitol, xylitol, sugar alcohol, polysaccharide In particular, raffinose is preferable.

  The positive electrode of the enzyme fuel cell further includes an electron mediator as necessary. A conventionally known electron mediator can be used as the electron mediator, and is selected according to need. Most preferably, ABTS can be used.

  Enzyme fuel cells can be used for almost anything that requires electric power and can be of any size. For example, electronic devices, mobile objects (automobiles, motorcycles, aircraft, rockets, spacecrafts, etc.), power units, construction machinery, It can be used for machine tools, power generation systems, cogeneration systems, etc. The output, size, shape, type of fuel, etc. are determined depending on the application.

  Electronic devices may be basically any type, and include both portable and stationary devices. Specific examples include mobile phones, mobile devices (portable information terminals). (PDA, etc.), robots, personal computers (including both desktop and notebook computers), game machines, camera-integrated VTRs (video tape recorders), in-vehicle devices, home appliances, industrial products, and the like.

In addition, this disclosure
A positive electrode;
A negative electrode,
A proton conductor provided between the positive electrode and the negative electrode;
The positive electrode is
Carbon particles,
A hydrophilic binder,
Oxidoreductase,
It is an enzyme fuel cell having ABTS.

In addition, this disclosure
Having at least one enzyme fuel cell;
The enzyme fuel cell is
A positive electrode;
A negative electrode,
A proton conductor provided between the positive electrode and the negative electrode;
The positive electrode is
Carbon particles,
A hydrophilic binder,
Oxidoreductase,
This is an electronic device having ABTS.

In addition, this disclosure
Carbon particles,
A hydrophilic binder,
Oxidoreductase,
ABTS,
An enzyme ink containing at least a solvent containing water.

  Here, ABTS in these enzyme fuel cells, electronic devices and enzyme inks act as promoter molecules or electron mediators or both.

  As far as these enzyme fuel cells, electronic devices and enzyme inks are not contrary to their properties, what has already been described in relation to the enzyme fuel cells, electronic devices and enzyme inks is valid.

  As described above, in the present disclosure, the enzyme ink includes carbon particles, a hydrophilic binder, a redox enzyme, a promoter molecule that promotes electron transfer between the carbon particles and the redox enzyme, and at least water. Containing a solvent. In this case, uniform water miscibility can be obtained by the carbon particles, the hydrophilic binder, and water, and the promoter molecules and the oxidoreductase can be uniformly dispersed.

  According to the present disclosure, hydrophilic oxidoreductase and promoter molecules can be mixed and used uniformly, high current density can be obtained by direct electron transfer reaction, and elution of promoter molecules can be suppressed. It is possible to obtain an enzyme ink capable of producing an enzyme electrode capable of By using this enzyme ink, it is possible to produce an enzyme electrode that can obtain a high current density by direct electron transfer reaction and can suppress elution of promoter molecules. A high-performance enzyme fuel cell can be realized using this enzyme electrode. Further, by using this enzyme fuel cell, a high-performance electronic device or the like can be realized.

It is sectional drawing for demonstrating the enzyme electrode by 1st Embodiment, and its manufacturing method. It is a basic diagram which shows the enzyme electrode by 1st Embodiment. It is a basic diagram which shows the experimental result of Examples 1-8. It is a basic diagram which shows the experimental result of Examples 1-8 and Comparative Examples 1-5. 6 is a schematic diagram showing the results of cyclic voltammetry measurement performed using the enzyme electrode of Example 20. FIG. It is an approximate line figure showing the enzyme electrode by a 1st embodiment typically. It is a basic diagram which shows the result of the experiment conducted in order to demonstrate the effect of raffinose addition to enzyme ink. It is a basic diagram which shows the result of the experiment conducted in order to demonstrate the effect of raffinose addition to enzyme ink. It is a basic diagram which shows the result of the experiment conducted in order to demonstrate the effect of raffinose addition to enzyme ink. It is a basic diagram which shows the enzyme fuel cell by 3rd Embodiment. Details of the configuration of the negative electrode of the enzyme fuel cell according to the third embodiment, an example of an enzyme group and a coenzyme immobilized on the negative electrode, and a schematic line schematically showing an electron transfer reaction by the enzyme group and the coenzyme FIG. It is a basic diagram which shows the specific structural example of the enzyme fuel cell by 3rd Embodiment. It is a basic diagram which shows the bioreactor by 4th Embodiment. It is a basic diagram which shows the result of the cyclic voltammetry measurement performed using the electrode A produced in order to perform the fundamental examination of the enzyme ink used for manufacture of the enzyme electrode by 6th Embodiment. It is a basic diagram which shows the result of the cyclic voltammetry measurement performed using the electrode B produced in order to perform the fundamental examination of the enzyme ink used for manufacture of the enzyme electrode by 6th Embodiment. It is a basic diagram which shows the result of the cyclic voltammetry measurement performed using the electrode C produced in order to perform the fundamental examination of the enzyme ink used for manufacture of the enzyme electrode by 6th Embodiment. It is a basic diagram which shows the result of the cyclic voltammetry measurement performed using the electrode D produced in order to perform the fundamental examination of the enzyme ink used for manufacture of the enzyme electrode by 6th Embodiment. It is a basic diagram which shows the result of the cyclic voltammetry measurement performed using the ABTS fixed electrode E produced in order to perform the fundamental examination of the enzyme ink used for manufacture of the enzyme electrode by 6th Embodiment. FIG. 19 is a schematic diagram in which peak current values obtained from the results of cyclic voltammetry measurement shown in FIG. 18 are plotted with respect to the number of cycles. It is a basic diagram which shows the result of the cyclic voltammetry measurement performed using the FeCN fixed electrode F produced in order to perform the fundamental examination of the enzyme ink used for manufacture of the enzyme electrode by 6th Embodiment. FIG. 21 is a schematic diagram in which peak current values obtained from the results of cyclic voltammetry measurement shown in FIG. 20 are plotted with respect to the number of cycles. It is a basic diagram which shows the result of having performed the constant potential measurement of the enzyme electrode of Examples 24-26. It is a basic diagram which shows the result of the voltage measurement of the enzyme electrode of Example 26, and the FeCN fixed enzyme electrode for a comparison.

  Hereinafter, modes for carrying out the invention (hereinafter referred to as “embodiments”) will be described. The description will be given in the following order.

1. First embodiment (enzyme ink and method for producing the same)
2. Second embodiment (enzyme electrode and method for producing the same)
3. Third embodiment (enzyme fuel cell)
4). Fourth embodiment (bioreactor)
5. Fifth embodiment (enzyme ink and method for producing the same)
6). Sixth embodiment (enzyme electrode and method for producing the same)

<1. First Embodiment>
[Enzyme ink]
The enzyme ink according to the first embodiment includes carbon particles, a hydrophilic (or water-soluble) binder, an oxidoreductase, a promoter molecule that promotes electron transfer between the carbon particles and the oxidoreductase, And a solvent containing at least water. The content of each component in the enzyme ink is 30% to 85% by weight for carbon particles, 1% to 65% by weight for the binder, and 5% to 15% by weight for the promoter molecule. This enzyme ink typically has a total of 100% by weight of carbon particles, binder, promoter molecule, and solvent, but when other components are also included, the total is 100 including these other components. It is made to become weight%. The enzyme ink contains saccharides such as raffinose as necessary, and the content thereof is 3 wt% or more and 85 wt% or less. When the enzyme ink contains saccharides such as raffinose, the total amount of carbon particles, binder, promoter molecule, saccharide and solvent is 100% by weight. Including the above components, the total amount is 100% by weight.

The carbon particles include, for example, at least one selected from the group consisting of carbon black, biocarbon, and vapor grown carbon fiber, but other than these, for example, activated carbon may be used. Examples of carbon black include furnace black, acetylene black, channel black, thermal black, ketjen black, and the like. Among these, ketjen black is preferable. Biocarbon is made from a plant-derived material with a silicon (silicon) content of 5% by weight or more. The specific surface area by nitrogen BET method is 10 m ² / g or more, and the silicon content is 1% by weight or less. A porous carbon material having a pore volume of 0.1 cm ³ / g or more by the BJH method and the MP method (see Patent Document 15). Specifically, biocarbon is produced as follows, for example. That is, first, the crushed rice husk (produced in Kagoshima Prefecture, Isehikari rice husk) is carbonized by heating in a nitrogen stream at 500 ° C. for 5 hours to obtain a carbide. Thereafter, 10 g of this carbide is put in an alumina crucible and heated to 1000 ° C. at a rate of 5 ° C./min in a nitrogen stream (10 liters / min). And after carbonizing at 1000 degreeC for 5 hours and converting into a carbonaceous substance (porous carbon material precursor), it cools to room temperature. In addition, nitrogen gas was kept flowing during carbonization and cooling. Next, this porous carbon material precursor is subjected to an acid treatment by immersing it overnight in a 46% by volume hydrofluoric acid aqueous solution, and then washed with water and ethyl alcohol until pH 7 is reached. And the porous carbon material, ie, biocarbon, is obtained by making it dry at the end. The vapor grown carbon fiber is, for example, VGDF (registered trademark of Showa Denko KK). Examples of the activated carbon include wood charcoal such as oak charcoal, kunugi charcoal, cedar charcoal, oak charcoal, hinoki charcoal, rubber charcoal, bamboo charcoal, oga charcoal, and coconut shell charcoal.

  In some cases, carbon particles whose surfaces are modified with carboxylic acid may be used for at least some of the carbon particles contained in the enzyme ink. By using carbon particles surface-modified with such a carboxylic acid, it is possible to expand the range of promoter molecules that can be used, or to facilitate the binding of promoter molecules to carbon particles.

  Hydrophilic (or water-soluble) binders include, as natural products and derivatives thereof, celluloses and derivatives thereof (methylcellulose, carboxymethylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose (HPC), hydroxyethyl Methylcellulose (HEMC), hydroxypropylmethylcellulose (HPMC), hydroxybutylmethylcellulose (HBMC), hydroxyethylethylcellulose (HEEC), hydroxyethylhydroxypropylmethylcellulose (HEMPMC, etc.), protein systems (vegetable proteins such as wheat protein, soybean protein, etc.) , Animal proteins such as albumin, casein, gelatin), others (den Starch derivatives such as punphosphate, sodium starch glycolate, guar gum derivatives such as cationized guar gum, carboxymethylhydroxypropylated guar gum, hydroxypropylated guar gum, curdlan, xanthan gum, gellan gum, cyclodextrin, dextran, pullulan, etc. Production mucilages, alginic acid, agar, starch, chitosan, hyaluronic acid, pectin, carrageenan, polysaccharides such as laminarin), and mixtures, but not limited to these. Synthetic polymer binders include vinyl compounds, vinylidene compounds, polyester compounds, polyamide compounds, polyether compounds, polyglycol compounds, polyvinyl alcohol compounds, polyalkylene oxide compounds, polyacrylic acid compounds , And mixtures. For example, polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene glycol, aluminum / magnesium silicate, carboxyvinyl polymer, quince seed, polyacrylic acid, polyvinyl pyrrolidone / vinyl alcohol copolymer, polymethyl vinyl ether, propylene glycol alginate, and the like.

Examples of the oxidoreductase include glucose oxidase, alcohol oxidase, glucose dehydrogenase, diaphorase, NADH dehydrogenase, alcohol dehydrogenase, fructose dehydrogenase, gluconate dehydrogenase, cellobiose dehydrogenase, aldehyde dehydrogenase, amine dehydrogenase, succinate dehydrogenase, hydrogenase, and p-cresol. methyl hydroxylase, histamine dehydrogenase, fumarate reductase, succinate dehydrogenase, carbon monoxide dehydrogenase, nitrate reductase, arsenate reductase, sulfite reductase, DMSO reductase, cytochrome C _3, multi-copper oxidase (laccase, Fet3p, bilirubin oxidase, Cu O, CotA, ascorbate oxidase, stellacyanin, tyrosinase, nitrite reductase, etc.), catalase, peroxidase, cytochrome C, hemoglobin, myoglobin, cytochrome P450, and ferritin. It is appropriately selected depending on whether the enzyme electrode produced using is used as a positive electrode or a negative electrode.

  The promoter molecule is typically an organic acid. The organic acid is preferably sparingly soluble in water. The organic acid is preferably a carboxylic acid. The carboxylic acid is at least one selected from the group consisting of a compound having at least one benzene ring, a compound having at least one heterocyclic ring, a fatty acid and citric acid. The carboxylic acid is an anthracene derivative, pyrene derivative or naphthalene derivative. Examples of the anthracene derivative include 2-anthracene carboxylic acid and 9-anthracene carboxylic acid. Pyrene derivatives include, for example, amine group, sulfone group, sulfhydryl group, carboxyl group, hydroxyl group, azide group, azo group, nitro group, nitrile group, cyano group, allene group, isonitrile group, urea group, aldehyde group, ketone group, It has functional groups and reactive groups such as NHS ester, imide ester, maleimide, pyridyldithiol, allyl azide, haloacetate, isocyanate, carbodiimide, allyl azide, diazirine, hydrazide, psoralen, iodo, pyridine disulfide, vinyl sulfone. Specific examples of the pyrene derivative include 1-pyrenecarboxylic acid. Examples of the naphthalene carboxylic acid include 2-naphthalene acetic acid and 2,6-naphthalenedicarboxylic acid.

[Method for producing enzyme ink]
A method for producing the enzyme ink will be described.

  In the first production method, first, a predetermined amount of carbon particles, a hydrophilic binder, and an oxidoreductase are mixed, and then a solvent containing at least water, typically water, is added and mixed. Next, a predetermined amount of promoter molecules is added to the mixture and mixed to form an ink. In this way, enzyme ink is manufactured.

  In the second production method, first, a predetermined amount of carbon particles, a hydrophilic binder, and an oxidoreductase are mixed. Next, a predetermined amount of promoter molecules is added to the mixture and mixed. Next, a solvent containing at least water, typically water, is added to the mixture and mixed to form an ink. In this way, enzyme ink is manufactured.

  In the third production method, first, a predetermined amount of carbon particles and a hydrophilic binder are mixed, and then a solvent containing at least water, typically water, is added thereto and mixed. Next, a predetermined amount of promoter molecules is added to the mixture and mixed. Next, a predetermined amount of oxidoreductase is added to the mixture and mixed to form an ink. In this way, enzyme ink is manufactured.

  In the first, second, and third production methods, when the enzyme ink contains a saccharide such as raffinose, the saccharide is added in any step during the production.

<Example 1>
An enzyme ink was produced as follows.
Ketjen black 0.5 g, VGCF (registered trademark) 0.5 g, carboxymethylcellulose (CMC) 0.05 g and bilirubin oxidase (BOD) 0.05 g are mixed in a powder state.

To this mixture, 0.175 g of 2-anthracenecarboxylic acid was mixed in a powder state, and then 8 ml of water was added and kneaded to form an ink. An enzyme ink was thus produced. The structure of 2-anthracenecarboxylic acid is as follows.

<Example 2>
An enzyme ink was produced in the same manner as in Example 1 except that 9-anthracenecarboxylic acid was used instead of 2-anthracenecarboxylic acid. The structure of 9-anthracenecarboxylic acid is as follows.

<Example 3>
An enzyme ink was produced in the same manner as in Example 1 except that 1-pyrenecarboxylic acid was used instead of 2-anthracenecarboxylic acid. The structure of 1-pyrenecarboxylic acid is as follows.

<Example 4>
An enzyme ink was produced in the same manner as in Example 1 except that 2-naphthaleneacetic acid was used instead of 2-anthracenecarboxylic acid. The structure of 2-naphthalene acetic acid is as follows.

<Example 5>
An enzyme ink was produced in the same manner as in Example 1 except that 2,6-naphthalenedicarboxylic acid was used instead of 2-anthracenecarboxylic acid. The structure of 2,6-naphthalenedicarboxylic acid is as follows.

<Example 6>
An enzyme ink was produced in the same manner as in Example 1 except that benzoic acid was used instead of 2-anthracenecarboxylic acid. The structure of benzoic acid is as follows.

<Example 7>
An enzyme ink was produced in the same manner as in Example 1 except that lauric acid was used instead of 2-anthracenecarboxylic acid. The structure of lauric acid is as follows.

<Example 8>
An enzyme ink was produced in the same manner as in Example 1 except that citric acid was used instead of 2-anthracenecarboxylic acid. The structure of citric acid is as follows.

<Example 9>
An enzyme ink was produced in the same manner as in Example 1 except that PQQ was used instead of 2-anthracenecarboxylic acid. The structure of PQQ is as follows.

<Example 10>
An enzyme ink was produced in the same manner as in Example 1 except that CueO was used instead of bilirubin oxidase as the oxidoreductase.

  As described above, according to the first embodiment, the enzyme ink includes carbon particles, a hydrophilic (or water-soluble) binder, an oxidoreductase, and a carbon particle and an oxidoreductase. A promoter molecule that promotes electron transfer and a solvent containing at least water are contained. In this case, uniform water miscibility can be obtained by the carbon particles, the hydrophilic binder, and water, and the promoter molecules and the oxidoreductase can be uniformly dispersed.

<2. Second Embodiment>
[Enzyme electrode and production method thereof]
1A to 1C show a method for producing an enzyme electrode according to a second embodiment.

  As shown in FIG. 1A, a substrate 11 is prepared. Although the board | substrate 11 is suitably selected according to the use, function, etc. of an enzyme electrode, a nonwoven fabric can be used suitably. As the material for the nonwoven fabric, for example, various organic polymer compounds such as polyolefin, polyester, cellulose, polyacrylamide and the like can be used, but the material is not limited thereto.

  Next, as shown in FIG. 1B, the enzyme ink 12 according to the first embodiment is applied or printed to a predetermined thickness on one main surface of the substrate 11. There is no particular limitation on the method of applying or printing the enzyme ink 12, and a conventionally known method can be used. Specifically, as a coating method, for example, a dipping method, a spray method, a wire bar method, a spin coating method, a roller coating method, a blade coating method, a gravure coating method, or the like can be used. Moreover, as a printing method, a relief printing method, an offset printing method, a gravure printing method, an intaglio printing method, a rubber plate printing method, a screen printing method, etc. can be used.

  Next, the substrate 11 on which the enzyme ink 12 has been applied or printed is heated or kept at room temperature to dry and remove the solvent in the enzyme ink 12 to solidify. Thus, as shown in FIG. 1C, an enzyme electrode 13 made of carbon particles, a hydrophilic binder, an oxidoreductase and a promoter molecule, or further containing a saccharide such as raffinose is obtained on the substrate 11. Thereafter, if necessary, both surfaces of the substrate 11 on which the enzyme electrode 13 is formed are cleaned by ozone treatment or the like.

  Depending on the material of the substrate 11, the enzyme ink 12 may permeate the substrate 11. In this case, as shown by the alternate long and short dash line in FIG. 1C, the enzyme electrode 13 is formed in a state where the lower part thereof is buried in the substrate 11.

  When the enzyme electrode 13 has water repellency, for example, a water repellent containing a water repellent material is formed on the surface of the enzyme electrode 13. For example, this water repellent is applied to the surface of the enzyme electrode 13, or the enzyme electrode 13 is dipped (immersed) in the water repellent. Various water repellents can be used and are selected as necessary. For example, a water repellent material, particularly a fine water repellent material dispersed in an organic solvent can be used. . The ratio of the water repellent material in the water repellent may be extremely small. As the water repellent, preferably used is one containing at least a water repellent material and an organic solvent having a sufficiently low solubility of the enzyme, for example, a solubility of 10 mg / ml or less, preferably 1 mg / ml or less. As the organic solvent, for example, methyl isobutyl ketone can be used. If necessary, the water repellent contains a binder in addition to the water repellent material and the organic solvent. Various binders can be used as this binder. For example, polyvinyl butyral can be used. The ratio of the binder in the water repellent is, for example, 0.01 to 10%, but is not limited thereto. In the case where the binder has water repellency such as polyvinylidene fluoride (PVDF), the binder itself can be used as the water repellent material. As the water repellent material, various materials can be used. For example, a carbon-based material, preferably carbon powder can be used. As the carbon powder, for example, graphite such as natural graphite, activated carbon, carbon nanofiber (gas phase carbon fiber) used for an additive of a lithium ion battery, ketjen black and the like can be used.

<Example 11>
A non-woven fabric was used as the substrate 11, and the enzyme ink 12 produced in Example 1 was applied to the non-woven fabric by a thickness of 50 μm using a coater, and then heated on a hot plate at 75 ° C. for 2 hours to dry and remove water.

  Thus, the enzyme electrode 13 made of ketjen black, VGCF (registered trademark), carboxymethylcellulose, and bilirubin oxidase was formed on the substrate 11 made of nonwoven fabric. At this time, the enzyme electrode 13 was formed in a state where the lower part was buried in the substrate 11 made of a nonwoven fabric.

<Example 12>
An enzyme electrode 13 was produced in the same manner as in Example 11 except that the enzyme ink of Example 2 was used instead of the enzyme ink of Example 1.

<Example 13>
An enzyme electrode 13 was produced in the same manner as in Example 11 except that the enzyme ink of Example 3 was used instead of the enzyme ink of Example 1.

<Example 14>
An enzyme electrode 13 was produced in the same manner as in Example 11 except that the enzyme ink of Example 4 was used instead of the enzyme ink of Example 1.

<Example 15>
An enzyme electrode 13 was produced in the same manner as in Example 11 except that the enzyme ink of Example 5 was used instead of the enzyme ink of Example 1.

<Example 16>
An enzyme electrode 13 was produced in the same manner as in Example 11 except that the enzyme ink of Example 6 was used instead of the enzyme ink of Example 1.

<Example 17>
An enzyme electrode 13 was produced in the same manner as in Example 11 except that the enzyme ink of Example 7 was used instead of the enzyme ink of Example 1.

<Example 18>
An enzyme electrode 13 was produced in the same manner as in Example 11 except that the enzyme ink of Example 8 was used instead of the enzyme ink of Example 1.

<Example 19>
An enzyme electrode 13 was produced in the same manner as in Example 11 except that the enzyme ink of Example 9 was used instead of the enzyme ink of Example 1.

<Example 20>
An enzyme electrode 13 was produced in the same manner as in Example 11 except that the enzyme ink of Example 10 was used instead of the enzyme ink of Example 1.

<Comparative example 1>
An enzyme ink was produced in the same manner as in Example 1 except that 2-anthracenecarboxylic acid was not added, and an enzyme electrode was produced in the same manner as in Example 11 using this enzyme ink.

<Comparative example 2>
Instead of the enzyme ink of Example 1 to which 2-anthracene carboxylic acid was added, an enzyme electrode to which 2-aminoanthracene was added instead of 2-anthracene carboxylic acid was used in the same manner as in Example 11 to form an enzyme electrode. Formed. The structure of 2-aminoanthracene is as follows.

<Comparative Example 3>
Instead of the enzyme ink of Example 1 to which 2-anthracenecarboxylic acid was added, an enzyme ink to which 2- (hydroxymethyl) anthracene was added instead of 2-anthracenecarboxylic acid was used in the same manner as in Example 11. An enzyme electrode was produced. The structure of 2- (hydroxymethyl) anthracene is as follows.

<Comparative example 4>
An enzyme electrode was produced in the same manner as in Example 11 using an enzyme ink to which anthracene was added instead of 2-anthracenecarboxylic acid instead of the enzyme ink of Example 1 to which 2-anthracenecarboxylic acid was added. The structure of anthracene is as follows.

<Comparative Example 5>
An enzyme electrode was formed in the same manner as in Example 11 using an enzyme ink to which phthalocyanine was added instead of 2-anthracenecarboxylic acid instead of the enzyme ink of Example 1 to which 2-anthracenecarboxylic acid was added. The structure of phthalocyanine is as follows.

  The enzyme electrodes of Examples 11 to 19 and Comparative Examples 1 to 5 were measured at a constant potential (0.25 V vs Ag | AgCl () in a solution of 1.0 M phosphate buffer pH 5.0 (containing 0.8 M glucose). The measurement results of Examples 11 to 18 are shown in Fig. 3. The measurement results of Comparative Examples 1 to 5 are shown in Fig. 4 together with the measurement results of Examples 11 to 19.

  As can be seen from FIGS. 3 and 4, the promoter molecule includes 2-anthracenecarboxylic acid, 9-anthracenecarboxylic acid, 1-pyrenecarboxylic acid, 2-naphthaleneacetic acid, 2,6-naphthalenedicarboxylic acid, benzoic acid, lauric acid. In any of Examples 11 to 19 using citric acid or PQQ, the current value was higher than that of Comparative Example 1 (indicated as Ctrl in FIGS. 3 and 4) using an enzyme ink containing no promoter molecule. Is obtained.

  Electrode performance was evaluated using the enzyme electrode 13 of Example 10 using CueO. Therefore, cyclic voltammetry evaluation was performed using hexacyanoferrate ions. The result is shown in FIG. As apparent from FIG. 5, the enzyme electrode of Example 11 manufactured using the enzyme ink of Example 1 to which 2-anthracenecarboxylic acid was added was obtained by using the enzyme ink to which 2-anthracenecarboxylic acid was not added. Compared with the enzyme electrode of Comparative Example 1 produced by using, a very good electrochemical response was shown.

[Model of enzyme electrode]
FIG. 6 shows an enzyme electrode model. This enzyme electrode is a positive electrode. As shown in FIG. 6, an enzyme is immobilized on an electrode made of a material containing carbon particles via a promoter molecule. Electrons move from the electrode through the promoter molecule to the enzyme, receive protons and react with O ₂ to produce H ₂ O.

[Effects obtained by including raffinose in enzyme ink]
Here, the result of verifying the effect obtained by including raffinose in the enzyme ink will be described. The structure of raffinose is as follows.

In order to perform verification, a positive electrode was produced as follows and constant potential measurement was performed.
First, commercially available bilirubin oxidase (BOD) (manufactured by Amano Enzyme) and BOD in which sucrose previously contained in the commercially available BOD is replaced with raffinose or trehalose are prepared.

20 μl of 25 mg / ml BOD solution, 20 μl of 2% poly-L-lysine (PLL) aqueous solution, and 20 μl of 20 mM aqueous potassium ferricyanide solution were mixed and coated on a 1 cm ² carbon electrode. Then, it was dried at 30 ° C. for 2 hours.

  After drying, 40 μl of water repellent was applied and dried for 2 hours. This water repellent contains 13 to 18% natural graphite as a water repellent material, 3 to 8% polyvinyl butyral as a binder, 8.4% carbon black, and 69.48% methyl isobutyl ketone as an organic solvent. It is.

  The positive electrode thus produced was stored in an atmosphere of 50 ° C. (43% relative humidity), and the relationship between the storage period and the deterioration of the electrode performance was measured in a solution of 2.0M imidazole pH 7.0 at a constant potential (0.25 V vs. Ag | AgCl (sat. KCl)).

  The result of plotting the current value after 1 hour is shown in FIG. FIG. 7 shows that heat resistance is improved by adding trehalose or raffinose to the positive electrode. When comparing trehalose and raffinose, it was found that raffinose had a higher performance maintenance rate.

  Next, the results of examining the relationship between the amount of raffinose added to the positive electrode, the improvement in current density, and the improvement in heat resistance will be described.

For this purpose, a positive electrode was produced as follows.
20 μl of 25 mg / ml BOD solution, 20 μl of 2% poly-L-lysine (PLL) aqueous solution, 20 μl of 20 mM potassium ferricyanide aqueous solution, 0-8 mg of raffinose (8 mg or more is insoluble) were mixed and coated on a 1 cm ² carbon electrode. . Then, it was dried at 30 ° C. for 2 hours.

  After drying, 40 μl of water repellent was applied and dried for 2 hours. This water repellent contains 13 to 18% natural graphite as a water repellent material, 3 to 8% polyvinyl butyral as a binder, 8.4% carbon black, and 69.48% methyl isobutyl ketone as an organic solvent. It is.

  The positive electrode thus prepared was stored in an atmosphere at 80 ° C. (dry conditions), and the relationship between the storage period and the performance deterioration of the electrode was determined in a solution of 1.0 M phosphate buffer pH 7.0 (containing 0.8 M glucose). And evaluated at a constant potential measurement (0.25 V vs Ag | AgCl (sat. KCl)).

  FIG. 8 shows the results of measuring the current density immediately after producing the positive electrode (after 1 hour) with respect to the amount of raffinose added. In FIG. 8, A shows the case where a commercially available BOD powder is used, and R shows the case where an enzyme powder in which sucrose contained in a commercially available BOD is substituted with raffinose is used. From FIG. 8, it was found that the current density of the positive electrode improved as the amount of raffinose added increased to 2.0 mg.

The result of the storage test at 80 ° C. (drying conditions) is shown in FIG. From FIG. 9, it was found that the heat resistance of the positive electrode was improved as the amount of raffinose added was increased, and 8 mg / cm ² was the highest.

  As described above, according to the second embodiment, the carbon particles, the hydrophilic (or water-soluble) binder, the redox enzyme, and the electron transfer between the carbon particles and the redox enzyme are promoted. The enzyme electrode 13 is manufactured by applying an enzyme ink 12 containing a promoter molecule and a solvent containing at least water on the substrate 11. The enzyme electrode 13 can obtain a high current density by direct electron transfer reaction. Moreover, elution of promoter molecules can be suppressed by using promoter molecules that are hardly soluble in water. In addition, many promoter molecules such as 2-anthracenecarboxylic acid do not have redox activity with the electrode, and therefore do not cause crossover due to elution from the electrode, and are effective for power generation by the enzyme fuel cell. Furthermore, many promoter molecules such as 2-anthracene carboxylic acid do not need to be covalently bonded to carbon particles, and are simply mixed with the enzyme ink, and the enzyme electrode 13 is simply manufactured by simply applying or printing the enzyme ink. be able to.

<3. Third Embodiment>
[Enzyme fuel cell]
Next, a third embodiment will be described. In the third embodiment, the enzyme electrode according to the second embodiment is used as the positive electrode of the enzyme fuel cell.

  FIG. 10 schematically shows this enzyme fuel cell. In this enzyme fuel cell, glucose is used as a fuel. FIG. 11 schematically shows details of the configuration of the negative electrode of the enzyme fuel cell, an example of an enzyme group and a coenzyme immobilized on the negative electrode, and an electron transfer reaction by the enzyme group and the coenzyme.

As shown in FIGS. 10 and 11, the enzyme fuel cell has a structure in which a negative electrode 21 and a positive electrode 22 face each other with an electrolyte layer 23 interposed therebetween. The negative electrode 21 decomposes glucose supplied as fuel with an enzyme to extract electrons and generate protons (H ⁺ ). The positive electrode 22 generates water by protons transported from the negative electrode 21 through the electrolyte layer 23, electrons sent from the negative electrode 21 through an external circuit, and oxygen in the air, for example.

  In the negative electrode 21, an enzyme involved in the decomposition of glucose, a coenzyme in which a reductant is generated by an oxidation reaction in the glucose decomposition process, and a coenzyme that oxidizes the reductant of the coenzyme are formed on the electrode 11 made of carbon or the like. Oxidase is immobilized. If necessary, an electron mediator that receives electrons generated from the coenzyme oxidase accompanying the oxidation of the coenzyme and passes it to the electrode 11 is also immobilized on the negative electrode 21.

  As an enzyme involved in the degradation of glucose, for example, glucose dehydrogenase (GDH), preferably NAD-dependent glucose dehydrogenase can be used. In the presence of this oxidase, for example, β-D-glucose can be oxidized to D-glucono-δ-lactone.

  Furthermore, this D-glucono-δ-lactone can be decomposed into 2-keto-6-phospho-D-gluconate by the presence of two enzymes, gluconokinase and phosphogluconate dehydrogenase (PhGDH). Can do. That is, D-glucono-δ-lactone is converted to D-gluconate by hydrolysis, and D-gluconate is converted to adenosine triphosphate (ATP) and adenosine diphosphate (ADP) in the presence of gluconokinase. And then phosphorylated to 6-phospho-D-gluconate. This 6-phospho-D-gluconate is oxidized to 2-keto-6-phospho-D-gluconate by the action of the oxidase PhGDH.

In addition to the above decomposition process, glucose can also be decomposed to CO ₂ by utilizing sugar metabolism. The decomposition process utilizing sugar metabolism is roughly divided into glucose decomposition and pyruvic acid generation by a glycolysis system and a TCA cycle, which are widely known reaction systems.

The oxidation reaction in the monosaccharide decomposition process is accompanied by a coenzyme reduction reaction. This coenzyme is almost determined by the acting enzyme. In the case of GDH, NAD ⁺ is used as the coenzyme. That is, when β-D-glucose is oxidized to D-glucono-δ-lactone by the action of GDH, NAD ⁺ is reduced to NADH to generate H ⁺ .

The produced NADH is immediately oxidized to NAD ⁺ in the presence of diaphorase (DI), generating two electrons and H ⁺ . Therefore, two electrons and two H ⁺ are generated by one-step oxidation reaction per glucose molecule. In the two-stage oxidation reaction, a total of four electrons and four H ⁺ are generated.

Electrons generated in the above process are transferred from diaphorase to the electrode 11 of the negative electrode 21 through the electron mediator, and H ⁺ is transported to the positive electrode 22 through the electrolyte layer 23.

The above enzyme, coenzyme, and electron mediator are optimal for the enzyme by using a buffer solution such as a phosphate buffer solution or a Tris buffer solution contained in the electrolyte layer 23 so that the electrode reaction can be performed efficiently and constantly. It is preferably maintained at a low pH, for example, around pH 7. As the phosphate buffer, for example, NaH ₂ PO ₄ or KH ₂ PO ₄ is used. Further, the ionic strength (IS) is too large or too small to adversely affect the enzyme activity, but considering the electrochemical response, it should be an appropriate ionic strength, for example, about 0.3. Is preferred. However, pH and ionic strength have optimum values for each enzyme used, and are not limited to the values described above.

In FIG. 11, as an example, the enzyme involved in the degradation of glucose is glucose dehydrogenase (GDH), the coenzyme that produces a reductant in the oxidation reaction in the glucose degradation process is NAD ⁺ , and the coenzyme reductant. A case where the coenzyme oxidase that oxidizes certain NADH is diaphorase (DI), and the electron mediator that receives electrons generated from the coenzyme oxidase accompanying the oxidation of the coenzyme and passes it to the electrode 11 of the negative electrode 21 is ACNQ is illustrated. Yes.

  As the positive electrode 22, the enzyme electrode according to the second embodiment is used. For example, oxygen reductases such as bilirubin oxidase, laccase, and ascorbate oxidase are immobilized on the enzyme electrode. Preferably, in addition to the oxygen reductase, an electron mediator that transfers electrons to and from the positive electrode 22 is also immobilized on the positive electrode 22.

In the positive electrode 22, in the presence of the enzyme that decomposes oxygen, oxygen in the air is reduced by H ⁺ from the electrolyte layer 23 and electrons from the negative electrode 21 to generate water.

The electrolyte layer 23 is for transporting H ⁺ generated in the negative electrode 21 to the positive electrode 22, and is made of a material that does not have electron conductivity and can transport H ⁺ . Specific examples of the electrolyte layer 23 include those already mentioned, such as cellophane.

In the enzyme fuel cell configured as described above, when glucose is supplied to the negative electrode 21 side, the glucose is decomposed by a decomposing enzyme containing an oxidase. Since the oxidase is involved in the monosaccharide decomposition process, electrons and H ⁺ can be generated on the negative electrode 21 side, and a current can be generated between the negative electrode 21 and the positive electrode 22.

Next, a specific structural example of the enzyme fuel cell will be described.
As shown in FIGS. 12A and 12B, the enzyme fuel cell has a configuration in which a negative electrode 21 and a positive electrode 22 face each other with an electrolyte layer 23 interposed therebetween. In this case, Ti current collectors 41 and 42 are placed under the positive electrode 22 and the negative electrode 21, respectively, so that current collection can be performed easily. Reference numerals 43 and 44 denote fixed plates. The fixing plates 43 and 44 are fastened to each other by screws 45, and the positive electrode 22, the negative electrode 21, the electrolyte layer 23, and the Ti current collectors 41 and 42 are sandwiched between them. One surface (outer surface) of the fixing plate 43 is provided with a circular recess 43a for taking in air, and a plurality of holes 43b penetrating to the other surface are provided in the bottom surface of the recess 43a. These holes 43 b serve as air supply paths to the positive electrode 22. On the other hand, a circular recess 44a for fuel loading is provided on one surface (outer surface) of the fixing plate 44, and a number of holes 44b penetrating to the other surface are provided on the bottom surface of the recess 44a. These holes 44 b serve as a fuel supply path to the negative electrode 21. A spacer 46 is provided on the periphery of the other surface of the fixing plate 44, and when the fixing plates 43 and 44 are fastened to each other with screws 45, the interval between them becomes a predetermined interval. .

  As shown in FIG. 12B, a load 47 is connected between the Ti current collectors 41 and 42, and a glucose solution in which glucose is dissolved in, for example, a phosphate buffer solution is put into the recess 44a of the fixed plate 44 to generate electric power. .

  According to the third embodiment, a high-power enzyme fuel cell suitable for use as a power source for various electronic devices can be obtained.

<4. Fourth Embodiment>
[Bioreactor]
FIG. 13 shows a bioreactor according to the fourth embodiment.

  As shown in FIG. 13, in this bioreactor, an enzyme electrode 51 manufactured according to the second embodiment is installed in a column reactor. The oxidoreductase immobilized on the enzyme electrode 51 is appropriately selected according to the use of the bioreactor.

  In this bioreactor, a solution containing a reactant is passed through the column reactor and brought into contact with the enzyme electrode 51. Then, a product is obtained from the reaction product by an enzyme reaction with an enzyme immobilized on the enzyme electrode 51.

  In this bioreactor, other than the above are the same as those in the second embodiment unless contrary to the nature thereof.

  According to the fourth embodiment, a high-performance bioreactor can be realized.

<5. Fifth Embodiment>
[Enzyme ink]
The enzyme ink according to the fifth embodiment includes carbon particles, a hydrophilic (or water-soluble) binder, an oxidoreductase, ABTS as an electron mediator, and a solvent containing at least water. Here, ABTS can also function as a promoter molecule that promotes electron transfer between the carbon particles and the oxidoreductase. The carbon particles, binder, oxidoreductase and solvent are the same as those in the first embodiment. The content of each component in the enzyme ink is the same as that in the first embodiment.

[Method for producing enzyme ink]
The method for producing the enzyme ink is the same as that in the first embodiment.

<Example 21>
Ketjen Black 0.25 g, VGCF (registered trademark) 0.25 g, carboxymethylcellulose 0.1 g and bilirubin oxidase 0.3 g are mixed in a powder state.

  After ABTS 0.2g was mixed with this mixture in a powder state, 4 ml of water was added and kneaded to make an ink. An enzyme ink was thus produced.

<Example 22>
Ketjen Black 0.25 g, VGCF (registered trademark) 0.25 g, carboxymethylcellulose 0.1 g, bilirubin oxidase 0.3 g and raffinose 0.5 g are mixed in powder form.

  After ABTS 0.2g was mixed with this mixture in a powder state, 4 ml of water was added and kneaded to make an ink. An enzyme ink was thus produced.

<Example 23>
Ketjen Black 0.25 g, VGCF (registered trademark) 0.25 g, carboxymethylcellulose 0.1 g, bilirubin oxidase 0.3 g, raffinose 0.5 g and 2-6 naphthalenedicarboxylic acid 0.085 g are mixed in a powder state. .

  After ABTS 0.2g was mixed with this mixture in a powder state, 4 ml of water was added and kneaded to make an ink. An enzyme ink was thus produced.

  According to the fifth embodiment, the enzyme ink includes not only carbon particles, a hydrophilic (or water-soluble) binder, an oxidoreductase, and an electron mediator, but also carbon particles and an oxidoreductase. It contains ABTS that can function even as a promoter molecule that promotes electron transfer between them, and a solvent containing at least water. According to this enzyme ink, an ABTS-immobilized enzyme electrode that can prevent the elution of ABTS can be produced. Moreover, uniform water miscibility can be obtained by the carbon particles, the hydrophilic binder, and water, and ABTS and oxidoreductase can be uniformly dispersed.

<6. Sixth Embodiment>
[Enzyme electrode and production method thereof]
In the sixth embodiment, an enzyme electrode is manufactured in the same manner as in the second embodiment using the enzyme ink according to the fifth embodiment.

[Electronic mediator elution confirmation experiment]
An experiment was conducted to confirm the presence or absence of elution of the electron mediator immobilized on the electrode.

  Here, as components other than the oxidoreductase of the enzyme ink according to the fifth embodiment, Ketjen black and VGCF (registered trademark) as carbon particles, carboxymethylcellulose, ABTS and water as hydrophilic (or water-soluble) binders are used. Consider the case where First, the influence on the elution of ABTS by the presence or absence of each component was confirmed. For this purpose, four electrodes A, B, C and D were prepared. For electrode A, an ink containing 0.25 g of Ketjen Black, 0.25 g of VGCF (registered trademark), 0.1 g of carboxymethylcellulose and 4 ml of water was applied to the nonwoven fabric by a coater, and 20 μl of 25 mM ABTS was dipped, It was prepared by drying. The electrode B was similarly produced using the ink which made the carboxymethylcellulose 0g of the ink used for preparation of the electrode A. FIG. The electrode C was similarly manufactured using the ink which used the ketjen black of the ink used for preparation of the electrode A as 0 g, and VGCF (trademark) 0.5g. The electrode D was prepared in the same manner by using an ink having 0.5 g of ketjen black and 0 of VGCF (registered trademark) among the inks used to prepare the electrode A.

  Using these electrodes A, B, C and D, cyclic voltammetry (CV) measurement was performed in a solution of 1.0 M phosphate buffer pH 7.0. The potential sweep rate was 10 mV / s. The solution was discarded for each measurement, the electrode was washed with water, the solution was added again, and CV measurement was performed again for 5 cycles.

  The results of five CV measurements performed using electrodes A, B, C, and D are shown in FIGS. 14, 15, 16, and 17, respectively. As shown in FIGS. 14, 15, 16, and 17, current is obtained for all of electrodes A, B, C, and D, but the change in current value after 5 cycles of CV measurement is caused by electrode A From this, it can be seen that the inclusion of ketjen black, VGCF (registered trademark) and carboxymethylcellulose in the electrode preparation ink is effective in preventing ABTS elution.

  Next, the electron mediator immobilized on the electrode was changed to two types of ABTS or potassium ferricyanide (FeCN) (potassium hexacyanoferrate), and an experiment was conducted to confirm the presence or degree of their elution. As components other than the electron mediator of the ink for producing the electrode, among the electrodes A, B, C and D, the same components as the ink used for producing the electrode A having the best ABTS elution prevention performance That is, ketjen black, VGCF (registered trademark) and carboxymethylcellulose were used.

  In order to perform the experiment, two ink samples 1 and 2 were prepared. Ink sample 1 contains 0.25 g of Ketjen Black and 0.25 g of VGCF (registered trademark) as carbon particles as components other than the oxidoreductase of the enzyme ink according to the fifth embodiment, and is hydrophilic (or water-soluble). An ink containing 0.1 g of carboxymethyl cellulose, 0.1 g of ABTS, and 4 ml of water as a binder. Ink sample 2 contains 0.1 g of potassium ferricyanide (FeCN) instead of ABTS in ink sample 1, and is for comparison with ink sample 1. In addition, after mixing the components of the ink samples 1 and 2, foaming for 10 minutes was performed twice.

  A non-woven fabric was used as the substrate 11, and the above ink samples 1 and 2 were coated on the non-woven fabric by a coater with a thickness of 50 μm, and then heated on a hot plate at 40 ° C. for 2 hours to dry and remove water to produce an electrode. .

  The electrode produced using the ink sample 1 was designated as electrode E, and the electrode produced using the ink sample 2 was designated as electrode F.

  Using these electrodes E and F, CV measurement was performed in a solution of 1.0 M phosphate buffer pH 7.0. The potential sweep rate was 10 mV / s. The solution was discarded for each measurement, the electrode was washed with water, the solution was added again, and CV measurement was performed again for 5 cycles.

  The results of five CV measurements performed using electrode E are shown in FIG. FIG. 19 shows changes in the peak current value obtained by each CV measurement, with the peak current value obtained by the first measurement being 100%. Moreover, the result of 3 times of CV measurements performed using the electrode F is shown in FIG. FIG. 21 shows the change in the peak current value obtained by each CV measurement, with the peak current value obtained by the first measurement being 100%.

  As can be seen from FIGS. 18 to 21, in the electrode E in which ABTS is fixed, the peak current value hardly changes even when CV measurement is performed for five cycles, whereas in the electrode F in which FeCN is fixed, 2 The peak current value obtained by the first CV measurement is drastically reduced to about 30% of the peak current value obtained by the first CV measurement. That is, it can be seen that FeCN is easily eluted in the electrode F on which FeCN is immobilized, whereas ABTS is hardly eluted in the electrode E on which ABTS is immobilized. From this, it can be seen that ABTS is firmly fixed on the electrode E.

<Example 24>
A non-woven fabric was used as the substrate 11, and the enzyme ink 12 produced in Example 21 was applied to the non-woven fabric by a coater with a thickness of 50 μm, and then heated on a hot plate at 75 ° C. for 2 hours to dry and remove water.

  Thus, the enzyme electrode 13 made of ketjen black, VGCF (registered trademark), carboxymethylcellulose, ABTS, and bilirubin oxidase was formed on the substrate 11 made of nonwoven fabric.

<Example 25>
A non-woven fabric was used as the substrate 11, and the enzyme ink 12 produced in Example 22 was applied on the non-woven fabric by a coater to a thickness of 50 μm, and then heated on a hot plate at 75 ° C. for 2 hours to dry and remove water.

  Thus, the enzyme electrode 13 made of ketjen black, VGCF (registered trademark), carboxymethylcellulose, ABTS, bilirubin oxidase and raffinose was formed on the substrate 11 made of nonwoven fabric.

<Example 26>
A nonwoven fabric was used as the substrate 11, and the enzyme ink 12 produced in Example 23 was applied on the nonwoven fabric by a coater to a thickness of 50 μm, and then heated on a hot plate at 75 ° C. for 2 hours to dry and remove water.

  Thus, the enzyme electrode 13 made of ketjen black, VGCF (registered trademark), carboxymethylcellulose, ABTS, bilirubin oxidase, raffinose and 2-6 naphthalenedicarboxylic acid was formed on the substrate 11 made of nonwoven fabric.

  The enzyme electrodes of Examples 24-26 were subjected to constant potential measurement (0.4 V vs Ag | AgCl (sat. KCl)) in a solution of 1.0 M phosphate buffer pH 5.0 (containing 0.8 M glucose). The measurement results of Examples 24 to 26 are shown in Fig. 22. From Fig. 22, the enzyme electrode of Example 24 produced using the enzyme ink of Example 22 containing raffinose, the enzyme of Example 21 containing no raffinose. It can be seen that the current value is greatly improved as compared with the enzyme electrode of Example 25 produced using ink, and the enzyme ink of Example 23 containing 2-6 naphthalenedicarboxylic acid in addition to raffinose. In the enzyme electrode of Example 26 produced using the same, the current value was further improved compared to the enzyme electrode of Example 25 produced using the enzyme ink of Example 22 containing raffinose. In particular it is found that the initial current value is greatly improved.

  FIG. 23 shows the results of evaluating the voltage characteristics of the enzyme electrode of Example 26 and the enzyme electrode (control electrode) of Comparative Example 6 using FeCN as the electron mediator. The enzyme electrode of Comparative Example 6 is an enzyme electrode produced using an enzyme ink obtained by adding bilirubin oxidase to the ink sample 2 described above. In FIG. 23, constant potential measurement is performed at each potential, and the current value after one hour is plotted. From FIG. 23, it can be seen that ABTS has a higher oxidation-reduction potential than FeCN, and therefore the rise of the current value is shifted to the high voltage side.

  As described above, according to the sixth embodiment, the enzyme ink 12 includes carbon particles, a hydrophilic (or water-soluble) binder, an oxidoreductase, ABTS, and a solvent containing at least water. Is applied on the substrate 11 to produce the enzyme electrode 13. Since the enzyme electrode 13 can prevent the elution of ABTS, for example, when the enzyme electrode 13 is used as the positive electrode of the enzyme fuel cell, the output current can be maintained over a long period of time. Further, the enzyme electrode 13 can obtain a high current density by direct electron transfer reaction promoted by ABTS acting as a promoter molecule or ABTS acting as an electron mediator. ABTS does not need to be covalently bonded to the carbon particles, and the enzyme electrode 13 can be easily produced simply by mixing with enzyme ink and applying or printing this enzyme ink.

  Although the embodiments and examples have been specifically described above, the present disclosure is not limited to the above-described embodiments and examples, and various modifications can be made.

  For example, the numerical values, structures, configurations, shapes, materials, and the like given in the above-described embodiments and examples are merely examples, and different numerical values, structures, configurations, shapes, materials, etc. are used as necessary. Also good.

In addition, this technique can also take the following structures.
(1) It has a positive electrode, a negative electrode, and a proton conductor provided between the positive electrode and the negative electrode, and the positive electrode has carbon particles, a hydrophilic binder, an oxidoreductase, and the carbon. An enzyme fuel cell comprising a promoter molecule that promotes electron transfer between particles and the oxidoreductase.
(2) The enzyme fuel cell according to (1), wherein the promoter molecule has a site that binds to the carbon particles and a site that binds to the oxidoreductase.
(3) The enzyme fuel cell according to (1) or (2), wherein the promoter molecule is an organic acid.
(4) The enzyme fuel cell according to any one of (1) to (3), wherein the organic acid is hardly soluble in water.
(5) The enzyme fuel cell according to (3), wherein the organic acid is a carboxylic acid.
(6) The enzyme according to (5), wherein the carboxylic acid is at least one selected from the group consisting of a compound having at least one benzene ring, a compound having at least one heterocyclic ring, a fatty acid, and citric acid. Fuel cell.
(7) The enzyme fuel cell according to (6), wherein the carboxylic acid is an anthracene derivative, pyrene derivative or naphthalene derivative.
(8) The above carboxylic acids are 2-anthracene carboxylic acid, 9-anthracene carboxylic acid, 1-pyrene carboxylic acid, 2-naphthalene acetic acid, 2,6-naphthalenedicarboxylic acid, benzoic acid, lauric acid, citric acid and pyrroloquinoline. The enzyme fuel cell according to (6), wherein the enzyme fuel cell is at least one selected from the group consisting of quinones.
(9) The oxidoreductase is glucose oxidase, alcohol oxidase, glucose dehydrogenase, diaphorase, NADH dehydrogenase, alcohol dehydrogenase, fructose dehydrogenase, gluconate dehydrogenase, cellobiose dehydrogenase, aldehyde dehydrogenase, amine dehydrogenase, succinate dehydrogenase, hydrogenase, p- cresol methyl hydroxylase, histamine dehydrogenase, fumarate reductase, succinate dehydrogenase, carbon monoxide dehydrogenase, nitrate reductase, arsenate reductase, sulfite reductase, DMSO reductase, cytochrome C _3, multi-copper oxidase, catalase, peroxidase, cytochrome C, hemoglobin Myoglobin, enzyme fuel cell according to any one of (1) to at least one type selected from the group consisting of cytochrome P450 and ferritin (8).
(10) The enzyme fuel cell according to any one of (1) to (9), wherein the positive electrode further includes an electron mediator.
(11) The enzyme fuel cell according to (10), wherein the electron mediator is ABTS.
(12)
Having at least one enzyme fuel cell, the enzyme fuel cell has a positive electrode, a negative electrode, and a proton conductor provided between the positive electrode and the negative electrode, the positive electrode comprising carbon particles; An electronic device comprising a hydrophilic binder, an oxidoreductase, and a promoter molecule that promotes electron transfer between the carbon particles and the oxidoreductase.
(13) Enzyme ink containing carbon particles, a hydrophilic binder, an oxidoreductase, a promoter molecule that promotes electron transfer between the carbon particles and the oxidoreductase, and a solvent containing at least water. .
(14) The carbon particle content is 30% by weight to 85% by weight, the binder content is 1% by weight to 65% by weight, and the promoter molecule content is 5% by weight to 15% by weight. The enzyme ink according to (13) above.
(15) The enzyme ink according to (13) or (14), further containing at least one kind of sugar.
(16) The enzyme ink according to (15), wherein the content of the saccharide is 3% by weight or more and 85% by weight or less.

  DESCRIPTION OF SYMBOLS 11 ... Board | substrate, 12 ... Enzyme ink, 13 ... Enzyme electrode, 21 ... Negative electrode, 22 ... Positive electrode, 23 ... Electrolyte layer, 41, 42 ... Ti collector, 43, 44 ... Fixed plate

Claims (19)

A positive electrode;
A negative electrode,
A proton conductor provided between the positive electrode and the negative electrode;
The positive electrode is
Carbon particles,
A hydrophilic binder,
Oxidoreductase,
An enzyme fuel cell comprising a promoter molecule that promotes electron transfer between the carbon particles and the oxidoreductase.   The enzyme fuel cell according to claim 1, wherein the promoter molecule has a site that binds to the carbon particles and a site that binds to the oxidoreductase.   The enzyme fuel cell according to claim 2, wherein the promoter molecule is an organic acid.   The enzyme fuel cell according to claim 2, wherein the organic acid is hardly soluble in water.   The enzyme fuel cell according to claim 3, wherein the organic acid is a carboxylic acid.   6. The enzyme fuel cell according to claim 5, wherein the carboxylic acid is at least one selected from the group consisting of a compound having at least one benzene ring, a compound having at least one heterocyclic ring, a fatty acid and citric acid.   The enzyme fuel cell according to claim 6, wherein the carboxylic acid is an anthracene derivative, a pyrene derivative or a naphthalene derivative.   The carboxylic acid comprises 2-anthracene carboxylic acid, 9-anthracene carboxylic acid, 1-pyrene carboxylic acid, 2-naphthalene acetic acid, 2,6-naphthalenedicarboxylic acid, benzoic acid, lauric acid, citric acid and pyrroloquinoline quinone. The enzyme fuel cell according to claim 6, which is at least one selected from the group. The oxidoreductase is glucose oxidase, alcohol oxidase, glucose dehydrogenase, diaphorase, NADH dehydrogenase, alcohol dehydrogenase, fructose dehydrogenase, gluconate dehydrogenase, cellobiose dehydrogenase, aldehyde dehydrogenase, amine dehydrogenase, succinate dehydrogenase, hydrogenase, p-cresol methylhydroxyl. hydrolase, histamine dehydrogenase, fumarate reductase, succinate dehydrogenase, carbon monoxide dehydrogenase, nitrate reductase, arsenate reductase, sulfite reductase, DMSO reductase, cytochrome C _3, multi-copper oxidase, catalase, peroxidase, cytochrome C, hemoglobin, myo Robin, enzyme fuel cell according to claim 1, wherein at least one kind selected from the group consisting of cytochrome P450 and ferritin.   The enzyme fuel cell according to claim 1, wherein the positive electrode further comprises an electron mediator.   The enzyme fuel cell according to claim 10, wherein the electron mediator is ABTS. Having at least one enzyme fuel cell;
The enzyme fuel cell is
A positive electrode;
A negative electrode,
A proton conductor provided between the positive electrode and the negative electrode;
The positive electrode is
Carbon particles,
A hydrophilic binder,
Oxidoreductase,
An electronic device having a promoter molecule that promotes electron transfer between the carbon particles and the oxidoreductase. Carbon particles,
A hydrophilic binder,
Oxidoreductase,
A promoter molecule that promotes electron transfer between the carbon particles and the redox enzyme;
An enzyme ink containing a solvent containing at least water.   The carbon particle content is 30% by weight to 85% by weight, the binder content is 1% by weight to 65% by weight, and the promoter molecule content is 5% by weight to 15% by weight. 13. The enzyme ink according to 13.   The enzyme ink according to claim 13, further comprising at least one kind of sugar.   The enzyme ink according to claim 15, wherein the content of the saccharide is 3 wt% or more and 85 wt% or less. A positive electrode;
A negative electrode,
A proton conductor provided between the positive electrode and the negative electrode;
The positive electrode is
Carbon particles,
A hydrophilic binder,
Oxidoreductase,
An enzyme fuel cell comprising ABTS. Having at least one enzyme fuel cell;
The enzyme fuel cell is
A positive electrode;
A negative electrode,
A proton conductor provided between the positive electrode and the negative electrode;
The positive electrode is
Carbon particles,
A hydrophilic binder,
Oxidoreductase,
Electronic equipment having ABTS. Carbon particles,
A hydrophilic binder,
Oxidoreductase,
ABTS,
An enzyme ink containing a solvent containing at least water.