JP2010267459A - Fuel cell, its manufacturing method, enzyme fixed electrode, its manufacturing method, and electronic equipment - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fuel cell for preventing deterioration of output characteristics, lifetime, and efficiency or the like and for improving maintenance rate of current by preventing elution of an electronic mediator fixed to a positive electrode together with an enzyme, and its manufacturing method. <P>SOLUTION: A positive electrode 2 of a bio-fuel cell is manufactured by contacting a solution, in which a hydrophobic electronic mediator such as ferrocene is dissolved in an organic solvent having phase separation with water, on the surface of an electrode on which an enzyme is fixed, after the enzyme is fixed on the surface of the electrode with gaps inside, and fixing the hydrophobic electronic mediator on the surface of the electrode. As the organic solvent which carries out phase separation with water, methyl-isobutyl ketone or the like is used. By containing a water repellent material in this solution, the water repellent material is formed on the surface of the electrode to make it water repellent. Carbon powder or the like is used as the water repellent material. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a fuel cell, a manufacturing method thereof, an enzyme-immobilized electrode, a manufacturing method thereof, and an electronic device. More specifically, the present invention is suitable for application to a fuel cell in which an enzyme and an electron mediator are immobilized on a positive electrode (cathode) and a method for producing the same. Furthermore, the present invention relates to an enzyme-immobilized electrode suitable for use in the fuel cell, a method for producing the same, and an electronic device using the fuel cell.

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

  Generally, an electron mediator is immobilized on the positive electrode of this biofuel cell together with an enzyme that reduces oxygen. As an electron mediator, potassium hexacyanoferrate (potassium ferricyanide), potassium octacyanotungstate, and the like have been conventionally used. It is used.

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 2007-87627 A

However, according to the knowledge obtained as a result of various experiments conducted by the present inventors, electron mediators such as potassium hexacyanoferrate and potassium octacyanotungstate immobilized on the positive electrode together with the enzyme are used in biofuel cells. Elution from the positive electrode is easy. For this reason, there existed a problem that the output of a biofuel cell, lifetime, efficiency, etc. will fall.
Accordingly, the problem to be solved by the present invention is to provide a fuel cell and a method for manufacturing the same, in which the retention rate of the current is improved by preventing the elution of the electron mediator immobilized together with the enzyme on the positive electrode. .
Another problem to be solved by the present invention is to provide an enzyme-immobilized electrode suitable for application to a positive electrode of a fuel cell in which an electron mediator is immobilized together with an enzyme, and a method for producing the same.
Still another problem to be solved by the present invention is to provide an electronic device using the above excellent fuel cell.

  In order to solve the above-described problems of the prior art, the present inventors considered using a hydrophobic electron mediator such as ferrocene as an electron mediator that is immobilized on the positive electrode together with the enzyme. However, since the highly hydrophobic electron mediator is insoluble in water, a mixed solution with an enzyme or the like cannot be prepared and applied to an electrode made of carbon or the like. On the other hand, ferrocene dissolves in organic solvents mixed with water such as acetone and ethanol. However, when a solution in which ferrocene is dissolved in these organic solvents is applied to an electrode on which the enzyme is immobilized, these organic solvents lose the enzyme. I will make it live. Therefore, as a result of intensive studies, the inventors fixed the hydrophobic electron mediator to the electrode by, for example, applying a solution in which the hydrophobic electron mediator is dissolved in an organic solvent that hardly dissolves the enzyme to the surface of the electrode. It came to the conclusion that it is effective. According to this method, the hydrophobic electron mediator can be immobilized on the electrode surface without deactivating the enzyme previously immobilized on the electrode surface.

On the other hand, the electrode material for the positive electrode of the above-described biofuel cell is generally a material having voids inside such as porous carbon for supplying oxygen. In the positive electrode using such a material having voids inside, water that moves from the fuel solution supplied to the negative electrode through the electrolyte to the positive electrode side, and H ⁺ supplied from the negative electrode through the electrolyte are externally supplied. Water generated by reacting with supplied oxygen and electrons sent from the negative electrode through an external circuit, or water generated by osmotic pressure from an electrolyte containing a buffer solution fills the void inside the positive electrode. As a result, the inside of the positive electrode may be submerged. When the inside of the positive electrode is submerged in this way, it becomes difficult to supply oxygen to the positive electrode, and the current obtained from the biofuel cell is greatly reduced. For this reason, it is important to control the amount of water contained in the positive electrode, but no effective method has been proposed so far. As a result of intensive studies to solve this problem, the present inventors have found that the positive electrode is composed of an electrode on which an enzyme is immobilized, and this electrode has voids inside porous carbon or the like. The inventors have found that the water content contained in the positive electrode can be maintained in the optimum range by making water repellent at least a part of the surface of the electrode including the inner surface of the void. In this case, the surface of the electrode can be made water-repellent while maintaining the activity of the enzyme. As a result of further investigation, it is concluded that this water repellent technology is also effective when the negative electrode is composed of an electrode on which an enzyme is immobilized, and this electrode has a void inside porous carbon or the like. It came to.

That is, in order to solve the above problems, the present invention provides:
A step of immobilizing an enzyme on the surface of an electrode having a void inside;
The hydrophobic electron mediator is immobilized on the surface of the electrode by bringing the surface of the electrode on which the enzyme is immobilized into contact with a solution in which the hydrophobic electron mediator is dissolved in an organic solvent that is phase-separated from water. The manufacturing method of the fuel cell which has a process.
Here, as the organic solvent to be phase-separated from water, typically, 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 is used. The electrode on which the enzyme and the hydrophobic electron mediator are immobilized is typically a positive electrode, but may be a negative electrode.

In addition, this invention
Having a structure in which the positive electrode and the negative electrode are opposed to each other via a proton conductor;
The positive electrode is a fuel cell in which an enzyme and a hydrophobic electron mediator are immobilized on the surface of an electrode having voids inside.

In addition, this invention
Using one or more fuel cells,
At least one of the fuel cells is
Having a structure in which the positive electrode and the negative electrode are opposed to each other via a proton conductor;
The positive electrode is an electronic device in which an enzyme and a hydrophobic electron mediator are immobilized on the surface of an electrode having a void inside.

  In each of the above inventions, in order to make the surface of the electrode water repellent, for example, a water repellent material is formed on the surface of the electrode. Specifically, for example, a solution in which a hydrophobic electron mediator is dissolved in a water-repellent material dispersed in an organic solvent that is phase-separated from water is applied to the surface of the electrode, or the electrode is soaked. Or dipping. As the water repellent material, a particulate water repellent material is preferably used, and the particulate water repellent material is dispersed in an organic solvent that is phase-separated from water. The ratio of the water repellent material in this solution may be extremely small.

  As a material having a void inside, which is used for a positive electrode or a negative electrode, a porous material is generally used. For example, many carbon-based materials such as porous carbon, carbon pellets, carbon felt, and carbon paper are used. Although used, other materials may be used.

  In this fuel cell, for example, the fuel solution is configured to contact a part of the positive electrode, or the fuel solution is configured to contact the outer peripheral surface of the negative electrode and the side surface of the positive electrode. It is not something. In the latter case, for example, a sheet made of a material that allows air to permeate but does not permeate the fuel solution is provided on the surface of the positive electrode opposite to the proton conductor.

The enzyme immobilized on the positive electrode and the negative electrode may be various, and is selected as necessary. When an enzyme is immobilized on the negative electrode, an electron mediator is preferably immobilized in addition to the enzyme.
The enzyme immobilized on the positive electrode typically includes an oxygen reductase. As this oxygen reductase, multi-copper oxidase can be used. However, multi-copper oxidase is a general term for enzymes having four copper ions at the active center and capable of reducing oxygen by four electrons, such as bilirubin oxidase, laccase, and ascorbate oxidase. In this case, examples of the hydrophobic electron mediator immobilized on the positive electrode together with the enzyme include the following.

Ferrocene FeCp ₂ (Cp: cyclopentadienyl ring; logPow = 3.28) and n, n′-substituted-ferrocene Fe (nX—Cp) (n′Y—Cp) (n, n ′ = 1 to 5) , X and Y are substituent groups), an electron donor for multi-copper oxidase, and anthraquinone- or naphthoquinone-based compounds that dissolve in an organic solvent that is phase-separated from water. Compounds that can become the body

  If necessary, a hydrophilic electron mediator such as potassium hexacyanoferrate or potassium octacyanotungstate may be immobilized on the positive electrode, if necessary, together with the above hydrophobic electron mediator.

For example, when a monosaccharide such as glucose is used as the fuel, the enzyme immobilized on the negative electrode includes an oxidase that promotes and decomposes the monosaccharide and is usually reduced by the oxidase. A coenzyme oxidase that returns the coenzyme to an oxidant. By the action of the coenzyme oxidase, electrons are generated when the coenzyme returns to the oxidized form, and the electrons are transferred from the coenzyme oxidase to the electrode via the electron mediator. For example, NAD ⁺ -dependent glucose dehydrogenase (GDH) is used as the oxidase, nicotinamide adenine dinucleotide (NAD ⁺ ) is used as the coenzyme, and diaphorase is used as the coenzyme oxidase.

  When using polysaccharides (a broadly defined polysaccharide, which refers to all carbohydrates that produce two or more monosaccharides by hydrolysis, including oligosaccharides such as disaccharides, trisaccharides, and tetrasaccharides) as fuel, Preferably, in addition to the above-mentioned oxidase, coenzyme oxidase, coenzyme and electron mediator, a degradation enzyme that promotes degradation such as hydrolysis of polysaccharides and produces monosaccharides such as glucose is also immobilized. . Specific examples of the polysaccharide include starch, amylose, amylopectin, glycogen, cellulose, maltose, sucrose, and lactose. These are a combination of two or more monosaccharides, and any polysaccharide contains glucose as a monosaccharide of the binding unit. Note that amylose and amylopectin are components contained in starch, and starch is a mixture of amylose and amylopectin. When glucoamylase is used as a polysaccharide degrading enzyme and glucose dehydrogenase is used as an oxidase degrading monosaccharides, polysaccharides that can be decomposed to glucose by glucoamylase, such as starch, amylose, amylopectin, glycogen As long as it contains any one of maltose, it is possible to generate electricity using this as fuel. Glucoamylase is a degrading enzyme that hydrolyzes α-glucan such as starch to produce glucose, and glucose dehydrogenase is an oxidase that oxidizes β-D-glucose to D-glucono-δ-lactone. Preferably, the degradation enzyme that decomposes the polysaccharide is also immobilized on the negative electrode, and the polysaccharide that will eventually become the fuel is also immobilized on the negative electrode.

  Further, when starch is used as fuel, it is possible to use a gelatinized fuel obtained by gelatinizing starch. In this case, it is preferable to use a method in which gelatinized starch is brought into contact with a negative electrode on which an enzyme or the like is immobilized, or is immobilized on the negative electrode together with the enzyme or the like. When such an electrode is used, the starch concentration on the negative electrode surface can be maintained at a higher level than when starch dissolved in the solution is used, the enzymatic degradation reaction becomes faster, and the output is improved. The fuel handling is easier than in the case of a solution, the fuel supply system can be simplified, and the fuel cell does not need to be used upside down, which is very advantageous when used for mobile devices, for example. .

When methanol is used as a fuel, alcohol dehydrogenase (ADH) that acts on methanol as a catalyst to oxidize to formaldehyde, formaldehyde dehydrogenase (FalDH) that acts on formaldehyde to oxidize to formic acid, and acts on formic acid to produce CO _2. It is decomposed to CO ₂ through a three-stage oxidation process with formate dehydrogenase (FateDH) that oxidizes to CO2. That is, three NADHs are generated per molecule of methanol, and six electrons are generated.

When ethanol is used as a fuel, a two-stage oxidation process is performed using alcohol dehydrogenase (ADH) that acts on ethanol as a catalyst to oxidize to acetaldehyde and aldehyde dehydrogenase (AlDH) that acts on acetaldehyde and oxidizes to acetic acid. Then it is decomposed to acetic acid. That is, a total of 4 electrons are generated by two-stage oxidation reaction per molecule of ethanol.
Incidentally, ethanol may take the method of decomposing to CO ₂ as well as methanol. In this case, acetaldehyde dehydrogenase (AalDH) is allowed to act on acetaldehyde to form acetyl CoA, which is then passed to the TCA circuit. Further electrons are generated in the TCA circuit.

  As the electron mediator that is immobilized on the negative electrode together with the enzyme, basically any kind of electron mediator may be used. However, a compound having a quinone skeleton, particularly a compound having a naphthoquinone skeleton is preferably used. As the compound having a naphthoquinone skeleton, various naphthoquinone derivatives can be used. Specifically, for example, 2-amino-1,4-naphthoquinone (ANQ), 2-amino-3-methyl-1 2, 4-naphthoquinone (AMNQ), 2-methyl-1,4-naphthoquinone (VK3), 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ), and the like are used. As a compound having a quinone skeleton, for example, anthraquinone or a derivative thereof can be used in addition to a compound having a naphthoquinone skeleton. In addition to the compound having a quinone skeleton, the electron mediator may contain one or two or more other compounds that function as an electron mediator, if necessary. As a solvent used when a compound having a quinone skeleton, particularly a compound having a naphthoquinone skeleton, is immobilized on the negative electrode, acetone is preferably used. Thus, by using acetone as a solvent, the solubility of the compound having a quinone skeleton can be increased, and the compound having a quinone skeleton can be efficiently immobilized on the negative electrode. If necessary, the solvent may contain one or two or more other solvents other than acetone.

In one example, the negative electrode is 2-methyl-1,4-naphthoquinone (VK3) as an electron mediator, reduced nicotinamide adenine dinucleotide (NADH) as a coenzyme, glucose dehydrogenase and coenzyme oxidase as oxidases The diaphorase as is immobilized, and preferably 1.0 (mol): 0.33-1.0 (mol): (1.8-3.6) × 10 ⁶ (U): (0. It is fixed at a ratio of 85 to 1.7) × 10 ⁷ (U). However, U (unit) is an index indicating enzyme activity, and indicates the degree to which 1 μmol of substrate reacts per minute at a certain temperature and pH.
As an immobilizing material for immobilizing an enzyme, a coenzyme, an electron mediator or the like on the negative electrode or the positive electrode, various conventionally known materials can be used and are selected as necessary.

Various proton conductors can be used as long as they do not have electron conductivity and only conduct protons, and are selected as necessary. Specifically, examples of the proton conductor include the following.
・ Cellophane ・ Perfluorocarbon sulfonic acid (PFS) resin film ・ Copolymer film of trifluorostyrene derivative ・ Polybenzimidazole film impregnated with phosphoric acid ・ Aromatic polyether ketone sulfonic acid film ・ PSSA-PVA (polystyrene sulfone) Acid polyvinyl alcohol copolymer)
・ PSSA-EVOH (polystyrene sulfonate ethylene vinyl alcohol copolymer)
・ Ion exchange resins with fluorine-containing carbon sulfonic acid groups (Nafion (trade name, DuPont, USA), etc.)

In addition, this invention
A step of immobilizing an enzyme on the surface of an electrode having a void inside;
The hydrophobic electron mediator is immobilized on the surface of the electrode by bringing the surface of the electrode on which the enzyme is immobilized into contact with a solution in which the hydrophobic electron mediator is dissolved in an organic solvent that is phase-separated from water. And an enzyme-immobilized electrode manufacturing method.

In addition, this invention
An enzyme-immobilized electrode comprising an enzyme and a hydrophobic electron mediator immobilized on the surface of an electrode having a void inside.

  The method for producing an enzyme-immobilized electrode and the enzyme-immobilized electrode according to the above invention have a structure in which a positive electrode and a negative electrode face each other via a proton conductor, and the positive electrode has an enzyme and It is suitable for application to the positive electrode of a fuel cell comprising an electron mediator immobilized. In the above-described method for producing an enzyme-immobilized electrode and the invention for an enzyme-immobilized electrode, the matters other than those described above relate to the method for producing a fuel cell and the invention for a fuel cell according to the above-described invention, as long as they do not contradict their properties. The explanation is valid.

  In the present invention configured as described above, an enzyme is immobilized on the surface of an electrode having a void inside, and an organic solvent that phase-separates a hydrophobic electron mediator from water on the surface of the electrode on which the enzyme is immobilized. The solution dissolved in is brought into contact. For this reason, a hydrophobic electron mediator can be immobilized on the surface of the electrode without inactivating the enzyme. Since this hydrophobic electron mediator is not soluble in water, it hardly elutes in a buffer solution containing water.

  According to the present invention, the elution of the electron mediator immobilized on the positive electrode together with the enzyme can be prevented, whereby the current maintenance rate can be improved, and a fuel cell having excellent performance can be obtained. By using such an excellent fuel cell, a high-performance electronic device can be realized.

1 is a schematic diagram showing a biofuel cell according to a first embodiment of the present invention. FIG. 2 is a schematic diagram schematically showing a configuration of a negative electrode of the biofuel cell according to the first embodiment of the present invention, an example of an enzyme group immobilized on the negative electrode, and an electron transfer reaction by the enzyme group. . It is a basic diagram which shows an example of the whole structure of the biofuel cell by 1st Embodiment of this invention. It is a basic diagram which shows the other example of the whole structure of the biofuel cell by 1st Embodiment of this invention. It is a basic diagram which shows the measurement system used for the measurement of the chronoamperometry performed for evaluation of the biofuel cell by 1st Embodiment of this invention. It is a basic diagram which shows the result of the chronoamperometry performed for evaluation of the biofuel cell by 1st Embodiment of this invention. It is a basic diagram which shows the result of the linear sweep voltammetry performed for evaluation of the biofuel cell by 1st Embodiment of this invention. It is a basic diagram which shows the result of the chronoamperometry performed for evaluation of the biofuel cell by 1st Embodiment of this invention. It is a basic diagram which shows the result of the chronoamperometry performed for evaluation of the biofuel cell by the 2nd Embodiment of this invention.

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 2. FIG. Second embodiment

<1. First Embodiment>
FIG. 1 schematically shows a biofuel cell according to a first embodiment. In this biofuel cell, glucose is used as the fuel. FIG. 2 schematically shows details of the configuration of the negative electrode of the biofuel cell, an example of an enzyme group immobilized on the negative electrode, and an electron transfer reaction by the enzyme group.

As shown in FIG. 1, this biofuel cell has a structure in which a negative electrode 1 and a positive electrode 2 are opposed to each other through an electrolyte layer 3 that does not have electronic conductivity and conducts only protons. The negative electrode 1 decomposes glucose supplied as fuel with an enzyme to extract electrons and generate protons (H ⁺ ). The positive electrode 2 generates water by protons transported from the negative electrode 1 through the electrolyte layer 3, electrons transmitted from the negative electrode 1 through an external circuit, and oxygen in the air, for example.

The negative electrode 1 is formed on a material having voids inside, for example, an electrode 11 made of porous carbon or the like (see FIG. 2), in addition to an oxidase involved in glucose decomposition, a coenzyme, a coenzyme oxidase, an electron mediator, etc. Is fixed by a fixing material (not shown). Here, the oxidase involved in the degradation of glucose is glucose dehydrogenase (GDH). A coenzyme is a product in which a reductant is generated in association with an oxidation reaction in a glucose decomposition process, such as NAD ⁺ and NADP ⁺ . The coenzyme oxidase oxidizes a reduced form of coenzyme (for example, NADH, NADPH, etc.) and is diaphorase (DI). The electron mediator is for receiving electrons generated from the diaphorase accompanying the oxidation of the coenzyme and passing them to the electrode 11.

By allowing glucose dehydrogenase (GDH) as an enzyme involved in the degradation of glucose, 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. D-gluconate is phosphorylated by hydrolyzing adenosine triphosphate (ATP) into adenosine diphosphate (ADP) and phosphate in the presence of gluconokinase to give 6-phospho-D-gluconate. Become. 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.
The electrons generated in the above process are transferred from diaphorase to the electrode 11 through the electron mediator, and H ⁺ is transported to the positive electrode 2 through the electrolyte layer 3.

  The electron mediator exchanges electrons with the electrode 11, and the output voltage of the biofuel cell depends on the redox potential of the electron mediator. That is, in order to obtain a higher output voltage, it is preferable to select an electron mediator having a more negative potential on the negative electrode 1 side. However, the reaction affinity of the electron mediator with respect to the enzyme, the rate of electron exchange with the electrode 11, the structural stability with respect to inhibitors (light, oxygen, etc.) must also be considered. From such a viewpoint, 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ), vitamin K3 (VK3), and the like are preferable as the electron mediator immobilized on the negative electrode 1. In addition, for example, a compound having a quinone skeleton, a metal complex such as osmium (Os), ruthenium (Ru), iron (Fe), and cobalt (Co) can also be used as an electron mediator. Furthermore, a viologen compound such as benzyl viologen, a compound having a nicotinamide structure, a compound having a riboflavin structure, a compound having a nucleotide-phosphate structure, and the like can also be used as an electron mediator.

The electrolyte layer 3 is a proton conductor that transports H ⁺ generated in the negative electrode 1 to the positive electrode 2, and is made of a material that does not have electronic conductivity and can transport H ⁺ . As the electrolyte layer 3, for example, one appropriately selected from those already mentioned can be used. In this case, the electrolyte layer 3 includes a buffer substance (buffer solution). When the electrolyte layer 3 containing a buffer substance (buffer solution) is used in this way, even when the increase or decrease in protons occurs in the electrode or in the immobilized membrane of the enzyme due to an enzyme reaction via protons during high output operation. It is desirable that a sufficient buffering action can be obtained. By being able to obtain a sufficient buffering action in this way, it is possible to suppress the deviation in pH from the optimum pH to a sufficiently small level and to fully demonstrate the ability of the enzyme. Can do. For this purpose, it is effective that the concentration of the buffer substance contained in the electrolyte is 0.2 M or more and 2.5 M or less, preferably 0.2 M or more and 2 M or less, more preferably 0.4 M or more and 2 M or less. More preferably, it is 0.8M or more and 1.2M or less. Buffer substances are generally as long as _a pK _a of 5 to 9 may be used What. A specific example is as follows.

· Dihydrogen phosphate ion (H ₂ PO ₄ ^-)
2-amino-2-hydroxymethyl-1,3-propanediol (abbreviation Tris)
2- (N-morpholino) ethanesulfonic acid (MES)
・ Cacodylic acid ・ Carbonic acid (H ₂ CO ₃ )
・ Hydrogen citrate ion ・ N- (2-acetamido) iminodiacetic acid (ADA)
Piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES)
N- (2-acetamido) -2-aminoethanesulfonic acid (ACES)
3- (N-morpholino) propanesulfonic acid (MOPS)
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)
N-2-hydroxyethylpiperazine-N′-3-propanesulfonic acid (HEPPS)
N- [Tris (hydroxymethyl) methyl] glycine (abbreviation tricine)
・ Glycylglycine ・ N, N-bis (2-hydroxyethyl) glycine (abbreviation: bicine)
Examples of the substance that generates dihydrogen phosphate ions (H ₂ PO ₄ ⁻ ) include sodium dihydrogen phosphate (NaH ₂ PO ₄ ) and potassium dihydrogen phosphate (KH ₂ PO ₄ ).

As the buffer substance, a compound containing an imidazole ring is also preferable. Specific examples of the compound containing the imidazole ring are as follows.
-Imidazole, triazole, pyridine derivatives, bipyridine derivatives, imidazole derivatives Specific examples of imidazole derivatives are as follows.
・ Histidine, 1-methylimidazole, 2-methylimidazole, 4-methylimidazole, 2-ethylimidazole, ethyl imidazole-2-carboxylate, imidazole-2-carboxaldehyde, imidazole-4-carboxylic acid, imidazole-4,5 -Dicarboxylic acid, imidazol-1-yl-acetic acid, 2-acetylbenzimidazole, 1-acetylimidazole, N-acetylimidazole, 2-aminobenzimidazole, N- (3-aminopropyl) imidazole, 5-amino-2- (Trifluoromethyl) benzimidazole, 4-azabenzimidazole, 4-aza-2-mercaptobenzimidazole, benzimidazole, 1-benzylimidazole, 1-butylimidazole Imidazo as the above buffer substance The concentration of the compound having the Le ring is selected as necessary, preferably in a 3M following concentrations above 0.2 M.
In addition to those listed above, 2-aminoethanol, triethanolamine, TES (N-Tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid), BES (N, N-Bis (2-hydroxyethyl)- 2-aminoethanesulfonic acid) may also be used.

In addition to the above buffer substance, for example, at least one acid selected from the group consisting of hydrochloric acid (HCl), acetic acid (CH ₃ COOH), phosphoric acid (H ₃ PO ₄ ) and sulfuric acid (H ₂ SO ₄ ) It may be added as a neutralizing agent. By doing so, the activity of the enzyme can be maintained higher. The pH of the electrolyte containing the buffer substance is preferably around 7, but may generally be any of 1-14.

  The ionic strength (I.S.) of the buffer substance has an adverse effect on the enzyme activity if it is too large or too small, but considering the electrochemical responsiveness, it is a moderate ionic strength, for example, about 0.3. It is preferable. However, pH and ionic strength have optimum values for each enzyme used, and are not limited to the values described above.

  The enzyme, coenzyme, and electron mediator are preferably immobilized on the electrode 11 using an immobilizing material in order to efficiently capture an enzyme reaction phenomenon occurring in the vicinity of the electrode as an electric signal. Furthermore, the enzyme reaction system of the negative electrode 1 can be stabilized by immobilizing the enzyme and the coenzyme for decomposing the fuel on the electrode 11. Specific examples of such an immobilizing material include poly-L-lysine (PLL), glutaraldehyde (GA), a combination of poly-L-lysine and glutaraldehyde, and a polyion complex. be able to. The polyion complex is formed using, for example, a polycation such as poly-L-lysine or a salt thereof and a polyanion such as polyacrylic acid (for example, sodium polyacrylate (PAAcNa)) or a salt thereof. Is.

In FIG. 2, for example, glucose dehydrogenase (GDH) is an enzyme involved in the degradation of glucose, NAD ⁺ is a coenzyme that produces a reductant in the oxidation reaction in the glucose degradation process, and a reductant of coenzyme. The case where the coenzyme oxidase that oxidizes a certain NADH is diaphorase (DI), and the electron mediator that receives electrons from the coenzyme oxidase accompanying the oxidation of the coenzyme and passes it to the electrode 11 is ACNQ is shown.

  In the positive electrode 2, an oxygen reductase and an electron mediator that transfers electrons to and from this electrode are immobilized on an electrode made of a material having voids inside, for example, porous carbon. As the oxygen reductase, for example, bilirubin oxidase (BOD), laccase, ascorbate oxidase and the like can be used. As the electron mediator, at least one kind of hydrophobic electron mediator is used, and if necessary, further potassium hexacyanoferrate (more specifically, hexacyanoferrate ion generated by ionization of potassium hexacyanoferrate), etc. At least one hydrophilic electron mediator is used.

In the positive electrode 2, in the presence of oxygen reductase, oxygen in the air is reduced by H ⁺ from the electrolyte layer 3 and electrons from the negative electrode 1 to generate water.
When glucose is supplied to the negative electrode 1 side during operation (in use) of the fuel cell configured as described above, the glucose is decomposed by a decomposing enzyme including an oxidase. Since oxidase is involved in the monosaccharide decomposition process, electrons and H ⁺ can be generated on the negative electrode 1 side, and a current can be generated between the negative electrode 1 and the positive electrode 2.

  In this biofuel cell, at least a part, preferably most of the surface of the electrode used for the positive electrode 2 having voids inside is made water-repellent. Here, the surface of the electrode includes the entire outer surface of the electrode and the inner surface of the void inside the electrode. Specifically, for example, a water repellent material is formed by forming a water repellent material on at least a part of the surface of the electrode. In order to form this water repellent material on the inner surface of the gap inside the electrode, the water repellent material is in the form of fine particles (powder) sufficiently smaller than the size of the gap, and most of the space inside the gap is the water repellent material. It is necessary not to be occupied by the 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 as an additive for a lithium ion battery, ketjen black, etc. can be used. Is preferably used. A water-repellent polymer can also be used as the water-repellent material. As such a water-repellent polymer, for example, various fluorine-based polymers can be used in addition to polyvinyl butyral. Specific examples of the fluoropolymer include tetrafluoroethylene-hexafluoropropylene copolymer (FEP), polytetrafluoroethylene (PTFE), polyvinylidene fluoride, tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer, polyfluoride. Vinyl, perfluoroalkoxy resin, tetrafluoroethylene-ethylene copolymer, polychlorotrifluoroethylene, polyether sulfone and the like are not limited thereto.

  In order to form the water-repellent material on at least a part of the surface of the electrode as described above, for example, the water-repellent material is dispersed in an organic solvent that is phase-separated from water, and at least one hydrophobic electron mediator is used. A solution in which is dissolved is applied to the surface of the electrode, and the electrode is impregnated through an internal gap, and then the organic solvent is removed.

  As the organic solvent for phase separation from the above water, methyl isobutyl ketone, heptane, hexane, toluene, isooctane, diethyl ether and the like are preferably used. As the organic solvent, various organic solvents such as aliphatic, alicyclic or aromatic hydrocarbon solvents, ether solvents, and halides thereof can be generally used. More specifically, examples of the organic solvent include butanes, pentanes, hexanes, heptanes, octanes, nonanes, decanes, dodecanes, cyclohexane, cyclopentane, benzene, xylenes, butanol, Benzene system substituted with one or more of pentanol, methyl ether, ethyl ether, isopropyl ether, methylene chloride, methyl chloroform, carbon tetrachloride, dichlorodifluoromethane, perchloroethylene, chlorine atom, bromine atom and / or iodine atom Although a solvent, a toluene type solvent, etc. are mentioned, it is not limited to these, Moreover, these may be used independently and may use 2 or more types together.

  Examples of the organic solvent for phase separation from water include methylene chloride, 1,2-dichloroethane, chloroform, monochlorobenzene, 1,2-dichlorobenzene, 2-chlorotoluene, 3-chlorotoluene, 4-chlorotoluene, 2-chloro- m-xylene, 2-chloro-p-xylene, 4-chloro-o-xylene, 2,3-dichlorotoluene, 2,4-dichlorotoluene, 2,5-dichlorotoluene, 2,6-dichlorotoluene, 3, Other examples include halogenated hydrocarbon solvents such as 4-dichlorotoluene and monofluorobenzene, and hydrocarbon solvents such as nitrobenzene and benzene. Examples of the organic solvent that is phase-separated from water include cyclohexane, normal hexane, cyclohexanone, 1-methoxyisopropanol acetate, ethyl acetate, butyl acetate, petroleum ether, and silicone oil.

  An ionic liquid can also be used as the organic solvent that is phase-separated from water. Examples of the ionic liquid include those containing at least one anion selected from the group consisting of a fluoroalkyl sulfate anion, a fluorocycloalkyl sulfate anion, and a fluorobenzyl sulfate anion as anions.

The solution in which the water repellent material is dispersed in an organic solvent that is phase-separated from water and the hydrophobic electron mediator is dissolved may contain a binder or the like, if necessary. Various binders can be used as the binder, but it is more preferable to use a binder having high water repellency such as polyvinyl butyral. Although the ratio of the binder in this solution is 0.01 to 10%, for example, it is not limited to this. 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.
When using the above solution, it is important not to inactivate the enzyme immobilized on the positive electrode 2, but the inactivation of the enzyme can be prevented by using those already mentioned as the organic solvent. .

Two examples of the overall configuration of this biofuel cell are shown in FIGS.
The biofuel cell shown in FIG. 3 has a structure in which the negative electrode 1 and the positive electrode 2 face each other with the electrolyte layer 3 interposed therebetween, and air passes through the surface of the positive electrode 2 on the side opposite to the electrolyte layer 3. A sheet 13 made of a material that does not transmit the solution 12 is attached. The fuel solution 12 (the container for containing the fuel solution 12 is not shown) on the entire outer surface (upper surface and side surface) of the negative electrode 1 and on the negative electrode 1, the side surface of the positive electrode 2, and the electrolyte layer 3 that protrudes outside the positive electrode 2. Is touching. For example, a nonwoven fabric is used as the electrolyte layer 3, but is not limited thereto. Moreover, as the sheet | seat 13, although a PTFE (polytetrafluoroethylene) membrane is used, for example, it is not limited to this. In this biofuel cell, since the side surface of the positive electrode 2 and the fuel solution 12 are in contact with each other, water generated in the positive electrode 2 as the cell reaction proceeds passes through the side surface of the positive electrode 2 and the fuel solution. 12 is returned. For this reason, the concentration of the fuel solution 12 can be kept substantially constant.

  The biofuel cell shown in FIG. 4 has a structure in which the negative electrode 1 and the positive electrode 2 face each other with the electrolyte layer 3 interposed therebetween. Then, the fuel solution 12 (illustration of the container for the fuel solution 12 is omitted) is in contact with the entire outer surface (upper surface and side surfaces) of the negative electrode 1 and the electrolyte layer 3 in a portion protruding from the negative electrode 1 and the positive electrode 2. Yes. For example, cellophane is used as the electrolyte layer 3, but is not limited thereto.

The result of evaluating the positive electrode 2 having a hydrophobic electron mediator immobilized as an electron mediator on the surface of the electrode will be described.
As the positive electrode 2, an enzyme / electron mediator fixed electrode prepared as follows was used. First, commercially available carbon felt (BORAY made by TORAY) was prepared as porous carbon. After firing this carbon felt, two sheets were stacked and cut into 1 cm squares. Next, 80 μl of poly-L-lysine (2 wt% aqueous solution) and 80 μl of BOD solution (100 mg / ml, 0.4 M imidazole / sulfate buffer) were mixed, and then soaked in the carbon felt and dried. Thus, the BOD is fixed to the carbon felt. Next, a solution in which 200 mM of ferrocene as a hydrophobic electron mediator is dissolved in methyl isobutyl ketone as an organic solvent containing carbon powder, silicon resin, fluorine resin, or the like as a water repellent material is prepared. Next, the carbon felt in which BOD was fixed in this solution was dipped, and this solution was applied to the surface of the carbon felt. Thereafter, drying is performed to remove the organic solvent contained in the solution. Thus, a water repellent layer was formed on the surface of the carbon felt to make it water repellent, and ferrocene was uniformly dispersed in the carbon felt. The enzyme / electron mediator immobilized electrode thus obtained has a thickness of 0.35 mm and an area of 1 cm ² .

  In order to investigate the effect of the solution containing the above graphite powder or the like on the enzyme immobilized on the positive electrode 2, ie, BOD, methyl isobutyl ketone, which is an organic solvent contained in this solution, and BOD solution (5 mg / ml 50 mM phosphoric acid) Buffer) and the ABTS solution were mixed, and it was confirmed that phase separation into methyl isobutyl ketone and water occurred. In this case, it was confirmed that the activity of BOD was maintained. This is because BOD is not easily deactivated because it exists in the aqueous phase. Here, the solubility of methyl isobutyl ketone in water is 1.91 g / 100 mL.

  Further, when heptane, hexane, toluene, isooctane and diethyl ether were used as organic solvents, and these organic solvents, BOD solution and ABTS solution were mixed, it was confirmed that the phases were separated into these organic solvents and water. . Also in these cases, it was confirmed that the activity of BOD was maintained. Here, heptane, toluene and isooctane are insoluble in water, the solubility of hexane in water is 13 mg / L, and the solubility of diethyl ether in water is 6.9 g / 100 mL.

  On the other hand, when tetrahydrofuran (THF), acetone, ethanol, N, N-dimethylformamide (DMF) is used as the organic solvent and these organic solvent, BOD solution, and ABTS solution are mixed, these mixed solutions become cloudy. Was confirmed. This shows that BOD has denatured. That is, when these organic solvents are used, BOD is deactivated. Here, these tetrahydrofuran, acetone, ethanol and N, N-dimethylformamide are all miscible with water.

  Next, the water repellency of the carbon felt surface was confirmed using a solution containing 13 to 18% of natural graphite as carbon powder in methyl isobutyl ketone as described above. Specifically, a carbon felt whose surface is water-repellent with the above solution and a carbon felt whose surface is not water-repellent are prepared. Then, when these carbon felts are left at room temperature and when kept at a temperature of 25 ° C. and a humidity of 100% for 6 hours, the water content contained in these carbon felts is determined according to the Karl Fischer water content meter (Diamond Co., Ltd.). VA-100 type manufactured by Instrument). The results are shown below.

Carbon felt without water repellency (1) Room temperature 1st 632.5
Second time 718.9
Third time 645.1
Average 665.5
(2) Left at a temperature of 25 ° C. and a humidity of 100% for 6 hours 1st 18482.2
Second time 15434.4
3rd 12549.1
Average 15488.6

Water-repellent carbon felt (1) Room temperature 1st 1481.7
Second time 756.6
Third time 698.1
4th 1338.1
Average 1068.6
(2) Left at a temperature of 25 ° C. and a humidity of 100% for 6 hours 1st 4943.8
Second time 3516.8
Third time 7280.8
Average 5247.1

  From the above results, the amount of water contained in carbon felt whose surface is water-repellent with a solution containing graphite powder as a water-repellent material in methyl isobutyl ketone is compared to the amount of water contained in carbon felt whose surface is not water-repellent. It was found that the carbon felt having a water repellent surface with a water repellent material certainly has water repellency.

  The electrochemical characteristics of the positive electrode 2 comprising the enzyme / electron mediator immobilized electrode prepared as described above were measured. The measurement system used is shown in FIG. As shown in FIG. 5, the positive electrode 2 was used as a working electrode, this was placed on a gas-permeable PTFE membrane 14 and pressed, and the measurement was performed with the buffer solution 15 in contact with the positive electrode 2. The counter electrode 16 and the reference electrode 17 were immersed in the buffer solution 15, and an electrochemical measurement device (not shown) was connected to the positive electrode 2, the counter electrode 16, and the reference electrode 17 as working electrodes. Pt wire was used as the counter electrode 16 and Ag | AgCl was used as the reference electrode 17. The measurement was performed in the atmosphere, and the measurement temperature was 25 ° C. As buffer solution 15, 2.0 M imidazole / sulfate buffer solution (pH 7.0) was used.

  Chronoamperometry was performed for 900 seconds using the measurement system shown in FIG. The applied voltage in chronoamperometry was 0.25 V vs. Ag | AgCl. The measurement result is shown in the curve (1) in FIG. Moreover, the result of having performed the same measurement under argon is shown in the curve (2) of FIG. Furthermore, linear sweep voltammetry was also performed using the measurement system shown in FIG. In linear sweep voltammetry, a potential region of 0.6 to −0.30 V vs. Ag | AgCl was scanned from 0.65 V to the negative side at a rate of 1 mV / s. The result is shown in FIG.

As shown by curve (1) in FIG. 6, a current value of about 3 mA / cm ² is stably observed over 900 seconds in the atmosphere. On the other hand, as shown by the curve (2) in FIG. 6, only a current value of about 0.5 mA / cm ² is observed under argon. From this, it can be seen that the current value observed in the atmosphere is a catalyst current by the BOD fixed to the positive electrode 2.

  FIG. 8 shows a case where the electron mediator immobilized on the positive electrode 2 is potassium hexacyanoferrate (abbreviated as FeCN) and a hydrophobic ferrocene (abbreviated as Fc) using the measurement system shown in FIG. The result of performing amperometry for 900 seconds is shown. Conventionally, potassium hexacyanoferrate used as an electron mediator has a very high solubility in water. Accordingly, potassium hexacyanoferrate has an advantage that it can be easily applied as a high-concentration aqueous solution at the time of immobilization, but is easily eluted from the positive electrode 2 into the buffer solution during the electrochemical measurement. . For this reason, as shown by the curve (1) in FIG. 8, a high current value is obtained at the beginning of the measurement, but the maintenance ratio is low. On the other hand, since ferrocene is insoluble in water, it hardly elutes from the positive electrode 2 into the buffer during the measurement. Therefore, as shown by the curve (2) in FIG. 8, although the current value itself is lower than that when potassium hexacyanoferrate is used, the maintenance ratio is high and a stable catalyst current is obtained.

  As described above, according to the first embodiment, the surface of the electrode of the positive electrode 2 is contacted with a solution in which a water repellent material is dispersed in an organic solvent that is phase-separated from water and a hydrophobic electron mediator is dissolved. Then, the organic solvent is dried. Thereby, a hydrophobic electron mediator can be immobilized on this surface while maintaining the activity of the immobilized enzyme. For this reason, since it is possible to prevent the elution of the electron mediator from the positive electrode 2 during use of the biofuel cell, it is possible to prevent a decrease in output characteristics, lifetime, efficiency, etc., and to improve the current maintenance rate. Can be planned. In addition, since the water repellent material is formed on the surface of the positive electrode 2 to make it water repellent, the amount of water contained in the positive electrode 2 can be maintained in the optimum range, thereby obtaining a high catalyst current, and consequently A high current value can be continuously obtained in the biofuel cell.

  In addition, when the electrolyte layer 3 contains a buffer substance such as a compound containing an imidazole ring at an appropriate concentration, a sufficient buffer capacity can be obtained. For this reason, at the time of high output operation of the biofuel cell, even if the increase or decrease of protons occurs in the proton electrode or in the enzyme immobilization membrane due to the proton-mediated enzyme reaction, sufficient buffer capacity can be obtained, Deviation from the optimum pH of the electrolyte surrounding the enzyme can be suppressed sufficiently small. For this reason, the ability inherent to the enzyme can be sufficiently exerted, and the electrode reaction by the enzyme, coenzyme, electron mediator and the like can be performed efficiently and constantly. As a result, a high-performance biofuel cell capable of high output operation can be realized.

  This biofuel cell can be used for anything requiring electric power, and can be of any size. Specifically, this fuel cell is used for, for example, an electronic device, a moving body (automobile, motorcycle, aircraft, rocket, spacecraft, etc.), power unit, construction machine, machine tool, power generation system, cogeneration system, etc. Can do. In this case, the output, size, shape, type of fuel, etc. of the fuel cell are determined depending on the application. The electronic device may basically be anything, and includes both a portable type and a stationary type. Specific examples include, for example, a mobile phone, a mobile device, a robot, a personal computer (including both a desktop type and a notebook type), a game device, a camera-integrated VTR (video tape recorder), an in-vehicle device, a home appliance, Industrial products. The mobile device is a personal digital assistant (PDA) or the like.

<2. Second Embodiment>
In the first embodiment, only the hydrophobic electron mediator is immobilized on the electrode of the positive electrode 2, whereas in the second embodiment, the hydrophobic electron mediator and the electrode of the positive electrode 2 are conventionally used. Immobilize both the hydrophilic electron mediator used. Others are the same as in the first embodiment.

FIG. 9 shows the result of performing chronoamperometry for 1500 seconds using the measurement system shown in FIG. 5 in the case where both potassium hexacyanoferrate (FeCN) and ferrocene (Fc) are immobilized on the electrode of the positive electrode 2 ( Curve (3)) is shown. However, 40 μL of 200 mM potassium hexacyanoferrate was applied and immobilized, and 70 mL of 200 mM ferrocene (Fc) was applied and immobilized. For comparison, FIG. 9 shows a case where only potassium hexacyanoferrate is immobilized on the electrode of the positive electrode 2 (curve (1)) and a case where only ferrocene is immobilized (curve (2)). The result of having performed chronoamperometry for 1500 second using the measuring system is shown. However, potassium hexacyanoferrate was immobilized by applying 80 μL of 200 mM. Ferrocene was immobilized by applying 140 mL of 200 mM. Curves (4) to (6) in FIG. 9 are standard deviations (stdev) of the current values of the curves (1) to (3), respectively. As shown by the curve (3) in FIG. 9, the current value and the retention rate when both potassium hexacyanoferrate (FeCN) and ferrocene (Fc) are immobilized on the electrode of the positive electrode 2 are as follows. The current value in the case where only potassium acid is immobilized and the maintenance ratio thereof, and the current value in the case where only ferrocene is immobilized and the maintenance ratio thereof are shown in an almost intermediate value. Therefore, even when ferrocene is immobilized on the electrode of the positive electrode 2 in addition to potassium hexacyanoferrate, the current value and its value are compared with the case where only potassium hexacyanoferrate is immobilized on the electrode of the positive electrode 2 as in the prior art. It can be seen that the maintenance rate can be improved.
According to the second embodiment, advantages similar to those of the first embodiment can be obtained.

Although the embodiment of the present invention has been specifically described above, the present invention is not limited to the above-described embodiment, and various modifications based on the technical idea of the present invention are possible.
For example, the numerical values, structures, configurations, shapes, materials, and the like given in the above-described embodiments are merely examples, and different numerical values, structures, configurations, shapes, materials, and the like may be used as necessary.

  DESCRIPTION OF SYMBOLS 1 ... Negative electrode, 2 ... Positive electrode, 3 ... Electrolyte layer, 11 ... Electrode

Claims (19)

A step of immobilizing an enzyme on the surface of an electrode having a void inside;
The hydrophobic electron mediator is immobilized on the surface of the electrode by bringing the surface of the electrode on which the enzyme is immobilized into contact with a solution in which the hydrophobic electron mediator is dissolved in an organic solvent that is phase-separated from water. A method for producing a fuel cell comprising the steps of:   The method for producing a fuel cell according to claim 1, wherein the organic solvent has a solubility of the enzyme of 10 mg / ml or less.   The method for producing a fuel cell according to claim 2, wherein the solution further contains a water repellent material.   The method for producing a fuel cell according to claim 1, wherein the electrode is made of a porous material.   The method for producing a fuel cell according to claim 1, wherein the electrode on which the enzyme and the hydrophobic electron mediator are immobilized is a positive electrode.   The method for producing a fuel cell according to claim 1, wherein the enzyme contains an oxygen reductase.   The method for producing a fuel cell according to claim 6, wherein the oxygen reductase is bilirubin oxidase.   4. The method for producing a fuel cell according to claim 3, wherein the water repellent material is carbon powder, silicon resin or fluorine resin, and the organic solvent is methyl isobutyl ketone, heptane, hexane, toluene, isooctane or diethyl ether. Having a structure in which the positive electrode and the negative electrode are opposed to each other via a proton conductor;
A fuel cell comprising the positive electrode, wherein an enzyme and a hydrophobic electron mediator are immobilized on the surface of an electrode having a void inside.   The fuel cell according to claim 9, wherein a water repellent material is further formed on a surface of the electrode.   The fuel cell according to claim 9, wherein the electrode is made of a porous material.   The fuel cell according to claim 9, wherein the enzyme comprises an oxygen reductase.   The fuel cell according to claim 12, wherein the oxygen reductase is bilirubin oxidase.   10. The fuel cell according to claim 9, wherein the water repellent material is carbon powder, silicon resin or fluorine resin, and the organic solvent is methyl isobutyl ketone, heptane, hexane, toluene, isooctane or diethyl ether. Using one or more fuel cells,
At least one of the fuel cells is
Having a structure in which the positive electrode and the negative electrode are opposed to each other via a proton conductor;
An electronic device in which the positive electrode is formed by immobilizing an enzyme and a hydrophobic electron mediator on the surface of an electrode having voids inside. A step of immobilizing an enzyme on the surface of an electrode having a void inside;
The hydrophobic electron mediator is immobilized on the surface of the electrode by bringing the surface of the electrode on which the enzyme is immobilized into contact with a solution in which the hydrophobic electron mediator is dissolved in an organic solvent that is phase-separated from water. And a method for producing an enzyme-immobilized electrode.   The method for producing an enzyme-immobilized electrode according to claim 16, wherein the solution further contains a water repellent material.   An enzyme-immobilized electrode comprising an enzyme and a hydrophobic electron mediator immobilized on the surface of an electrode having a void inside.   The enzyme-immobilized electrode according to claim 18, wherein a water-repellent material is further formed on the surface of the electrode.

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