JP2007280944A - Enzyme immobilization electrode and its manufacturing method, fuel cell and its manufacturing method, electronic apparatus, and manufacturing method of electrode reaction utilization apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a manufacturing method capable of sufficiently utilizing an effective surface area of an electrode and obtaining a highly efficient enzyme immobilization electrode by easily penetrating and immobilizing an enzyme in an inside of the electrode with a complicated structure. <P>SOLUTION: When the enzyme is immobilized in the electrode with the complicated structure such as a porus carbon electrode, an organic solvent such as ethanol is contained in a solution used in immobilizing the enzyme in the electrode. The enzyme immobilization electrode manufactured as described above is used for a cathode electrode 22 or the like of a biofuel cell. In other case, the organic solvent is contained in fuel. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  TECHNICAL FIELD The present invention relates to an enzyme-immobilized electrode, a method for producing the same, a fuel cell, a method for producing the same, an electronic device, and a method for producing an electrode reaction utilization device, and in particular, a biofuel cell using an enzyme, and the biofuel cell It is suitable for application to various devices, devices, systems, and the like, as well as biosensors and bioreactors.

  Biological metabolism carried out in living organisms has a high substrate selectivity, is an extremely efficient reaction mechanism, and has a feature that the reaction proceeds in a relatively mild atmosphere at room temperature and neutrality. The biological metabolism mentioned here includes respiration and photosynthesis that convert various nutrients such as oxygen, sugars, fats, and proteins into energy necessary for the growth of microorganisms and cells.

  In such in vivo reactions, biocatalysts consisting of proteins, that is, enzymes, are greatly involved. The idea of utilizing the catalytic action of this enzyme has been practiced for a long time with the history of mankind. In particular, the concept and method of immobilizing an enzyme has been studied for a long time. By immobilizing an enzyme at a high density, an enzyme having high catalytic performance with a small amount of enzyme and a high specificity is generally used. It can be handled in the same manner as a solid catalyst used in the chemical reaction of, and is very useful as an enzyme utilization method. The range of applications covers a wide range of industries such as brewing, fermentation, textile industry, leather industry, food industry, and pharmaceutical industry. Application to the above has been studied and put into practical use (for example, see Patent Documents 1 to 4).

  The same can be said for the application of enzymes to electrode systems. By immobilizing enzymes on electrodes at high density, it is possible to efficiently capture enzyme reaction phenomena occurring near the electrodes as electrical signals. Become. By the way, when examining an electrode system, electron mediation is generally difficult between an enzyme, which is a protein, and an electrode, and an electron mediator (electron acceptor compound) that is an electron transfer medium is required. The electron mediator is preferably immobilized in the same manner as the enzyme.

JP 2000-133297 A JP 2003-282124 A JP 2004-71559 A JP 2005-13210 A

  However, according to the study by the present inventors, an electrode having a complicated structure and a large effective surface area (for example, a porous carbon electrode, a carbon pellet electrode, a carbon felt electrode, a carbon paper electrode, etc.) or a highly water-repellent electrode However, since enzymes and electron mediators do not penetrate into the electrode, it is difficult to immobilize them on the surface inside the electrode, and the effective surface area of the electrode cannot be fully utilized. In biofuel cells, when an electrode having a complicated structure as described above is used as a cathode electrode or an anode electrode, it is difficult to obtain a current value that reflects the effective surface area of these electrodes.

Therefore, the problem to be solved by the present invention is that, for example, an enzyme or an electron mediator can be easily infiltrated and immobilized in an electrode having a complicated structure such as a porous carbon electrode, and the effective surface area of the electrode is sufficiently increased. A highly efficient enzyme-immobilized electrode manufacturing method, a high-efficiency enzyme-immobilized electrode manufactured by this method, a fuel cell manufacturing method using this enzyme-immobilized electrode, and a method manufactured by this method And a method for producing a highly efficient electrode reaction utilizing apparatus using the enzyme-immobilized electrode.
The above and other problems will become apparent from the following description of the present specification with reference to the accompanying drawings.

As a result of intensive studies to solve the above problems, the present inventors have added an organic solvent to a solution used when immobilizing an enzyme on an electrode having a complicated structure such as a porous carbon electrode, thereby It was found that the enzyme can easily penetrate into the electrode and can be immobilized on the surface inside the electrode at a high density in a three-dimensional manner without impairing the activity. Moreover, in the fuel cell, it discovered that the same effect could be acquired by including the organic solvent in a fuel.
This invention has been devised based on the above knowledge obtained by the present inventors.

That is, in order to solve the above problem, the first invention
A method for producing an enzyme-immobilized electrode in which an enzyme is immobilized on the electrode,
An organic solvent is contained in the solution used when the enzyme is immobilized on the electrode.
The second invention is
An enzyme-immobilized electrode in which an enzyme is immobilized on the electrode,
It is produced using a solution in which an organic solvent is included in a solution used when the enzyme is immobilized on the electrode.

  In the first and second inventions, the enzyme-immobilized electrode may be an enzyme such as an electrode made of a carbon-based material such as a porous carbon electrode, a carbon pellet electrode, a carbon felt electrode, or a carbon paper electrode, or an electrode with high water repellency. Is preferably immobilized, and preferably an electron mediator is also immobilized in addition to the enzyme. The enzyme is, for example, a decomposing enzyme, particularly a fuel decomposing enzyme, and optionally contains one or more other enzymes.

  A solution (enzyme solution) used for immobilizing an enzyme on an electrode is usually a solution obtained by dissolving an enzyme in a conventionally known buffer solution such as a phosphate buffer solution or a Tris buffer solution. Include. The concentration of the organic solvent in the enzyme solution can be selected as necessary, but in order to increase the catalyst current value, the concentration of the organic solvent is preferably in the range of, for example, 5% to 65%, and more preferably. Is in the range of 10% to 60%, more preferably 20% to 40%. Various organic solvents can be used, which may be either polar solvents or nonpolar solvents, may be either protic solvents or aprotic solvents, and two or more organic solvents may be used. It may be used. Specific examples of the organic solvent include alcohols, ethers, ketones (such as acetone), aromatic hydrocarbons, chlorinated hydrocarbon compounds, and the like, but are not limited thereto. Basically, any alcohol may be used, and it may be a monohydric alcohol or a polyhydric alcohol, and may be a saturated alcohol or an unsaturated alcohol. Specific examples of the alcohol include methanol, ethanol, 1-propanol, 2-propanol (isopropanol), 1-butanol, 2-butanol (sec-butanol), 2-methyl-1-propanol (isobutanol), 2 -Methyl-2-propanol (tert-butanol) and the like are exemplified, but not limited thereto.

The third invention is
A method for producing a fuel cell, wherein a cathode electrode and an anode electrode have a structure facing each other via a proton conductor, and an enzyme is immobilized on the cathode electrode and / or the anode electrode,
An organic solvent is included in a solution used when the enzyme is immobilized on the cathode electrode and / or the anode electrode.

The fourth invention is:
A fuel cell having a structure in which a cathode electrode and an anode electrode are opposed to each other via a proton conductor, and an enzyme is immobilized on the cathode electrode and / or the anode electrode,
The cathode electrode and / or the anode electrode is produced by using an organic solvent contained in a solution used when the enzyme is immobilized on the cathode electrode and / or the anode electrode. It is.

  The cathode electrode and / or anode electrode on which the enzyme is immobilized is preferably an electrode made of a carbon-based material such as a porous carbon electrode, a carbon pellet electrode, a carbon felt electrode, a carbon paper electrode, or a highly water-repellent electrode. An enzyme is immobilized on an electrode or the like, and preferably an electron mediator is also immobilized in addition to the enzyme. The enzyme is, for example, a decomposing enzyme, particularly a fuel decomposing enzyme, and optionally contains one or more other enzymes.

Various types of fuels can be used and are selected as necessary. Typical examples include methanol, ethanol, monosaccharides, polysaccharides, and the like. When monosaccharides, polysaccharides and the like are used as fuel, they are typically used in the form of a fuel solution in which these are dissolved in a conventionally known buffer solution such as a phosphate buffer or a Tris buffer.
For example, when a monosaccharide such as glucose is used as fuel, it is preferable that the enzyme is an oxidase that promotes and decomposes monosaccharides and a coenzyme that is reduced by the oxidase is returned to the oxidized form. Enzyme oxidase is immobilized. 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, glucose dehydrogenase (GDH) is used as the oxidase, nicotinamide adenine dinucleotide (NAD ⁺ ) or nicotinamide adenine dinucleotide phosphate (NADP ⁺ ) is used as the coenzyme, and diaphorase (DI) is used as the coenzyme oxidase. ) Is used.

When the above-mentioned electron mediator, reduced nicotinamide adenine dinucleotide (NADH) as a coenzyme, glucose dehydrogenase as an oxidase, and diaphorase as a coenzyme oxidase are immobilized on the anode electrode, most preferably 1.0 (mol): 0.33-1.0 (mol): (1.8-3.6) × 10 ⁶ (U): (0.85-1.7) × 10 ⁷ (U) Immobilize by ratio. 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.

  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 that degrades monosaccharides, polysaccharides that can be degraded 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.

  In a fuel cell using cellulase as a degrading enzyme and glucose dehydrogenase as an oxidase, cellulose that can be decomposed into glucose by cellulase can be used as a fuel. More specifically, the cellulase may be any of cellulase (EC 3.2.1.4), exocellobiohydrase (EC 3.2.1.91), β-glucosidase (EC 3.2.1.21) and the like. Or at least one kind. In addition, glucoamylase and cellulase may be mixed and used as the degrading enzyme. In this case, most of the polysaccharides produced in nature can be decomposed, so those containing a large amount of these, for example, garbage Etc. can be used as fuel.

In a fuel cell using α-glucosidase as a degrading enzyme and glucose dehydrogenase as an oxidase, maltose that is decomposed into glucose by α-glucosidase can be used as a fuel.
In a fuel cell using sucrose as a decomposing enzyme and glucose dehydrogenase as an oxidase, sucrose that is decomposed into glucose and fructose by sucrose can be used as a fuel. More specifically, sucrase is α-glucosidase (EC 3.2.1.20), sucrose-α-glucosidase (EC 3.2.1.48), β-fructofuranosidase (EC 3.2.1. 26) or the like.
In a fuel cell using β-galactosidase as a degrading enzyme and glucose dehydrogenase as an oxidase, lactose that is decomposed into glucose and galactose by β-galactosidase can be used as a fuel.
If necessary, these polysaccharides serving as fuels may also be immobilized on the anode electrode.

  In particular, in a fuel cell using starch as a fuel, a gelatinized solid fuel obtained by gelatinizing starch can also be used. In this case, the gelatinized starch can be brought into contact with an anode electrode on which an enzyme or the like is immobilized, or can be immobilized on the anode electrode together with the enzyme or the like. When such an electrode is used, the starch concentration on the surface of the anode electrode can be maintained at a higher level than when starch dissolved in the solution is used, the enzymatic decomposition reaction is faster, and the output is improved. At the same time, handling of the fuel is easier than in the case of the 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 in a mobile device. .

When an enzyme is immobilized on the cathode electrode, the enzyme typically includes an enzyme that reduces oxygen. Examples of the enzyme that reduces oxygen include bilirubin oxidase, laccase, and ascorbate oxidase. In this case, an electron mediator is preferably immobilized on the cathode electrode in addition to the enzyme. As the electron mediator, for example, potassium hexacyanoferrate, potassium ferricyanide, potassium octacyanotungstate and the like are used. The electron mediator is preferably immobilized at a sufficiently high concentration, for example, 0.64 × 10 ⁻⁶ mol / mm ² or more on average.

  As the anode electrode or the cathode electrode, the above-described electrode can be used, and a skeleton made of a porous material and a material mainly composed of a carbon-based material that covers at least a part of the surface of the skeleton are included. A porous conductive material can be used. This porous conductive material can be obtained by coating at least a part of the surface of the skeleton made of the porous material with a material mainly composed of a carbon-based material. The porous material constituting the skeleton of the porous conductive material may be basically any material as long as the skeleton can be stably maintained even if the porosity is high. It does not matter whether or not there is sex. As the porous material, a material having high porosity and high conductivity is preferably used. Specifically, as such a porous material having a high porosity and high conductivity, a metal material (metal or alloy), a carbon-based material with a strong skeleton (improved brittleness), or the like is used. Can do. When a metal material is used as the porous material, various options are conceivable because the metal material has different state stability depending on the usage environment such as pH and potential of the solution. For example, nickel, copper, silver, gold A foam metal such as nickel-chromium alloy and stainless steel or a foam alloy is one of the easily available materials. As the porous material, a resin material (for example, a sponge-like material) can be used in addition to the metal material and the carbon-based material. The porosity and pore diameter (minimum pore diameter) of this porous material are in balance with the thickness of the material mainly composed of a carbon-based material that is coated on the surface of the skeleton made of this porous material. Is determined according to the required porosity and pore diameter. The pore diameter of the porous material is generally 10 nm to 1 mm, typically 10 nm to 600 μm. On the other hand, it is necessary to use a material that covers the surface of the skeleton and has conductivity and is stable at an assumed operating potential. Here, a material mainly composed of a carbon-based material is used as such a material. Carbon-based materials generally have a wide potential window, and many are chemically stable. Specifically, the carbon-based material is mainly composed of a carbon-based material, and the carbon-based material is the main component, and the secondary material is selected according to the characteristics required for the porous conductive material. Some materials contain a small amount of material. Specific examples of the latter material include a material whose electrical conductivity has been improved by adding a metal or other highly conductive material to the carbon-based material, or a polytetrafluoroethylene-based material or the like added to the carbon-based material. Thus, it is a material imparted with a function other than conductivity, such as imparting surface water repellency. There are various types of carbon-based materials, but any carbon-based material may be used. In addition to carbon alone, carbon may be added with other elements. The carbon-based material is particularly preferably a fine powder carbon material having high conductivity and a high surface area. Specific examples of the carbon-based material include materials imparted with high conductivity such as KB (Ketjen Black) and functional carbon materials such as carbon nanotubes and fullerenes. As a coating method of the material mainly composed of the carbon-based material, any coating method can be used as long as the surface of the skeleton made of the porous material can be coated by using an appropriate binder as necessary. Also good. The pore diameter of the porous conductive material is selected to a size that allows a solution containing a substrate or the like to easily enter and exit through the pores, and is generally 9 nm to 1 mm, more typically 1 μm to 1 mm, and more generally Specifically, it is 1 to 600 μm. A state in which at least a part of the surface of the skeleton made of a porous material is coated with a material mainly composed of a carbon-based material, or a surface of at least a part of the skeleton made of a porous material is mainly composed of a carbon-based material In the state coated with the material, it is desirable to prevent all the holes from communicating with each other or clogging with a material mainly composed of a carbon-based material.

When an electrolyte containing a buffer substance (buffer solution) is used as the proton conductor, sufficient buffer capacity can be obtained during high output operation, and the ability inherent to the enzyme can be fully demonstrated. Therefore, 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, in general, as long as _a pK _a of 6 to 9, may it be used What Specific examples and, dihydrogen phosphate ion (H ₂ PO ₄ ^- ), 2-amino-2-hydroxymethyl-1,3-propanediol (abbreviated to Tris), 2- (N-morpholino) ethanesulfonic acid (MES), cacodylic acid, carbonic acid (H ₂ CO ₃ ), hydrogen citrate Ions, 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- '-3-propanesulfonic acid (HEPPS), N- [tris (hydroxymethyl) methyl] glycine (abbreviation tricine), glycylglycine, N, N-bis (2-hydroxyethyl) glycine (abbreviation bicine), etc. . 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 ). The pH of the electrolyte containing the buffer substance is preferably around 7, but may generally be any of 1-14.

This fuel cell 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 devices, 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.
In the third and fourth inventions, the matters other than the above are explained in relation to the first and second inventions unless they are contrary to the nature.

The fifth invention is:
A fuel cell having a structure in which a cathode electrode and an anode electrode are opposed to each other via a proton conductor, and an enzyme is immobilized on the cathode electrode and / or the anode electrode,
It is characterized by containing an organic solvent in the fuel.

When using monosaccharides, polysaccharides, or the like for the fuel, they are typically used in the form of a fuel solution in which these are dissolved in a conventionally known buffer solution such as a phosphate buffer solution or a Tris buffer solution. The volume ratio of the organic solvent to the buffer solution in the fuel solution can be selected as necessary, but is generally greater than 0% and 50% or less, preferably 5% or more and 30% or less, more preferably Is 5% or more and 15% or less.
In the fifth invention, the matters other than those described above are explained in relation to the first to fourth inventions unless they are contrary to the nature.

The sixth invention is:
In an electronic device using one or more fuel cells,
At least one of the fuel cells is
A fuel cell having a structure in which a cathode electrode and an anode electrode are opposed to each other via a proton conductor, and an enzyme is immobilized on the cathode electrode and / or the anode electrode,
The cathode electrode and / or the anode electrode is produced by using an organic solvent contained in a solution used when the enzyme is immobilized on the cathode electrode and / or the anode electrode. It is what.

The electronic device according to the sixth invention may be basically any device, and includes both a portable device and a stationary device. Specific examples include a mobile phone and a mobile device. (Personal digital assistants (PDAs), etc.), robots, personal computers (including both desktop and notebook types), game machines, camera-integrated VTRs (video tape recorders), in-vehicle devices, home appliances, industrial products, etc. It is.
In the sixth aspect of the present invention, what has been described in relation to the first to fourth aspects of the invention is valid as long as not against its nature.

The seventh invention
In an electronic device using one or more fuel cells,
At least one of the fuel cells is
A fuel cell having a structure in which a cathode electrode and an anode electrode are opposed to each other via a proton conductor, and an enzyme is immobilized on the cathode electrode and / or the anode electrode,
It is characterized by containing an organic solvent in the fuel.
In the seventh invention, the matters other than those described above are explained in relation to the first to sixth inventions unless they are contrary to the nature.

The eighth invention
A method for producing an enzyme reaction utilization device having an enzyme-immobilized electrode in which an enzyme is immobilized on the electrode,
An organic solvent is contained in the solution used when the enzyme is immobilized on the electrode.

In the eighth invention, the electrode reaction utilization device includes a biosensor (such as a glucose sensor), a bioreactor, etc. in addition to the above fuel cell, that is, a biofuel cell. Is used.
In the eighth invention, what is described in relation to the first to seventh inventions holds true for matters other than those described above, unless they are contrary to the nature thereof.
The ninth invention
This is an enzyme-immobilized electrode in which an aggregate containing an enzyme is immobilized on the electrode.
In the enzyme-immobilized electrode, preferably, the aggregate further contains an electron mediator, a coenzyme, a phospholipid or a derivative thereof, or two or more thereof. Basically, any phospholipid may be used, and either glycerophospholipid or sphingophospholipid may be used. Examples of the glycerophospholipid include, but are not limited to, phosphatidic acid, phosphatidylcholine (lecithin), phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, and diphosphatidylglycerol (cardiolipin). Sphingophospholipids include, but are not limited to, sphingomyelin. A representative example of phosphatidylcholine is dimyristoyl phosphatidylcholine (DMPC). Aggregates typically consist of fine particles. The fine particles are formed of, for example, a polyion complex, but are not limited thereto. The fine particles are typically substantially spherical, and the diameter thereof is selected as necessary, but is, for example, 1 μm or less.
The tenth invention is
The fuel cell has a structure in which a cathode electrode and an anode electrode face each other via a proton conductor, and an aggregate containing an enzyme is immobilized on the cathode electrode and / or the anode electrode.
In the ninth and tenth inventions, what has been described in relation to the first to seventh inventions holds true for matters other than those described above, unless they are contrary to their properties.
The eleventh invention is
A layer containing an enzyme is fixed to the electrode, a void is provided in the layer, and a reactant or oxygen is supplied to the enzyme through the void.
In this enzyme-immobilized electrode, the layer containing the enzyme exists, for example, uniformly on the electrode. The voids provided in this layer are typically substantially spherical and have a diameter of 1 μm or less, but are not limited thereto. In addition, this layer preferably further contains an electron mediator, or a coenzyme, phospholipid or a derivative thereof, or two or more thereof.
In the eleventh invention, the matters other than those described above are explained in relation to the first to tenth inventions unless they are contrary to their properties.

In the present invention configured as described above, when the electrode has a complicated structure, such as a porous carbon electrode, due to the action of the organic solvent contained in the solution or fuel used when the enzyme is immobilized on the electrode. Even when the electrode has high water repellency, the enzyme easily penetrates into the electrode.
In addition, an enzyme-immobilized electrode in which an enzyme-containing aggregate is immobilized on an electrode or an enzyme-immobilized electrode in which a layer containing an enzyme is immobilized on this electrode and a void is provided in this layer reacts through the void in the aggregate. The supply of the product and the discharge of the product can be performed easily and smoothly.

According to this invention, since the penetration of the enzyme into the electrode is facilitated, the enzyme can be immobilized on the entire inner surface of the electrode having a complicated structure. For this reason, it is possible to immobilize the enzyme three-dimensionally at a high density by fully utilizing the effective surface area of the electrode having a complicated structure. For this reason, the enzyme metabolism reaction on the electrode can be performed with high efficiency, and a highly efficient enzyme-immobilized electrode can be obtained. By using this enzyme-immobilized electrode, a highly efficient fuel cell or electrode reaction utilizing device can be realized. By using such a highly efficient fuel cell, a high-performance electronic device can be realized.
In addition, high-efficiency enzyme immobilization is possible with an enzyme-immobilized electrode in which an enzyme-containing aggregate is immobilized on the electrode or an enzyme-immobilized electrode in which a layer containing the enzyme is immobilized on this electrode and a void is provided in this layer An electrode can be obtained, and by using this enzyme-immobilized electrode, a highly efficient fuel cell or an electrode reaction utilization device can be realized. By using such a highly efficient fuel cell, a high-performance electronic device can be realized.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. In all the drawings of the embodiments, the same or corresponding parts are denoted by the same reference numerals.
In the first embodiment, a case where an enzyme or an enzyme and an electron mediator are immobilized on an electrode having a complicated structure to produce an enzyme-immobilized electrode will be described.
The enzyme-immobilized electrode is manufactured as follows.
As an electrode for immobilizing an enzyme or an enzyme and an electron mediator, for example, a porous carbon electrode, a carbon pellet electrode, a carbon felt electrode, a carbon paper electrode, or an electrode having high water repellency is used. However, the electrodes are not limited to these.

The enzyme solution containing the enzyme to be immobilized on the electrode is mixed with an organic solvent such as the alcohol as described above, and the concentration of the organic solvent in the enzyme solution is, for example, 5% to 65%, preferably 10% to 60%. More preferably, it is contained so as to be 20% or more and 40% or less. And after immersing the said electrode in this solution, it wash | cleans with water and produces an enzyme fixed electrode. When the electron mediator is immobilized on the electrode in addition to the enzyme, a solution in which the electron mediator is dissolved in an organic solvent is used.
According to the first embodiment, even in an electrode having a complicated structure, the action of the organic solvent facilitates the penetration of the enzyme in the enzyme solution into the electrode, and as a result, the enzyme is applied to the entire surface inside the electrode. The enzyme can be immobilized at a high density three-dimensionally. For this reason, this enzyme fixed electrode is highly efficient, and can obtain a large catalyst current value.

[Example 1]
An electrode for immobilizing the enzyme was produced as follows. A commercially available ketjen black and polyvinylidene fluoride (PVdF) were well dispersed in NMP (N-methyl-2-pyrrolidone) (1.2 ml) in a 7: 3 ratio (Ketjen black 20 mg, PVdF 10 mg). It was. On the other hand, as shown in FIG. 1A, a commercially available glassy carbon electrode 11 (electrode diameter 3 mm) is prepared. Then, as shown in FIG. 1B, 10 μl (Ketjen Black 0.17 mg) of the dispersion solution 13 was dropped onto the glassy carbon electrode 11 with a microsyringe 12 and sufficiently dried at 100 ° C. Thus, as shown in FIG. 1C, an electrode 14 for immobilizing the enzyme was produced.

This electrode 14 was subjected to cyclic voltammetry (CV) in a phosphate buffer (0.1 M NaH ₂ PO ₄ , ionic strength = 0.3, pH = 7) using Ag | AgCl as a reference electrode. . The result is shown as curve 1 in FIG. 2A. The electrode 14 was washed in ethanol and completely dried, and then CV measurement was performed. The results are shown in curve 2 of FIGS. 2A and B. Further, this electrode 14 was immersed in ethanol, washed with water, and CV measurement was performed. The result is shown as curve 3 in FIG. 2B. As can be seen from FIGS. 2A and 2B, the electrode 14 was washed with ethanol and then dried, so that the background current became about 10 times that in the case where the electrode 14 was not washed with ethanol. Further, when the electrode 14 was not dried after being washed in ethanol, a background current 50 times higher than that obtained when the electrode 14 was not washed was obtained. This indicates that the reaction area of the electrode 14 is increased by the penetration of the phosphate buffer solution into the electrode 14 by ethanol.

Next, a solution (ethanol concentration 10%) is prepared by adding 0.1 ml of ethanol to an enzyme solution in which bilirubin oxidase is dissolved in 0.9 ml of phosphate buffer as an enzyme that reduces oxygen as a substrate. To do. And after immersing the electrode 14 in this solution, it wash | cleaned immediately and performed CV measurement. The result is shown in curve 1 of FIGS. For comparison, the electrode 14 was immersed in an enzyme solution containing no ethanol, and then immediately washed and CV measurement was performed. The results are shown in curve 2 in FIGS. 3A and B. However, FIG. 3A is a measurement result when only dissolved oxygen in the buffer solution becomes a substrate (when deoxidizing), and FIG. 3B is a measurement result when the buffer solution is stirred to include a saturated amount of oxygen (when oxygen saturation is stirred). is there. As can be seen from FIGS. 3A and B, when the electrode 14 is immersed in an enzyme solution containing ethanol, an extremely large catalyst current value is obtained as compared with the case where the electrode 14 is immersed in an enzyme solution not containing ethanol. Yes.
FIG. 4 shows changes in the catalyst current value (catalyst limit current (potential is 0 V) under oxygen saturation stirring conditions) when the ethanol concentration in the enzyme solution is changed. As can be seen from FIG. 4, an increase in the catalyst current value is clearly observed when the ethanol concentration is in the range of 5% to 65%, and a remarkable increase in the catalyst current value is observed particularly in the range of 10% to 60%. In particular, in the range of 20% to 40%, a significant increase in the catalyst current value is observed.

Next explained is the second embodiment of the invention. In the second embodiment, the same method as in the first embodiment is used when an enzyme is immobilized on the cathode electrode of a biofuel cell.
FIG. 5 schematically shows the biofuel cell according to the first embodiment. In this biofuel cell, glucose is used as the fuel. FIG. 6 schematically shows details of the configuration of the anode electrode of the biofuel cell, an example of an enzyme group immobilized on the anode electrode, and an electron transfer reaction by the enzyme group.
As shown in FIG. 5, this biofuel cell has a structure in which an anode electrode 21 and a cathode electrode 22 face each other with a proton conductor 23 interposed therebetween. The anode electrode 21 decomposes glucose supplied as a fuel with an enzyme to extract electrons and generate protons (H ⁺ ). The cathode electrode 22 generates water from protons transported from the anode electrode 21 through the proton conductor 23, electrons sent from the anode electrode 21 through an external circuit, and oxygen in the air, for example.

The anode electrode 21 includes, for example, an enzyme involved in the decomposition of glucose on an electrode 21a (see FIG. 6) made of, for example, porous carbon, and a coenzyme in which a reductant is generated by an oxidation reaction in the glucose decomposition process (for example, , NAD ⁺ ), a coenzyme oxidase (eg, diaphorase) that oxidizes a reduced form of the coenzyme (eg, NADH), and an electron generated from the coenzyme oxidase accompanying the oxidation of the coenzyme to the electrode 21a. The passing electron mediator (for example, VK3) uses an immobilizing material (not shown) (for example, a polycation such as poly-L-lysine (PLL) and a polyanion such as sodium polyacrylate (PAAcNa)). The polyion complex is formed by immobilization.

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.
The electrons generated in the above process are transferred from diaphorase to the electrode 21 a via the electron mediator, and H ⁺ is transported to the cathode electrode 22 through the proton conductor 23.

The enzyme, coenzyme, and electron mediator described above are used for the enzyme by a buffer solution such as a phosphate buffer solution or a Tris buffer solution contained in the proton conductor 23 in order to perform the electrode reaction efficiently and constantly. It is preferably maintained at an optimum 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. 6, 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 21a is VK3.

The cathode electrode 22 performs an enzyme immobilization treatment using an organic solvent on a porous carbon electrode, a carbon pellet electrode, a carbon felt electrode, a carbon paper electrode, or a highly water-repellent electrode as in the first embodiment. Thus, for example, an enzyme capable of decomposing oxygen such as bilirubin oxidase, laccase, ascorbate oxidase is immobilized. The portion outside the cathode electrode 22 (the portion opposite to the proton conductor 23) is usually formed by a gas diffusion layer made of porous carbon. Preferably, in addition to the enzyme, an electron mediator that exchanges electrons with the cathode electrode 22 is also immobilized on the cathode electrode 22.
In the cathode electrode 22, oxygen in the air is reduced by H ⁺ from the proton conductor 23 and electrons from the anode electrode 21 in the presence of the enzyme that decomposes oxygen to generate water.

The proton conductor 23 is for transporting H ⁺ generated at the anode electrode 21 to the cathode electrode 22 and is made of a material that does not have electron conductivity and can transport H ⁺ . Specific examples of the proton conductor 23 include a perfluorocarbon sulfonic acid (PFS) resin film, a copolymer film of a trifluorostyrene derivative, a polybenzimidazole film impregnated with phosphoric acid, and an aromatic polyether. Examples thereof include a ketone sulfonic acid film, PSSA-PVA (polystyrene sulfonate polyvinyl alcohol copolymer) and PSSA-EVOH (polystyrene sulfonate ethylene vinyl alcohol copolymer). Among these, those made of an ion exchange resin having a fluorine-containing carbon sulfonic acid group are preferable, and specifically, Nafion (trade name, DuPont, USA) is used.

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

Next, a specific structural example of the biofuel cell will be described.
As shown in FIGS. 7A and B, this biofuel cell includes an enzyme / coenzyme / electron mediator in which an enzyme, a coenzyme, and an electron mediator are immobilized on a carbon felt (for example, an area of 0.25 cm ² ) with an immobilizing material. An anode electrode 21 composed of an immobilized carbon electrode, and a cathode electrode composed of an enzyme / electron mediator-immobilized carbon electrode in which an enzyme or an electron mediator is immobilized on a carbon felt (for example, an area of 0.25 cm ² ) with an immobilizing material. 22 is opposed to the proton conductor 23 via the proton conductor 23. In this case, Ti current collectors 31 and 32 are respectively placed under the cathode electrode 22 and the anode electrode 21 so that current collection can be performed easily. Reference numerals 33 and 34 denote fixed plates. The fixing plates 33 and 34 are fastened to each other by screws 35, and the cathode electrode 22, the anode electrode 21, the proton conductor 23, and the Ti current collectors 33 and 34 are sandwiched between them. One surface (outer surface) of the fixing plate 33 is provided with a circular recess 33a for taking in air, and a plurality of holes 33b penetrating to the other surface are provided on the bottom surface of the recess 33a. These holes 33 b serve as air supply paths to the cathode electrode 22. On the other hand, a circular recess 34a for fuel loading is provided on one surface (outer surface) of the fixed plate 34, and a number of holes 34b penetrating to the other surface are provided on the bottom surface of the recess 34a. These holes 34 b serve as fuel supply paths to the anode electrode 21. A spacer 36 is provided on the periphery of the other surface of the fixing plate 34, and when the fixing plates 33 and 34 are fastened to each other with screws 35, the interval between them becomes a predetermined interval. .
As shown in FIG. 7B, a load 37 is connected between the Ti current collectors 31 and 32, and a glucose solution in which glucose is dissolved in a phosphate buffer, for example, is put into the recess 34a of the fixed plate 34 to generate power. .

Next explained is a biofuel cell according to the third embodiment of the invention.
The biofuel cell according to the third embodiment has the same configuration as that of the biofuel cell according to the second embodiment, but a fuel such as a monosaccharide or polysaccharide is used as a buffer solution such as a phosphate buffer or a Tris buffer. The difference is that an organic solvent is included in the fuel solution dissolved in the solution. The volume ratio of the organic solvent to the buffer solution in the fuel solution is generally greater than 0% and 50% or less, preferably 5% or more and 30% or less, and more preferably 5% or more and 15% or less.
In this biofuel cell, immediately after the enzyme is immobilized on the cathode electrode 22 and the anode electrode 21, even when the enzyme does not permeate into the inside of the cathode electrode 22 and the anode electrode 21, By contacting with the fuel solution containing the organic solvent, the buffer solution easily penetrates into the cathode electrode 22 and the anode electrode 21 due to the action of the organic solvent, and the enzyme easily penetrates into the inside in a form prompted by this. It is fixed on the internal surface in a three-dimensional and high-density manner. For this reason, a highly efficient biofuel cell is realizable similarly to 2nd Embodiment.

[Example 2]
An electrode 14 for immobilizing an enzyme was produced in the same manner as in Example 1. Then, bilirubin oxidase was added at a rate of 1 mg to 1 ml of the phosphate buffer in the same phosphate buffer as in Example 1, and CV measurement was performed with the electrode 14. The result is shown in FIG. 8A. Next, ethanol was added to the phosphate buffer so that the volume ratio was 10%, and the same CV measurement was performed. The result is shown in FIG. 8B. As can be seen from FIGS. 8A and 8B, when ethanol is added to the phosphate buffer, an extremely large catalyst current value about 100 times that obtained when ethanol is not added to the phosphate buffer is obtained. . This is due to the action of ethanol in the phosphate buffer, which facilitates the penetration of the phosphate buffer into the inside of the electrode 14, and is facilitated by this to the inside of the electrode 14 of the bilirubin oxidase in the phosphate buffer. It is thought that this is the result of the easy penetration of.

Next explained is a biofuel cell according to the fourth embodiment of the invention.
The biofuel cell according to the fourth embodiment is the same as the biofuel cell according to the second embodiment except that a porous conductive material as shown in FIG. 9 is used as the material of the electrode 21a of the anode electrode 21. It has a configuration.
FIG. 9A schematically shows the structure of the porous conductive material, and FIG. 9B is a cross-sectional view of the skeleton of the porous conductive material. As shown in FIGS. 9A and 9B, this porous conductive material includes a skeleton 41 made of a porous material having a three-dimensional network structure, and a carbon-based material 42 that covers the surface of the skeleton 41. This porous conductive material has a three-dimensional network structure in which a large number of holes 43 surrounded by the carbon-based material 42 correspond to a network. In this case, these holes 43 communicate with each other. The form of the carbon-based material 42 is not limited and may be any of a fibrous shape (needle shape) and a granular shape.

As the skeleton 41 made of a porous material, a foam metal or a foam alloy such as nickel foam is used. The porosity of the skeleton 41 is generally 85% or more, more typically 90% or more, and the pore diameter is typically, for example, 10 nm to 1 mm, more typically 10 nm to 600 μm, and more generally 1 to 600 μm, typically 50 to 300 μm, more typically 100 to 250 μm. The carbon material 42 is preferably a highly conductive material such as ketjen black, but a functional carbon material such as carbon nanotube or fullerene may be used.
The porosity of the porous conductive material is generally 80% or more, more typically 90% or more, and the diameter of the hole 43 is typically, for example, 9 nm to 1 mm, more generally 9 nm to It is 600 μm, more generally 1 to 600 μm, typically 30 to 400 μm, more typically 80 to 230 μm.

Next, a method for producing this porous conductive material will be described.
As shown in FIG. 10A, first, a skeleton 41 made of a foam metal or a foam alloy (for example, foam nickel) is prepared.
Next, as shown in FIG. 10B, a carbon-based material 42 is coated on the surface of the skeleton 41 made of the foam metal or foam alloy. As this coating method, a conventionally known method can be used. For example, the carbon-based material 42 is coated by spraying an emulsion containing carbon powder, an appropriate binder, or the like onto the surface of the skeleton 41 by spraying. The coating thickness of the carbon-based material 42 is determined according to the porosity and the pore diameter required for the porous conductive material in consideration of the porosity and the pore diameter of the skeleton 41 made of the foam metal or foam alloy. In this coating, a large number of holes 43 surrounded by the carbon-based material 42 are communicated with each other.
In this way, the target porous conductive material is manufactured.

  According to the fourth embodiment, in addition to the advantages similar to those of the second embodiment, the following advantages can be obtained. That is, the porous conductive material in which the surface of the skeleton 41 made of foam metal or foam alloy is coated with the carbon-based material 42 has a sufficiently large diameter of the holes 43 and has a rough three-dimensional network structure, while having high strength. In addition, it has high conductivity and can provide a necessary and sufficient surface area. For this reason, the electrode 21a of the anode electrode 21 is formed using this porous conductive material, and the enzyme / coenzyme / electron mediator immobilized electrode obtained by immobilizing the enzyme, coenzyme and electron mediator on the electrode 21a is formed. The anode electrode 21 can perform the enzyme metabolism reaction on the anode electrode 21 with high efficiency, or can efficiently capture the enzyme reaction phenomenon occurring near the electrode 21a as an electric signal. A stable and high-performance biofuel cell can be realized regardless of the environment.

Next explained is a biofuel cell according to the fifth embodiment of the invention.
In the biofuel cell according to the fifth embodiment, the proton conductor 23 is composed of an electrolyte layer containing a buffer solution such as a phosphate buffer solution or a Tris buffer solution, and is immobilized on at least the anode electrode 21 and / or the cathode electrode 22. In the electrolyte layer around the enzyme formed, a buffer solution is 0.2M to 2.5M, preferably 0.2M to 2M, more preferably 0.4M to 2M, and even more preferably 0.8M. A concentration of 1.2 M or less is included. In this way, a high buffer capacity can be obtained, and even at the time of high output operation of the biofuel cell, it can be maintained at an optimum pH for the enzyme, for example, near pH 7, so that the original ability of the enzyme can be fully exhibited. be able to. As the phosphate buffer, for example, NaH ₂ PO ₄ or KH ₂ PO ₄ is used.
Other than the above are the same as in the second embodiment.

  As described above, according to the fifth embodiment, the concentration of the buffer substance (buffer solution) contained in the electrolyte layer constituting the proton conductor 23 is not less than 0.2M and not more than 2.5M. Therefore, even when the biofuel cell is operated at a high output, the pH of the electrolyte layer around the enzyme immobilized on the cathode electrode 22 and / or the anode electrode 21 is kept close to the optimum pH. Can be maintained, and the ability of the enzyme can be fully exerted. Thereby, a high-performance biofuel cell capable of high output operation can be realized.

Next explained is a biofuel cell according to the sixth embodiment of the invention.
In this biofuel cell, starch, which is a polysaccharide, is used as a fuel. In addition, with the use of starch as fuel, glucoamylase, which is a degrading enzyme that decomposes starch into glucose, is also immobilized on the anode electrode 21.
In this biofuel cell, when starch is supplied as fuel to the anode electrode 21 side, this starch is hydrolyzed into glucose by glucoamylase, and this glucose is further decomposed by glucose dehydrogenase. At the same time, NAD ⁺ is reduced to produce NADH, which is oxidized by diaphorase and separated into two electrons, NAD ⁺ and H ⁺ . Therefore, two electrons and two H ⁺ are generated by one-step oxidation reaction per molecule of glucose. In the two-stage oxidation reaction, a total of 4 electrons and 4 H ⁺ are generated. The electrons thus generated are transferred to the electrode 21 a of the anode electrode 21, and H ⁺ moves to the cathode electrode 22 through the proton conductor 23. In the cathode electrode 22, this H ⁺ reacts with oxygen supplied from outside and electrons sent from the anode electrode 21 through an external circuit to generate H ₂ O.
Other than the above, the biofuel cell according to the second embodiment is the same as the biofuel cell according to the second embodiment.
According to the sixth embodiment, the same advantages as those of the second embodiment can be obtained, and the amount of power generation can be increased by using starch as fuel, compared with the case where glucose is used as fuel. The advantage that it can be obtained.

Next explained is an enzyme-immobilized electrode according to the seventh embodiment of the invention.
In the seventh embodiment, as shown in FIG. 11, on the electrode 51 made of, for example, porous carbon, an enzyme involved in the decomposition of glucose and a reductant are generated in accordance with the oxidation reaction in the glucose decomposition process. A coenzyme that is oxidized, a coenzyme oxidase that oxidizes a reduced form of the coenzyme, and an electron mediator that receives electrons generated from the coenzyme oxidase accompanying the oxidation of the coenzyme and passes them to the electrode 51 are fixed by the fixing material An aggregate 53 in which a large number of microparticles 52 are aggregated is fixed. This immobilization material typically comprises a polyion complex formed using, for example, a polycation such as poly-L-lysine (PLL) and a polyanion such as sodium polyacrylate (PAAcNa). However, the present invention is not limited to this. When the fine particles 52 are formed of a polyion complex, the diameter of the fine particles 52 is determined by the drying conditions such as the molecular weight of the polycation and polyanion used to form the polyion complex, the drying temperature at which the polyion complex is formed, and the polyion complex. It can be controlled by selecting the composition of the polycation and polyanion to be formed. The fine particles 52 are preferably, for example, substantially spherical and have a diameter of 1 μm or less, but are not limited thereto.

  As shown in FIG. 11, in this enzyme-immobilized electrode, the surface and the inside of the fine particles 52 constituting the aggregate 53 serve as an enzyme reaction field. In this case, since the aggregate 53 includes many voids, the reactant (for example, fuel) is supplied through the voids to the enzyme in the reaction field and the product obtained by the reaction of the reactants is easily and smoothly discharged. In addition, the aggregate 53 has a large surface area, and the reactant can be easily supplied to the inside of the aggregate 53, so that the reaction rate can be improved as a whole. The electrons can be exchanged with the electrode 51 via an electron mediator immobilized on the aggregate 53 or between the electron mediators.

Example 3
An enzyme-immobilized electrode was prepared as follows.
Solution preparation First, various solutions were prepared as follows. As the buffer solution, 0.1M NaH ₂ PO ₄ —NaOH—NaCl (IS = 0.3, pH 7) was used.
・ GDH / DI / NADH enzyme ・ Coenzyme buffer solution ((1))
10 mg of diaphorase (DI) (EC 1.6.99.-, manufactured by Amano Enzyme, DH “Amano” 3) was weighed and dissolved in 200 μl of a buffer solution ((1) ′). The buffer solution for dissolving the enzyme is preferably refrigerated at 8 ° C. or less until immediately before, and the enzyme buffer solution is preferably refrigerated at 8 ° C. or less as much as possible.
13.8 mg of glucose dehydrogenase (GDH) (NAD-dependent, EC 1.1.1.47, Amano Enzyme, GDH-2) was weighed and dissolved in 180 μl of a buffer solution. To this, 20 μl of the above solution (1) ′ was added and mixed well to finally make 200 μl of GDH / DI = 4.2 / 20 U / μl solution.
-NADH buffer solution ((2))
64 mg of NADH (manufactured by Sigma-Aldrich, N-8129) was weighed and dissolved in 0.1 ml of a buffer solution (640 μg / μl).
・ PLL aqueous solution ((3))
An appropriate amount of poly-L-lysine hydrobromide (PLL) (manufactured by Sigma-Aldrich, P-2174) was weighed and dissolved in deionized water to 2 wt%.
・ ANQ acetone solution ((4))
10 mg of 2-amino-1,4-naphthoquinone (ANQ) was weighed and dissolved in 964 μl of an acetone solution to give 60 mM.
-PAAcNa aqueous solution ((5))
Sodium polyacrylate (PAAcNa) (manufactured by Sigma-Aldrich, 41,604-5) was weighed in an appropriate amount and prepared by adding deionized water to 0.022 wt%.
Preparation of Immobilized Membrane The various solutions prepared as described above were applied in the following order in the following order on the surface of a glassy carbon disk electrode (manufactured by BAS, 002012, 3 mmφ, 0.071 cm ² ). After dripping and mixing, an enzyme-immobilized electrode was prepared by appropriately drying at room temperature or 40 ° C. or lower.
GDH / DI enzyme buffer solution ((1)): 8 μl
NADH buffer solution (2): 2 μl
PLL aqueous solution ((3)): 10 μl
ANQ acetone solution ((4)): 18.7 μl
PAAcNa aqueous solution ((5)): 12 μl
FIG. 12 shows the result of observing the surface of the enzyme-immobilized electrode thus produced with a confocal microscope (Olympus Fluoview FV1000 × 100 lens zoom × 1). From FIG. 12, it is observed that fine particles containing an enzyme having a diameter of about 1 μm aggregate on the electrode surface to form an aggregate.

Example 4
An enzyme-immobilized electrode was prepared as follows.
Solution preparation Various solutions were prepared as follows. The same buffer solution as in Example 3 was used.
DMPC ethanol solution (6)
1 mg of dimyristoylphosphatidylcholine (DMPC) (manufactured by Wako Pure Chemical Industries, 163-12771) was dissolved in 1 ml of ethanol to obtain a 0.1% (w / v) ethanol solution.
GDH / DI enzyme buffer solution ((1)), NADH buffer solution ((2)), PLL aqueous solution ((3)), ANQ acetone solution (4) and PAAcNa aqueous solution ((5)) were the same as in Example 3. Prepared.
Preparation of Immobilized Membrane The various solutions prepared as described above were applied in the following order in the following order on the surface of a glassy carbon disk electrode (manufactured by BAS, 002012, 3 mmφ, 0.071 cm ² ). After dripping and mixing, an enzyme-immobilized electrode was prepared by appropriately drying at room temperature or 40 ° C. or lower.
GDH / DI enzyme buffer solution ((1)): 8 μl
NADH buffer solution ((2)): 2 μl
PLL aqueous solution ((3)): 10 μl
ANQ acetone solution ((4)): 18.7 μl
PAAcNa aqueous solution ((5)): 12 μl
DMPC ethanol solution (6): 10 μl
When the surface of the enzyme-immobilized electrode thus prepared was observed with a confocal microscope in the same manner as in Example 3, the same result as in Example 3 was obtained.
According to the seventh embodiment, a highly efficient enzyme-immobilized electrode can be realized. A highly efficient biofuel cell can be realized by using the enzyme-immobilized electrode as a cathode electrode or an anode electrode of the biofuel cell.

As mentioned above, although embodiment of this invention was described concretely, this invention is not limited to the above-mentioned embodiment, The various deformation | transformation based on the technical idea of this invention is 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.

It is a basic diagram for demonstrating the production method of the electrode which fix | immobilizes the enzyme for evaluation of the enzyme fixed electrode by 1st Embodiment of this invention. It is a basic diagram which shows the cyclic voltammogram of the electrode shown in FIG. It is a basic diagram which shows the cyclic voltammogram of the enzyme fixed electrode which fixed the enzyme to the electrode shown in FIG. It is a basic diagram which shows the change of the catalyst electric current value when changing the ethanol concentration in an enzyme solution. It is a basic diagram which shows the biofuel cell by 2nd Embodiment of this invention. FIG. 5 is a schematic diagram schematically showing a configuration of an anode electrode of a biofuel cell according to a second embodiment of the present invention, an example of an enzyme group immobilized on the anode electrode, and an electron transfer reaction by the enzyme group. is there. It is a basic diagram which shows the specific structural example of the biofuel cell by the 2nd Embodiment of this invention. It is a basic diagram which shows the cyclic voltammogram of the enzyme fixed electrode for evaluation of the biofuel cell by 3rd Embodiment of this invention. It is a basic diagram and sectional drawing for demonstrating the structure of the porous conductive material used for the electrode material of an anode electrode in the biofuel cell by the 4th Embodiment of this invention. It is a basic diagram for demonstrating the manufacturing method of the porous electroconductive material used for the electrode material of an anode electrode in the biofuel cell by the 4th Embodiment of this invention. It is a basic diagram which shows the enzyme fixed electrode by 7th Embodiment of this invention. It is a confocal microscope image of the surface of the enzyme fixed electrode produced by Example 3 of this invention.

Explanation of symbols

  21 ... Anode electrode, 21a ... Electrode, 22 ... Cathode electrode, 23 ... Proton conductor, 31, 32 ... Ti current collector, 33, 34 ... Fixed plate, 37 ... Load, 41 ... Skeleton, 42 ... Carbon-based material, 43 ... pores, 51 ... electrodes, 52 ... fine particles, 53 ... aggregates

Claims (20)

A method for producing an enzyme-immobilized electrode in which an enzyme is immobilized on the electrode,
A method for producing an enzyme-immobilized electrode, wherein an organic solvent is contained in a solution used when the enzyme is immobilized on the electrode.   2. The method for producing an enzyme-immobilized electrode according to claim 1, wherein the organic solvent is an alcohol.   The method for producing an enzyme-immobilized electrode according to claim 1, wherein an electron mediator is immobilized on the electrode in addition to the enzyme. An enzyme-immobilized electrode in which an enzyme is immobilized on the electrode,
An enzyme-immobilized electrode produced by using an organic solvent in a solution used when immobilizing the enzyme on the electrode. A method for producing a fuel cell, wherein a cathode electrode and an anode electrode have a structure facing each other via a proton conductor, and an enzyme is immobilized on the cathode electrode and / or the anode electrode,
A method for producing a fuel cell, characterized in that an organic solvent is included in a solution used when the enzyme is immobilized on the cathode electrode and / or the anode electrode. A fuel cell having a structure in which a cathode electrode and an anode electrode are opposed to each other via a proton conductor, and an enzyme is immobilized on the cathode electrode and / or the anode electrode,
A fuel characterized in that the cathode electrode and / or the anode electrode is produced by using an organic solvent contained in a solution used when the enzyme is immobilized on the cathode electrode and / or the anode electrode. battery. A fuel cell having a structure in which a cathode electrode and an anode electrode are opposed to each other via a proton conductor, and an enzyme is immobilized on the cathode electrode and / or the anode electrode,
A fuel cell characterized in that an organic solvent is contained in the fuel. In an electronic device using one or more fuel cells,
At least one of the fuel cells is
A fuel cell having a structure in which a cathode electrode and an anode electrode are opposed to each other via a proton conductor, and an enzyme is immobilized on the cathode electrode and / or the anode electrode,
The cathode electrode and / or the anode electrode is produced by using an organic solvent contained in a solution used when the enzyme is immobilized on the cathode electrode and / or the anode electrode. Electronic equipment. In an electronic device using one or more fuel cells,
At least one of the fuel cells is
A fuel cell having a structure in which a cathode electrode and an anode electrode are opposed to each other via a proton conductor, and an enzyme is immobilized on the cathode electrode and / or the anode electrode,
An electronic device characterized by containing an organic solvent in fuel. A method for producing an enzyme reaction utilization device having an enzyme-immobilized electrode in which an enzyme is immobilized on the electrode,
A method for producing an enzyme reaction utilization device, wherein an organic solvent is included in a solution used when the enzyme is immobilized on the electrode.   An enzyme-immobilized electrode in which an aggregate containing an enzyme is immobilized on the electrode.   The enzyme-immobilized electrode according to claim 11, wherein the aggregate further contains an electron mediator.   The enzyme-immobilized electrode according to claim 11, wherein the aggregate further contains a coenzyme.   The enzyme-immobilized electrode according to claim 11, wherein the aggregate further contains a phospholipid or a derivative thereof.   The enzyme-immobilized electrode according to claim 11, wherein the aggregate comprises substantially spherical fine particles.   The enzyme-immobilized electrode according to claim 15, wherein the fine particles have a diameter of 1 µm or less.   The enzyme-immobilized electrode according to claim 15, wherein the fine particles are formed of a polyion complex.   A fuel cell having a structure in which a cathode electrode and an anode electrode face each other via a proton conductor, and an aggregate containing an enzyme is immobilized on the cathode electrode and / or the anode electrode.   An enzyme-immobilized electrode in which a layer containing an enzyme is fixed to the electrode, a void is provided in the layer, and a reactant or oxygen is supplied to the enzyme through the void.   The enzyme-immobilized electrode according to claim 19, wherein the gap is substantially spherical and the diameter of the gap is 1 µm or less.

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