JP2007012281A - Fuel cell, electronic equipment, moving body, power generation system, and enzyme reaction utilization device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fuel cell capable of generating electric power efficiently by using an NADH solution. <P>SOLUTION: In the fuel cell having a structure in which a negative electrode 1 and a positive electrode 2 are opposed via a proton conductor 3, an enzyme/electron mediator fixing electrode in which diaphorase 12 and an electron mediator 13 are fixed on an electrode 11 such as carbon is employed as the negative electrode 1, and the NADH solution is used as fuel. The concentration of the NADH solution is 2 to 130 mM, and preferably 3 to 40 mM. Instead of the NADH solution, an NADPH solution or a solution containing NADH and NADPH may be used. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a fuel cell using an enzyme as a catalyst, an electronic device using the fuel cell, a moving body and a power generation system, and an enzyme reaction utilization device such as a fuel cell.

A fuel cell basically has a structure in which a negative electrode (fuel electrode) and a positive electrode (oxidant electrode) face each other with a proton conductor interposed therebetween. In a conventional fuel cell, the fuel (hydrogen) supplied to the negative electrode is oxidized and separated into electrons and protons (H ⁺ ), the electrons are passed to the negative electrode, and H ⁺ moves through the proton conductor to the positive electrode. To do. In the positive electrode, this H ⁺ reacts with oxygen supplied from the outside and electrons sent from the negative electrode through the external circuit to generate H ₂ O.

  In this way, a fuel cell is a highly efficient power generator that directly converts the chemical energy of fuel into electrical energy, and the chemical energy of fossil energy such as natural gas, oil, and coal can be used regardless of where and when it is used. Moreover, it can be extracted as electrical energy with high conversion efficiency. For this reason, research and development of fuel cells for large-scale power generation has been actively conducted. For example, a fuel cell is mounted on the space shuttle, and there is a track record of proving that it is possible to replenish crew water simultaneously with the generation of electric power and that it is a clean power generator.

  Furthermore, in recent years, fuel cells having a relatively low operating temperature range from room temperature to about 90 ° C., such as solid polymer fuel cells, have been developed and attracting attention. For this reason, not only large-scale power generation applications but also applications to small systems such as automobile power supplies and portable power supplies such as personal computers and mobile devices are being sought.

  Thus, the fuel cell can be used in a wide range of applications from large-scale power generation to small-scale power generation, and has attracted much attention as a highly efficient power generation device. However, in fuel cells, natural gas, petroleum, coal, etc. are usually used as fuel by converting them into hydrogen gas using a reformer, which consumes limited resources and needs to be heated to a high temperature. There are various problems such as the need for expensive noble metal catalysts such as (Pt). Even when hydrogen gas or methanol is used directly as a fuel, care must be taken when handling it.

  Accordingly, attention has been paid to the fact that biological metabolism performed in living organisms is a highly efficient energy conversion mechanism, and proposals have been made to apply this to fuel cells. The biological metabolism here includes respiration, photosynthesis and the like performed in microbial somatic cells. Biological metabolism has the characteristics that the power generation efficiency is extremely high and the reaction proceeds under mild conditions of about room temperature.

For example, respiration takes nutrients such as sugars, fats, and proteins into microorganisms or cells, and these chemical energies are converted to carbon dioxide (glycolytic and tricarboxylic acid (TCA) cycles through a number of enzymatic reaction steps. In the process of generating CO ₂ ), nicotinamide adenine dinucleotide (NAD ⁺ ) is reduced to reduced nicotinamide adenine dinucleotide (NADH) to convert it into redox energy, that is, electric energy, and further, an electron transfer system In this mechanism, the electric energy of NADH is directly converted into electric energy of proton gradient, and oxygen is reduced to generate water. The electrical energy obtained here produces ATP from adenosine diphosphate (ADP) via adenosine triphosphate (ATP) synthase, and this ATP is used for reactions necessary for the growth of microorganisms and cells. Used. Such energy conversion occurs in the cytosol and mitochondria.

In addition, photosynthesis captures light energy and reduces nicotinamide adenine dinucleotide phosphate (NADP ⁺ ) via an electron transfer system to reduce nicotinamide adenine dinucleotide phosphate (NADPH), thereby converting it into electrical energy. In the process of conversion, it is a mechanism that oxidizes water to produce oxygen. This electric energy takes in CO ₂ and is used for carbon fixation reaction, and is used for carbohydrate synthesis.

  As a technique for utilizing the above-described biological metabolism in a fuel cell, a microbial cell that obtains an electric current by taking out the electric energy generated in the microorganism through the electron mediator and passing the electrons to the electrode has been reported. (For example, refer to Patent Document 1).

  However, since microorganisms and cells have many unnecessary functions in addition to the intended reaction such as conversion from chemical energy to electrical energy, the above-described method consumes electrical energy for unwanted reactions, and sufficient energy conversion efficiency is achieved. Is not demonstrated.

Therefore, a fuel cell that performs only a desired reaction using an enzyme or an electron mediator has been proposed (see, for example, Patent Documents 2, 3, and 4). In this fuel cell, fuel is decomposed by an enzyme and separated into protons and electrons. In addition to alcohols such as methanol and ethanol and monosaccharides such as glucose, NADH is used as the fuel. Has been proposed (see, for example, Patent Document 4). NADH is advantageous because it can obtain a larger electric power than methanol or ethanol.
JP 2000-133297 A JP 2003-282124 A JP 2004-71559 A JP 2005-13210 A

However, when an NADH solution is used as a fuel in a fuel cell using an enzyme or an electron mediator, the optimum concentration of the NADH solution has not been known from the viewpoint of power generation efficiency.
Therefore, the problem to be solved by the present invention is that NADH or its related substance NADPH can be used for high-efficiency power generation, and it is not necessary to use limited fossil fuels, contributing to the realization of a resource recycling society. A fuel cell that can be used, and an electronic device, a moving body, and a power generation system using the fuel cell.
Another problem to be solved by the present invention is, more generally, to provide various enzyme reaction utilization apparatuses capable of high-efficiency operation using NADH or NADPH.

As a result of diligent research to solve the above-mentioned problems of the prior art, the inventors of the present invention used a diaphorase as an enzyme and a NADH solution as a fuel in a fuel cell that decomposes fuel with an enzyme to generate electricity. It has been found that a change in the reaction rate of the enzyme reaction when the concentration of the NADH solution is increased causes an unexpected specific behavior. Specifically, when the NADH concentration is increased, the reaction rate usually increases monotonously and tends to saturate (changes indicated by the Michaelis-Menten equation), but in reality. It has been found that a steep peak is taken at the NADH concentration, and that this value becomes substantially constant after the peak falls. In order to improve the power generation efficiency, it is normal to increase the NADH concentration. However, in reality, it is not so. If the NADH concentration is increased too much, the reaction rate decreases. It turns out that the efficiency decreases. This means that substrate inhibition occurs when the NADH concentration exceeds the NADH concentration corresponding to the above peak.
Thus, it was found that there is an optimum range for the NADH concentration from the viewpoint of improving the reaction rate and thus the power generation efficiency, and this optimum range was actually found.
On the other hand, diaphorase is an enzyme that oxidizes NADPH as a substrate in addition to NADH. NADH has a structure as shown in FIG. 9A, whereas NADPH has one phosphate group at the adenosine site of NADH as shown in FIG. 9B, and is very similar to the structure of NADH. Yes. For this reason, NADPH or a mixture of NADH and NADPH is also considered to cause substrate inhibition in the reaction with diaphorase, similarly to NADH.

The present invention has been devised based on the above research results.
That is, in order to solve the above problem,
The first invention is
A fuel cell having a structure in which a positive electrode and a negative electrode are opposed via a proton conductor, the negative electrode contains at least diaphorase and an electron mediator, and uses a reduced nicotinamide adenine dinucleotide solution as a fuel,
A curve showing a change in the reaction rate of the enzyme reaction by diaphorase with respect to the concentration of the reduced nicotinamide adenine dinucleotide solution has a peak shape,
The concentration of the reduced nicotinamide adenine dinucleotide solution is 2 mM or more, and is equal to or lower than the concentration of the reduced nicotinamide adenine dinucleotide solution where the peak falls.

Diaphorase and electron mediator may be present in the form of a solution together with reduced nicotinamide adenine dinucleotide, but in order to efficiently capture the enzyme reaction phenomenon occurring near the negative electrode as an electrical signal, it is preferable. Is immobilized on the negative electrode using an immobilizing material.
In addition to the reduced nicotinamide adenine dinucleotide, one containing one or two or more other fuels may be used as the fuel. In that case, one or two or more other fuels that decompose these other fuels may be used. The enzyme is included in the negative electrode. As another fuel, for example, glucose can be used. In this case, NAD ⁺ -dependent glucose dehydrogenase is included in the negative electrode as another enzyme. Polysaccharides can also be used as other fuels. In this case, glucoamylase and NAD ⁺ dependent glucose dehydrogenase, which are enzymes that decompose the polysaccharides into glucose, are included in the negative electrode as other enzymes. Polysaccharides (polysaccharides in a broad sense, which refers to all carbohydrates that produce two or more monosaccharides by hydrolysis, including oligosaccharides such as disaccharides, trisaccharides, and tetrasaccharides) include, for example, starch, amylose , Amylopectin, glycogen, cellulose, maltose, sucrose, lactose and the like. These are a combination of two or more monosaccharides, and in any polysaccharide, glucose is contained 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.

The second invention is
The negative electrode has a structure in which a positive electrode and a negative electrode are opposed via a proton conductor, and the negative electrode contains at least diaphorase and an electron mediator, and reduced nicotinamide adenine dinucleotide and / or reduced nicotinamide adenine dinucleotide phosphorus as a fuel A fuel cell using a solution containing an acid,
The concentration of the solution containing the reduced nicotinamide adenine dinucleotide and / or the reduced nicotinamide adenine dinucleotide phosphate is 2 mM or more and 130 mM or less.
Here, the concentration of the solution containing reduced nicotinamide adenine dinucleotide and / or reduced nicotinamide adenine dinucleotide phosphate is preferably from the viewpoint of suppressing fuel consumption and further improving power generation efficiency. 2 mM or more and 80 mM or less, more preferably 3 mM or more and 40 mM or less, and further preferably 5 mM or more and 20 mM or less.

  The fuel cells according to the first and second inventions can be used for almost all of the devices that require electric power, and may be of any size. For example, electronic devices, mobile objects (automobiles, motorcycles, airplanes, rockets, spacecrafts, etc.) ), Power equipment, construction machines, machine tools, power generation systems, cogeneration systems, and the like, and the output, size, shape, type of fuel, etc. are determined depending on the application.

The third invention is
In electronic devices using fuel cells,
The fuel cell is
A fuel cell having a structure in which a positive electrode and a negative electrode are opposed via a proton conductor, the negative electrode contains at least diaphorase and an electron mediator, and uses a reduced nicotinamide adenine dinucleotide solution as a fuel,
A curve showing a change in the reaction rate of the enzyme reaction by diaphorase with respect to the concentration of the reduced nicotinamide adenine dinucleotide solution has a peak shape,
The concentration of the reduced nicotinamide adenine dinucleotide solution is 2 mM or more, and is equal to or lower than the concentration of the reduced nicotinamide adenine dinucleotide solution where the peak falls.

The fourth invention is:
In electronic devices using fuel cells,
The fuel cell is
The negative electrode has a structure in which a positive electrode and a negative electrode are opposed via a proton conductor, and the negative electrode contains at least diaphorase and an electron mediator, and reduced nicotinamide adenine dinucleotide and / or reduced nicotinamide adenine dinucleotide phosphorus as a fuel A fuel cell using a solution containing an acid,
The concentration of the solution containing the reduced nicotinamide adenine dinucleotide and / or the reduced nicotinamide adenine dinucleotide phosphate is 2 mM or more and 130 mM or less.

The electronic device according to the third and fourth inventions may be basically any device, and includes both a portable type and a stationary type. For example, a mobile phone Mobile devices, robots, personal computers, game devices, in-vehicle devices, home appliances, industrial products, and the like.
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:
In a mobile body using a fuel cell,
The fuel cell is
A fuel cell having a structure in which a positive electrode and a negative electrode are opposed via a proton conductor, the negative electrode contains at least diaphorase and an electron mediator, and uses a reduced nicotinamide adenine dinucleotide solution as a fuel,
A curve showing a change in the reaction rate of the enzyme reaction by diaphorase with respect to the concentration of the reduced nicotinamide adenine dinucleotide solution has a peak shape,
The concentration of the reduced nicotinamide adenine dinucleotide solution is 2 mM or more, and is equal to or lower than the concentration of the reduced nicotinamide adenine dinucleotide solution where the peak falls.

The sixth invention is:
In a mobile body using a fuel cell,
The fuel cell is
The negative electrode has a structure in which a positive electrode and a negative electrode are opposed via a proton conductor, and the negative electrode contains at least diaphorase and an electron mediator, and reduced nicotinamide adenine dinucleotide and / or reduced nicotinamide adenine dinucleotide phosphorus as a fuel A fuel cell using a solution containing an acid,
The concentration of the solution containing the reduced nicotinamide adenine dinucleotide and / or the reduced nicotinamide adenine dinucleotide phosphate is 2 mM or more and 130 mM or less.

The mobile body according to the fifth and sixth inventions may be basically any type, and specific examples include automobiles, motorcycles, aircraft, rockets, spacecrafts, and the like.
In the fifth and sixth inventions, what has been described in relation to the first and second inventions holds true for matters other than those described above, unless they are contrary to their properties.

The seventh invention
In a power generation system using a fuel cell,
The fuel cell is
A fuel cell having a structure in which a positive electrode and a negative electrode are opposed via a proton conductor, the negative electrode contains at least diaphorase and an electron mediator, and uses a reduced nicotinamide adenine dinucleotide solution as a fuel,
A curve showing a change in the reaction rate of the enzyme reaction by diaphorase with respect to the concentration of the reduced nicotinamide adenine dinucleotide solution has a peak shape,
The concentration of the reduced nicotinamide adenine dinucleotide solution is 2 mM or more, and is equal to or lower than the concentration of the reduced nicotinamide adenine dinucleotide solution where the peak falls.

The eighth invention
In a power generation system using a fuel cell,
The fuel cell is
The negative electrode has a structure in which a positive electrode and a negative electrode are opposed via a proton conductor, and the negative electrode contains at least diaphorase and an electron mediator, and reduced nicotinamide adenine dinucleotide and / or reduced nicotinamide adenine dinucleotide phosphorus as a fuel A fuel cell using a solution containing an acid,
The concentration of the solution containing the reduced nicotinamide adenine dinucleotide and / or the reduced nicotinamide adenine dinucleotide phosphate is 2 mM or more and 130 mM or less.

The power generation system according to the seventh and eighth inventions may be basically any type, and its scale is not limited.
In the seventh and eighth inventions, what has been described in relation to the first and second inventions holds true for matters other than those described above, unless they are contrary to their properties.

The ninth invention
At least a reduced nicotinamide adenine dinucleotide solution, diaphorase and an electron mediator,
A curve showing a change in the reaction rate of the enzyme reaction by diaphorase with respect to the concentration of the reduced nicotinamide adenine dinucleotide solution has a peak shape,
The enzyme reaction utilization apparatus, wherein the concentration of the reduced nicotinamide adenine dinucleotide solution is 2 mM or more and is equal to or lower than the concentration of the reduced nicotinamide adenine dinucleotide solution at which the peak falls.

The tenth invention is
At least a solution containing reduced nicotinamide adenine dinucleotide and / or reduced nicotinamide adenine dinucleotide phosphate, diaphorase and an electron mediator,
The enzyme reaction utilization apparatus, wherein the concentration of the solution containing the reduced nicotinamide adenine dinucleotide and / or the reduced nicotinamide adenine dinucleotide phosphate is 2 mM or more and 130 mM or less.

In the ninth and tenth inventions, the enzyme reaction utilization device includes various devices utilizing an enzyme reaction, and specifically, is a fuel cell or a biosensor.
In the ninth and tenth inventions, what has been described in relation to the first and second inventions holds true for matters other than those described above, unless they are contrary to the nature thereof.

  In the present invention configured as described above, by setting the concentration of the NADH solution or NADPH solution or the solution containing both NADH and NADPH in the above range, electric energy can be efficiently extracted from this fuel. it can.

  According to the present invention, it is possible to obtain a fuel cell capable of generating power with high efficiency by using an enzyme as a catalyst and using a NADH solution or NADPH solution or a solution containing both of them as a fuel. According to this fuel cell, not only the amount of fuel consumed can be suppressed, but also limited fossil fuel can be used, which can contribute to the realization of a resource recycling society. By using such an excellent fuel cell, it is possible to realize a high-performance electronic device, a moving body, a power generation system, and the like.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 shows a fuel cell according to a first embodiment of the present invention.
As shown in FIG. 1, the fuel cell has a structure in which a negative electrode 1 and a positive electrode 2 face each other with a proton conductor 3 interposed therebetween. In the negative electrode 1, NADH supplied as fuel is oxidized to NAD ⁺ and two electrons and protons (H ⁺ ) are generated. The proton conductor 3 conducts only protons and transports protons generated at the negative electrode 1 to the positive electrode 2. In the positive electrode 2, water is generated by protons transported from the negative electrode 1 to the positive electrode 2, electrons sent from the negative electrode 2 through an external circuit, and oxygen (O ₂ ) in the air, for example.

  The negative electrode 1 includes, for example, an electrode made of carbon or the like, diaphorase that is an oxidase involved in the decomposition of NADH, and an electron mediator. The electron mediator transfers electrons to and from the electrode, and the voltage of the fuel cell depends on the redox potential of the electron mediator. In other words, in order to obtain a higher voltage, it is better to select an electron mediator having a more negative potential on the negative electrode 1 side. However, the reaction affinity of the electron mediator to the enzyme, the rate of electron exchange with the electrode, an inhibitor (light, oxygen, etc.) ) Structural stability must also be considered. From such a viewpoint, examples of the electron mediator that acts on the negative electrode 1 include 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ), 2-amino-1,4-naphthoquinone (ANQ), vitamin K3, and the like. preferable. In addition, for example, compounds having a quinone skeleton, metal complexes such as Os, Ru, Fe and Co, viologen compounds such as benzyl viologen, compounds having a nicotinamide structure, compounds having a riboflavin structure, compounds having a nucleotide-phosphate structure Etc. can also be used as electron mediators.

  The above-mentioned diaphorase and electron mediator are maintained at a pH optimum for the enzyme, for example, around pH 7, by a buffer solution such as Tris buffer or phosphate buffer so that the electrode reaction can be carried out efficiently and constantly. It is preferable. The ionic strength (I.S.) is too large or too small to adversely affect the enzyme activity. However, considering the electrochemical response, the ionic strength (IS) 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.

  The diaphorase and the electron mediator may be used in a state dissolved in a buffer solution. However, in order to efficiently capture the enzyme reaction phenomenon occurring in the vicinity of the electrode as an electric signal, the immobilization material is used on the negative electrode 1. It is preferably immobilized. FIG. 2 shows a state in which an electron mediator 12 and diaphorase 13 are immobilized on an electrode 11 made of, for example, carbon or the like by an immobilizing material (not shown). As such an immobilizing material, for example, a combination of glutaraldehyde (GA) and poly-L-lysine (PLL) or a combination of sodium polyacrylate (PAAcNa) and poly-L-lysine (PLL). These may be used, these may be used alone, or other polymers may be used. By using an immobilization material combining glutaraldehyde and poly-L-lysine, it becomes possible to greatly improve the enzyme immobilization ability of each, and to obtain an excellent enzyme immobilization ability as a whole immobilization material. Can do. In this case, the optimum composition ratio between glutaraldehyde and poly-L-lysine varies depending on the enzyme to be immobilized and the substrate of the enzyme, but generally an arbitrary composition ratio may be used. As a specific example, a glutaraldehyde aqueous solution (0.125%) and a poly-L-lysine aqueous solution (1%) are used, and the ratio thereof is 1: 1, 1: 2, 2: 1, or the like.

  The positive electrode 2 is made of, for example, carbon powder on which a catalyst is supported or catalyst particles that are not held by carbon. Examples of the catalyst include fine particles of platinum (Pt), fine particles of an alloy or oxide of a transition metal such as iron (Fe), nickel (Ni), cobalt (Co), or ruthenium (Ru) and platinum. Used. For example, the positive electrode 2 is formed in a structure in which a catalyst layer made of a catalyst or a carbon powder containing a catalyst and a gas diffusion layer made of a porous carbon material are laminated in order from the proton conductor 3 side. The positive electrode 2 may be configured to include an oxygen reductase as a catalyst. As this oxygen reductase, for example, bilirubin oxidase can be used. In this case, it is used in combination with an electron mediator that transfers electrons to and from the electrode. As this electron mediator, for example, hexacyanoferrate ion or the like can be used. Various materials can be used as the immobilizing material, and preferably, the above-described poly-L-lysine (PLL) is used.

The proton conductor 3 is a proton conductive membrane that transports H ⁺ generated in the negative electrode 1 to the positive electrode 2 and is made of a material that does not have electron conductivity and can transport H ⁺ . Examples of the proton conductor 3 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 ketone sulfonic acid film. , PSSA-PVA (polystyrene sulfonate polyvinyl alcohol copolymer), PSSA-EVOH (polystyrene sulfonate ethylene vinyl alcohol copolymer), and the like. Among these, those made of an ion exchange resin having a fluorine-containing carbon sulfonic acid group are preferable, and specifically, Nafion (trade name, US DuPont) is used.

3 and 4 show a specific configuration example of the fuel cell, and FIG. 4 is an exploded view of each component of the fuel cell.
As shown in FIGS. 3 and 4, this fuel cell includes a negative electrode 1 composed of an enzyme / electron mediator-immobilized electrode in which an enzyme (diaphorase) and an electron mediator are immobilized on a porous carbon or the like by an immobilizing material, For example, a structure in which a positive electrode 2 composed of an enzyme / electron mediator-immobilized electrode in which an enzyme (for example, bilirubin oxidase) and an electron mediator are immobilized on a porous carbon or the like is opposed via a proton conductor 3. Have.

  Current collectors 4 and 5 made of, for example, Ti are installed above and below the proton conductor 3, respectively, so that current collection can be performed easily. Reference numerals 6 and 7 denote fixed plates. These fixing plates 6 and 7 are fastened to each other by screws 8, and the whole of the negative electrode 1, the positive electrode 2, the proton conductor 3 and the current collectors 4 and 5 is sandwiched between them. A circular hole 6a for fuel loading is provided on one surface (outer surface) of the fixed plate 6. Reference numeral 6b denotes a hole into which the screw 8 is inserted. One surface (outer surface) of the fixing plate 7 is provided with a circular hole 7a for taking in air or oxygen. Reference numeral 7b indicates a hole into which the screw 8 is inserted. A spacer 9 is provided in the periphery of the other surface of the fixing plate 6, and when the fixing plates 6 and 7 are fastened to each other by the screws 8, the interval between them becomes a predetermined interval. A silicon plate or the like is used as the spacer 9.

In this fuel cell, as shown in FIG. 3, a load 10 is connected between the current collectors 4, 5, NADH solution is put as fuel in the hole 6 a of the fixing plate 6, and Or generate oxygen by taking in oxygen. In this case, in the negative electrode 1, NADH is oxidized by diaphorase and separated into two electrons, NAD ⁺ and H ⁺ , which are passed to the negative electrode 1, and H ⁺ passes through the proton conductor 3 and is positive electrode 2. Move up. In the positive electrode 2, this H ⁺ reacts with oxygen supplied from the outside and electrons sent from the negative electrode 1 through an external circuit to generate H ₂ O.

Next, the result of electrochemical measurement at a single electrode of the enzyme / electron mediator immobilized electrode used as the negative electrode 1 will be described.
The electrochemical measurement system used is shown in FIG. As shown in FIG. 5, the solution 52 was put in the container 51, the working electrode 53, the counter electrode 54, and the reference electrode 55 were immersed in the solution 52, and the electrochemical measurement device 56 was connected to the working electrode 53. The working electrode 53 is fixed on a glassy carbon disk electrode (3 mmφ, 0.071 cm ² ) using an immobilizing material in which diaphorase and ANQ are combined with glutaraldehyde (GA) and poly-L-lysine (PLL). What was made into was used. Pt line was used as the counter electrode 54 and Ag | AgCl was used as the reference electrode 55. The measurement was performed under atmospheric pressure, and the measurement temperature was 25 ° C. The amount of the solution 52 was 1.0 ml, and before the measurement, Ar gas was sent into the solution 52 by a bubbler 57 to perform sufficient bubbling to perform deoxygenation.

The enzyme / electron mediator immobilized electrode was prepared as follows.
First, various solutions were prepared as follows. As a buffer solution, a 100 mM sodium dihydrogen phosphate (NaH ₂ PO ₄ ) buffer solution (IS = 0.3, pH = 8.0) was used.
Diaphorase (abbreviated as DI) (EC1.6.99.-, Amanoenzyme, DH-3) was weighed 4.76 mg and dissolved in 1.0 ml of a buffer solution to obtain a DI enzyme buffer solution ((1)). The buffer solution for dissolving the enzyme is preferably refrigerated until just before, and the enzyme buffer solution is preferably stored as refrigerated as much as possible. A suitable amount of poly-L-lysine hydrobromide (abbreviated as PLL) (Aldrich, P-1399) was weighed and dissolved in ion-exchanged water so as to be 2.0 wt%, and an aqueous PLL solution ((2)) did. 10.4 mg of ANQ was weighed and dissolved in 1.0 ml of an acetone solution to obtain an ANQ acetone solution ((3)). An appropriate amount of glutaraldehyde (abbreviated as GA) (Kanto Chemical Co., Inc., 17026-02) was weighed and dissolved in ion-exchanged water so as to be 0.125 wt% to obtain an aqueous GA solution ((4)).

Various solutions prepared as described above were applied onto a glassy carbon disk electrode (3 mmφ, 0.071 cm ² ) with a microsyringe in the order from the top to the bottom of Table 1 below, mixed, and dried at room temperature. Two types of enzyme / electron mediator immobilized electrodes (samples 1 and 2) having different enzyme amounts were prepared.

On the other hand, 709 mg of NADH (Sigma Aldrich, N-8129) was weighed and dissolved in 1.0 ml of a buffer solution to obtain a NADH buffer solution ((5)).
Then, the above buffer solution is used as the solution 52, and the NADH buffer solution ((5)) is gradually added to the solution 52 under the condition that the enzyme / electron mediator immobilized electrode is the working electrode 53 and the electrode rotation speed is 500 rpm. Thus, cyclic voltammetry was performed with the NADH concentration varied from 0 to 167 mM. The scan speed was 10 mVs ⁻¹ . FIG. 6 shows a plot of the current value at 0.4 V, which is a potential sufficiently higher than the redox potential of the electron mediator, as a steady-state current value I from the cyclic voltammetry. The amount of enzyme was set at two levels of 5 U (sample 1) and 10 U (sample 2). 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.

  FIG. 6 shows that the steady-state current value of the enzyme / electron mediator immobilized electrode has a peak when the NADH concentration ([NADH]) is around 10 mM. It can also be seen that the steady-state current value gradually decreases when the NADH concentration is higher than 10 mM. This is considered to be due to substrate inhibition in which NADHs compete for the active site of diaphorase. In FIG. 6, the NADH concentration at which the peak has fallen, specifically, in the concentration range exceeding about 130 mM, the steady current value obtained is low, and it is useless to simply consume the fuel. Therefore, the NADH concentration Is preferably 130 mM or less. On the other hand, since the peak rises steeply, a sufficiently high steady-state current value can be obtained if the NADH concentration is 2 mM or more. In order to simultaneously suppress the fuel consumption while obtaining a steady current value of a certain level or more, the NADH concentration is preferably 2 to 80 mM, more preferably 3 to 40 mM, and even more preferably 5 to 20 mM.

  FIG. 7 shows cyclic voltammetry by changing the NADH concentration from 0 to 167 mM by gradually adding the NADH buffer solution ((5)) to the solution 52 under the condition where the electrode rotation speed is 0 rpm (condition where the electrode is stationary). The result of having performed is shown together with said result (FIG. 6) measured on the conditions of electrode rotation speed 500rpm. The conditions other than the electrode rotation speed are the same in both cases. From FIG. 7, it can be seen that at the electrode rotation speed of 0 rpm, the peak position is shifted to the higher concentration side by about 30 mM as compared with the electrode rotation speed of 500 rpm, but the peak shape is the same. In this case, the NADH concentration is preferably 130 mM or less, more preferably 7 mM or more. In order to simultaneously suppress the fuel consumption while obtaining a steady current value of a certain level or more, the NADH concentration is preferably 7 to 90 mM, more preferably 10 to 80 mM, and even more preferably 20 to 60 mM.

The results shown in FIG. 6 above are obtained when the enzyme / electron mediator-immobilized electrode is used, but similar results can be obtained when using a solution system containing the enzyme and electron mediator dissolved. This will be described.
A solution containing 87 ng / ml diaphorase and 2.2 mM ANQ was prepared using a 100 mM sodium dihydrogen phosphate buffer solution (I.S. = 0.3, pH = 8.0) as a solvent. In this solution, ANQ is present in a sufficient concentration (K _{M, ANQ} about 10 times). Prior to the measurement, Ar gas was sent into the solution by a bubbler and bubbling was performed for 15 minutes to sufficiently deoxygenate. Then, the NADH buffer solution ((5)) similar to the above is gradually added to the above solution to change the NADH concentration within the range of 0 to 500 mM, thereby reacting the oxidation reaction of NADH with diaphorase. The speed was determined from the change in absorbance of ANQ. The measurement was performed under atmospheric pressure, and the measurement temperature was 25 ° C. The measurement wavelength of absorbance was 520 nm. FIG. 8A shows a reaction rate (v ₀ ) -NADH concentration ([NADH] ₀ ) curve of the oxidation reaction of NADH by diaphorase thus measured, and FIG. 8B is an enlarged view of the low concentration portion (0 to 35 mM) of FIG. 8A. Is. 8A and 8B, it can be seen that in this solution system, the NADH concentration has a peak around 25 mM, and the reaction rate gradually decreases on the higher concentration side. From this, it was confirmed that NADH substrate inhibition also occurred for diaphorase in the solution system. In this case, the NADH concentration is preferably 2 to 300 mM, more preferably 5 to 200 mM, and still more preferably 10 to 100 mM in order to suppress the fuel consumption while obtaining a reaction rate of a certain level or more.

As described above, according to the first embodiment, by optimizing the concentration of the NADH solution, the efficiency of the fuel cell can be greatly improved, and the fuel consumption can be suppressed.
This fuel cell is suitable for use as a power source for various electronic devices such as mobile phones and other various devices or devices.

Next explained is a fuel cell according to the second embodiment of the invention.
In this fuel cell, glucose is used as fuel in addition to NADH. In addition, with the use of this glucose, the negative electrode 1 also contains NAD ⁺ -dependent glucose dehydrogenase, which is a glucose degrading enzyme. This NAD ⁺ -dependent glucose dehydrogenase is also preferably immobilized on the negative electrode 1.
In this fuel cell, when a NADH solution containing glucose is supplied to the negative electrode 1 side, this glucose is decomposed by NAD ⁺ -dependent glucose dehydrogenase, and NAD ⁺ is reduced along with an oxidation reaction in this decomposition process, and NADH This NADH 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 generated electrons are transferred to the negative electrode 1, and H ⁺ moves to the positive electrode 2 through the proton conductor 3. In the positive electrode 2, this H ⁺ reacts with oxygen supplied from the outside and electrons sent from the negative electrode 1 through an external circuit to generate H ₂ O.
The fuel cell other than the above is the same as the fuel cell according to the first embodiment.
According to the second embodiment, the same advantages as in the first embodiment can be obtained, and in addition to NADH, glucose is also used as fuel, so that only NADH is used as fuel. The advantage that the amount of power generation can be increased can be obtained.

Next explained is a fuel cell according to the third embodiment of the invention.
In this fuel cell, starch, which is a polysaccharide, is used as fuel in addition to NADH. In addition, with the use of starch, the negative electrode 1 includes glucoamylase, which is a starch degrading enzyme, and NAD ⁺ -dependent glucose dehydrogenase, which is a glucose degrading enzyme obtained by degrading starch by this glucoamylase. . These glucoamylase and NAD ⁺ -dependent glucose dehydrogenase are also preferably immobilized on the negative electrode 1.

In this fuel cell, when a NADH solution containing starch is supplied to the negative electrode 1 side, this starch is hydrolyzed into glucose by glucoamylase, and this glucose is further decomposed by NAD ⁺ -dependent glucose dehydrogenase. NAD ⁺ is reduced to produce NADH along with the oxidation reaction, and this NADH is oxidized by diaphorase to be 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 generated electrons are transferred to the negative electrode 1, and H ⁺ moves to the positive electrode 2 through the proton conductor 3. In the positive electrode 2, this H ⁺ reacts with oxygen supplied from the outside and electrons sent from the negative electrode 1 through an external circuit to generate H ₂ O.
The fuel cell other than the above is the same as the fuel cell according to the first embodiment.
According to the third embodiment, the same advantages as in the first embodiment can be obtained, and starch is also used as fuel in addition to NADH, so that only NADH is used as fuel. The advantage that the amount of power generation can be increased can be obtained.

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.

1 is a schematic diagram showing a fuel cell according to a first embodiment of the present invention. 1 is a schematic diagram showing a fuel cell according to a first embodiment of the present invention. It is a basic diagram which shows the specific structural example of the fuel cell by 1st Embodiment of this invention. It is a basic diagram which shows the specific structural example of the fuel cell by 1st Embodiment of this invention. It is a basic diagram which shows the measurement system used for the electrochemical measurement by the single electrode of the enzyme / electron mediator fixed electrode used for a negative electrode in the fuel cell by 1st Embodiment of this invention. It is a basic diagram which shows the NADH density | concentration dependence of the steady-state current value of the fuel cell by 1st Embodiment of this invention. It is a basic diagram which shows the NADH density | concentration dependence of the steady-state current value of the fuel cell by 1st Embodiment of this invention. It is a basic diagram which shows the NADH density | concentration dependence of the reaction rate of the oxidation reaction of NADH by a diaphorase in the case of using the NADH solution which diaphorase and the electron mediator melt | dissolved. It is a basic diagram which shows the structure of NADH and NADPH.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Negative electrode, 2 ... Positive electrode, 3 ... Proton conductor, 4, 5 ... Current collector, 6, 7 ... Fixed plate, 10 ... Load, 11 ... Electrode, 12 ... Electron mediator, 13 ... Diaphorase

Claims (15)

A fuel cell having a structure in which a positive electrode and a negative electrode are opposed via a proton conductor, the negative electrode contains at least diaphorase and an electron mediator, and uses a reduced nicotinamide adenine dinucleotide solution as a fuel,
A curve showing a change in the reaction rate of the enzyme reaction by diaphorase with respect to the concentration of the reduced nicotinamide adenine dinucleotide solution has a peak shape,
A fuel cell, wherein the concentration of the reduced nicotinamide adenine dinucleotide solution is 2 mM or more and not more than the concentration of the reduced nicotinamide adenine dinucleotide solution at which the peak falls.   The fuel cell according to claim 1, wherein the diaphorase and the electron mediator are immobilized on the negative electrode.   In addition to the reduced nicotinamide adenine dinucleotide, the fuel contains one or more other fuels and one or more other enzymes that decompose the fuel. Item 2. The fuel cell according to Item 1. 4. The fuel cell according to claim 3, wherein glucose is contained in the other fuel, and NAD ⁺ dependent glucose dehydrogenase is contained in the other enzyme. 4. The fuel cell according to claim 3, wherein the other fuel contains a polysaccharide, and the other enzyme contains an enzyme that decomposes the polysaccharide into glucose and an NAD ⁺ -dependent glucose dehydrogenase. The negative electrode has a structure in which a positive electrode and a negative electrode are opposed via a proton conductor, and the negative electrode contains at least diaphorase and an electron mediator, and reduced nicotinamide adenine dinucleotide and / or reduced nicotinamide adenine dinucleotide phosphorus as a fuel A fuel cell using a solution containing an acid,
A fuel cell, wherein the concentration of the solution containing reduced nicotinamide adenine dinucleotide and / or reduced nicotinamide adenine dinucleotide phosphate is 2 mM or more and 130 mM or less. In electronic devices using fuel cells,
The fuel cell is
A fuel cell having a structure in which a positive electrode and a negative electrode are opposed via a proton conductor, the negative electrode contains at least diaphorase and an electron mediator, and uses a reduced nicotinamide adenine dinucleotide solution as a fuel,
A curve showing a change in the reaction rate of the enzyme reaction by diaphorase with respect to the concentration of the reduced nicotinamide adenine dinucleotide solution has a peak shape,
An electronic device, wherein the concentration of the reduced nicotinamide adenine dinucleotide solution is 2 mM or more and is equal to or lower than the concentration of the reduced nicotinamide adenine dinucleotide solution at which the peak falls. In electronic devices using fuel cells,
The fuel cell is
The negative electrode has a structure in which a positive electrode and a negative electrode are opposed via a proton conductor, and the negative electrode contains at least diaphorase and an electron mediator, and reduced nicotinamide adenine dinucleotide and / or reduced nicotinamide adenine dinucleotide phosphorus as a fuel A fuel cell using a solution containing an acid,
An electronic device, wherein a concentration of the solution containing the reduced nicotinamide adenine dinucleotide and / or the reduced nicotinamide adenine dinucleotide phosphate is 2 mM or more and 130 mM or less. In a mobile body using a fuel cell,
The fuel cell is
A fuel cell having a structure in which a positive electrode and a negative electrode are opposed via a proton conductor, the negative electrode contains at least diaphorase and an electron mediator, and uses a reduced nicotinamide adenine dinucleotide solution as a fuel,
The curve showing the change in the reaction rate of the enzyme reaction by diaphorase with respect to the concentration of the reduced nicotinamide adenine dinucleotide container has a peak shape,
The moving body characterized in that the concentration of the reduced nicotinamide adenine dinucleotide solution is 2 mM or more and not more than the concentration of the reduced nicotinamide adenine dinucleotide solution at which the peak falls. In a mobile body using a fuel cell,
The fuel cell is
The negative electrode has a structure in which a positive electrode and a negative electrode are opposed via a proton conductor, and the negative electrode contains at least diaphorase and an electron mediator, and reduced nicotinamide adenine dinucleotide and / or reduced nicotinamide adenine dinucleotide phosphorus as a fuel A fuel cell using a solution containing an acid,
The moving body, wherein the concentration of the solution containing the reduced nicotinamide adenine dinucleotide and / or the reduced nicotinamide adenine dinucleotide phosphate is 2 mM or more and 130 mM or less. In a power generation system using a fuel cell,
The fuel cell is
A fuel cell having a structure in which a positive electrode and a negative electrode are opposed via a proton conductor, the negative electrode contains at least diaphorase and an electron mediator, and uses a reduced nicotinamide adenine dinucleotide solution as a fuel,
A curve showing a change in the reaction rate of the enzyme reaction by diaphorase with respect to the concentration of the reduced nicotinamide adenine dinucleotide solution has a peak shape,
A power generation system characterized in that the concentration of the reduced nicotinamide adenine dinucleotide solution is 2 mM or more and not more than the concentration of the reduced nicotinamide adenine dinucleotide solution where the peak falls. In a power generation system using a fuel cell,
The fuel cell is
The negative electrode has a structure in which a positive electrode and a negative electrode are opposed via a proton conductor, and the negative electrode contains at least diaphorase and an electron mediator, and reduced nicotinamide adenine dinucleotide and / or reduced nicotinamide adenine dinucleotide phosphorus as a fuel A fuel cell using a solution containing an acid,
The concentration of the solution containing the reduced nicotinamide adenine dinucleotide and / or the reduced nicotinamide adenine dinucleotide phosphate is 2 mM or more and 130 mM or less. At least a reduced nicotinamide adenine dinucleotide solution, diaphorase and an electron mediator,
A curve showing a change in the reaction rate of the enzyme reaction by diaphorase with respect to the concentration of the reduced nicotinamide adenine dinucleotide solution has a peak shape,
The enzyme reaction utilization apparatus, wherein the concentration of the reduced nicotinamide adenine dinucleotide solution is 2 mM or more and not more than the concentration of the reduced nicotinamide adenine dinucleotide solution at which the peak falls.   14. The enzyme reaction utilization apparatus according to claim 13, wherein the enzyme reaction utilization apparatus is a biosensor. At least a solution containing reduced nicotinamide adenine dinucleotide and / or reduced nicotinamide adenine dinucleotide phosphate, diaphorase and an electron mediator,
The enzyme reaction utilization apparatus, wherein the concentration of the solution containing the reduced nicotinamide adenine dinucleotide and / or the reduced nicotinamide adenine dinucleotide phosphate is 2 mM or more and 130 mM or less.

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