JP2012028181A - Enzyme electrode and fuel cell having the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an enzyme electrode and a biofuel cell using the same capable of enhancing efficiency of an enzyme reaction in the enzyme electrode and attaining higher output than the conventional one.SOLUTION: According to the present invention, an enzyme electrode and a fuel cell using the same are provided, in which a mixture including carbon fine particles, an enzyme, and a metal oxide including at least one of zirconium oxide and molybdenum oxide is fixed to a conductive substrate.

Description

  The present invention relates to an enzyme electrode and a fuel cell having an enzyme electrode on at least one of an anode electrode and a cathode electrode.

  In recent years, interest in fuel cells has increased as one of countermeasures against environmental problems and resource problems. A fuel cell is a device that directly converts chemical energy into electrical energy by supplying fuel and an oxidant to two electrically connected electrodes and causing the fuel to be oxidized electrochemically. A fuel cell that extracts energy electrochemically is not subject to the Carnot cycle, unlike thermal power generation, and thus exhibits high energy conversion efficiency. Commonly known types of fuel cells include polymer electrolyte fuel cells (PEFC), alkaline electrolyte fuel cells (AFC), phosphoric acid fuel cells (PAFC), direct methanol fuel cells (DMFC), etc. There is. These fuel cells often use platinum (Pt) as a catalyst. However, platinum is a very expensive material, which is one of the obstacles to the spread of fuel cells.

  On the other hand, biofuel cells are attracting attention as fuel cells that do not use platinum as a catalyst. A biofuel cell is a fuel cell that applies an in vivo metabolic mechanism. In a biofuel cell, an enzyme electrode in which an oxidoreductase is immobilized on at least one of an anode electrode and a cathode electrode is generally used. The enzyme is immobilized, for example, by applying a mixture of carbon fine particles and a binder to which an enzyme is added to a conductive electrode substrate.

Various attempts have been made to improve enzyme electrodes in order to improve the output of biofuel cells. For example, Patent Document 1 discloses that in a biocatalytic direct alcohol fuel cell, a metal oxide such as TiO ₂ is added to an enzyme electrode for the purpose of stabilizing an electron mediator that receives an electron generated by an enzyme reaction and transmits it to the electrode. Proposed.

JP2004165142A

  Carbon fine particles typified by carbon black have a large specific surface area and are useful as electrode materials, and are therefore often used for preparing enzyme electrodes. However, since carbon fine particles generally exhibit hydrophobicity and have a low affinity with an enzyme, it is difficult to carry a sufficient amount of the enzyme on the electrode as it is. Moreover, since the enzyme used for the electrode catalyst usually requires water for the reaction, it has been difficult to exhibit sufficient performance in a hydrophobic environment. Low output is listed as a problem of biofuel cells, and means for increasing the efficiency of enzyme reaction is required to improve the output of enzyme electrodes and biofuel cells.

The present inventor has found that in an enzyme electrode in which a mixture containing a specific metal oxide in addition to carbon fine particles and an enzyme is fixed to a conductive substrate, the enzyme reaction becomes efficient and the output is improved. The gist of the present invention is as follows.
(1) An enzyme electrode in which a mixture containing carbon fine particles, an enzyme, and a metal oxide containing at least one of zirconium oxide and molybdenum oxide is fixed to a conductive substrate.
(2) The enzyme electrode according to (1), comprising 15 to 130 mmol of metal oxide with respect to 1 g of carbon fine particles.

(3) Produced by preparing a slurry in which carbon fine particles and metal oxide are dispersed in a solvent, applying the slurry to a conductive substrate, and then immobilizing an enzyme. (1) or (2) The enzyme electrode according to 1.
(4) A fuel cell having the enzyme electrode according to any one of (1) to (3) on at least one of an anode electrode and a cathode electrode.
(5) preparing a slurry in which carbon fine particles and a metal oxide containing at least one of zirconium oxide and molybdenum oxide are dispersed in a solvent, applying the slurry to a conductive substrate, and then immobilizing an enzyme. A method for producing an enzyme electrode, comprising:

  ADVANTAGE OF THE INVENTION According to this invention, the enzyme electrode which the electrode output improved compared with the past can be provided. By using the enzyme electrode of the present invention, a biofuel cell with higher output than before can be provided.

It is the schematic of the fuel cell for evaluation used in order to evaluate an enzyme electrode. It is a graph which shows the result of the chronoamperometry measurement of the electrode of an Example and a comparative example. It is a graph which shows the relationship between the amount of enzyme fixed to the electrode, and the electric current value obtained by the chronoamperometry measurement. It is a graph which shows the result of the chronoamperometry measurement with and without an enzyme in an electrode containing molybdenum oxide. It is a graph which shows the relationship between molybdenum oxide mixing amount and output current.

  The enzyme electrode of the present invention is characterized in that a mixture containing carbon fine particles, an enzyme, and a metal oxide containing at least one of zirconium oxide and molybdenum oxide is fixed to a conductive substrate. The mixture may further contain a binder. As the binder, in addition to Teflon (registered trademark), a polymer organic compound solution typified by polyvinylidene fluoride (PVDF), or a dispersion solution of a resin having hydrogen ion conductivity such as Nafion (registered trademark) is used. Can be used. For example, in the case of an enzyme electrode using an enzyme having oxygen as a substrate, the binder preferably has water repellency (hydrophobic) in addition to the binding property for the purpose of securing an oxygen ventilation path. However, it is also possible to mix a hydrophilic binder with a substance exhibiting water repellency. In addition, the binder may be a substance showing conductivity or a substance showing non-conductivity. However, if an excessive amount of the binder is added, the carbon fine particles may be covered and micropores existing on the surface may be blocked, thereby inhibiting the adsorption of the enzyme. Therefore, it is preferable to minimize the amount used to improve the output of the enzyme electrode. The amount of the binder used is preferably 1 g or less, 0.9 g or less, 0.8 g or less, and more preferably 0.7 g or less with respect to 1 g of the carbon fine particles. In the mixture fixed on the conductive substrate, each component such as carbon fine particles, enzyme, and metal oxide may not be completely dispersed uniformly.

  Examples of the conductive base material used in the present invention include conductive carbonaceous materials such as graphite, carbon black, activated carbon, carbon cloth, carbon paper, and carbon felt, and metals such as gold, platinum, titanium, and nickel. Common things, such as a thing, can be used.

  Examples of the carbon fine particles used in the present invention include carbon black, activated carbon, nanoporous carbon having a nanoscale pore on the surface, carbon nanotube, carbon nanofiber, fullerene, and the like.

  As an enzyme used in the present invention, for example, a dehydrogenase or an oxidoreductase such as an oxidase can be used. Specific examples of the oxidoreductase include bilirubin oxidase (BOD), glucose dehydrogenase (GDH), fructose dehydrogenase (FDH), alcohol dehydrogenase (ADH), aldehyde dehydrogenase, glucose oxidase (GOD), alcohol oxidase (AOD), aldehyde oxidase , Formate dehydrogenase, formate oxidase, diaphorase, multi-copper oxidase and the like. Only 1 type may be used for an enzyme and it may use it in combination of 2 or more type.

  The enzyme electrode of the present invention can be used as either an anode or a cathode of a fuel cell. When the enzyme electrode of the present invention is used as an anode electrode, the enzyme is an enzyme that oxidizes a substrate. On the other hand, when the enzyme electrode of the present invention is used as a cathode electrode, the enzyme is an enzyme that reduces a substrate.

The metal oxide used in the present invention contains at least one of zirconium oxide (ZrO ₂ ) and molybdenum oxide (MoO ₂ or MoO ₃ ). The metal oxide used in the present invention may contain both zirconium oxide (ZrO ₂ ) and molybdenum oxide (MoO ₂ or MoO ₃ ), or may contain other metal oxides. Examples of metal oxides that can be contained in addition to zirconium oxide and molybdenum oxide include manganese oxide (MnO ₂ ), yttrium oxide (Y ₂ O ₃ ), titanium oxide (TiO ₂ ), iron oxide (Fe ₂ O ₃ ), and aluminum oxide. (Al ₂ O ₃ ) and the like. The metal oxide used in the present invention is preferably made of either zirconium oxide (ZrO ₂ ) and molybdenum oxide (MoO ₂ or MoO ₃ ), or from either zirconium oxide (ZrO ₂ ) or molybdenum oxide (MoO ₂ or MoO ₃ ). Become.

  When mixed with carbon fine particles, zirconium oxide and molybdenum oxide impart hydrophilicity (water retention) to carbon fine particles that are usually hydrophobic. As a result, the affinity between the electrode and the enzyme is improved, and the amount of enzyme immobilized on the electrode is considered to increase. Zirconium oxide and molybdenum oxide are considered to have an action of activating the enzyme fixed on the electrode and improving the output of the enzyme electrode. Zirconium oxide and molybdenum oxide, especially molybdenum oxide, are considered particularly suitable for enhancing the activity of oxidases, especially bilirubin oxidase (BOD). The metal oxide containing at least one of zirconium oxide and molybdenum oxide is used in the range of 15 to 130 mmol, more preferably 30 to 120 mmol, particularly 40 to 100 mmol, especially 50 to 80 mmol with respect to 1 g of carbon fine particles. It is preferable from the viewpoint.

  The enzyme electrode of the present invention is prepared, for example, by preparing a slurry in which carbon fine particles and metal oxide, and optionally a binder is dispersed in a solvent, applying the slurry to a conductive substrate, and evaporating the solvent. It can be produced by immobilizing an enzyme. Alternatively, if the enzyme can be prevented from being deactivated, the enzyme may be mixed with the slurry in advance, the enzyme-containing slurry is applied to the conductive substrate, and the solvent is evaporated to produce the enzyme electrode. . The solvent for preparing the slurry is not particularly limited as long as it can disperse carbon fine particles and can be removed by evaporation after applying the slurry to the conductive substrate. Specific examples of usable solvents include alcohol solvents such as 2-propanol, ethanol, and methanol, ketone solvents such as acetone, ether solvents such as tetrahydrofuran, aliphatic hydrocarbon solvents such as hexane, and toluene. And organic solvents such as aromatic hydrocarbon solvents such as water, and various aqueous buffer solutions such as sodium phosphate buffer and potassium phosphate buffer. Immobilization of the enzyme on the electrode is performed by applying a slurry containing carbon fine particles and a metal oxide to the conductive substrate, evaporating the solvent and drying the electrode, and then immersing the electrode in the enzyme solution. Alternatively, it can be carried out by applying or dropping the enzyme solution to the electrode and then allowing it to stand and physically adsorbing the enzyme contained in the solution to the electrode. Enzyme immobilization is not limited to physical adsorption, but is known as an enzyme immobilization method such as a chemical immobilization method using a cross-linking reaction using a chemical such as glutaraldehyde, or a polyion complex method using polyerucine or chitosan. Any technique that can be used. As the enzyme solution, for example, an enzyme solution dissolved in a buffer solution such as a potassium phosphate buffer can be used.

  The enzyme electrode of the present invention can be used as at least one of an anode electrode and a cathode electrode in a fuel cell. That is, the present invention also relates to a fuel cell (that is, a biofuel cell) having the enzyme electrode as described above on at least one of an anode and a cathode.

  When the enzyme electrode of the present invention is used as an anode electrode, a substance serving as a substrate for the reaction of the enzyme used for the electrode is supplied to the anode electrode side as a fuel. Specific examples of the fuel include alcohols such as methanol and ethanol, aldehydes such as acetaldehyde, carboxylic acids such as formic acid and acetic acid, and sugars such as glucose and fructose. The fuel can be appropriately selected according to the enzyme immobilized on the electrode, and is supplied into the system in the form of an aqueous solution, for example. On the other hand, the cathode electrode takes in oxygen from the atmosphere and produces water. The configuration of the fuel cell of the present invention can be a configuration known to those skilled in the art other than the enzyme electrode.

  The fuel cell of the present invention may further use an electronic mediator. The electron mediator means a substance that can exchange electrons with an enzyme or a coenzyme, and can also exchange electrons with a conductive substrate. Examples of compounds that can be used as electron mediators include, for example, metal elements such as Os, Fe, Ru, Co, Cu, Ni, V, Mo, Cr, Mn, Pt, and W or ions of these metals as central metals. Metal complexes (ferrocene, potassium ferricyanide, lithium ferricyanide, alkali metal ferricyanide such as sodium ferricyanide, or alkyl substitutions thereof (methyl substitution, ethyl substitution, propyl substitution, etc.), octacyano Quinones such as quinone, benzoquinone, anthraquinone and naphthoquinone; heterocyclic compounds such as viologen, methyl viologen, benzyl viologen, phenazine methosulfate, phenazine etsulfate, bipyridine and derivatives thereof; , 2,6-dichlorophenol indophenol, methylene blue, potassium β- naphthoquinone-4-sulfonic acid, and the like vitamins K. The electron mediator may be fixed to the enzyme electrode together with the enzyme, or may be added to the fuel solution.

  In the fuel cell of the present invention, a coenzyme that further assists the function of the enzyme may be used. Examples of coenzymes include nicotinamide adenine dinucleotide (oxidized form: NAD, reduced form: NADH, also referred to as NAD / NADH), nicotinamide adenine dinucleotide phosphate (oxidized form: NADP, reduced form: NADPH, NADP / NADPH). As well as cytochromes and quinones (for example, pyrroloquinoline quinone). Among these, NAD / NADH or NADP / NADPH (also collectively referred to as NAD (P) / NAD (P) H) that functions as a coenzyme for various enzymes is particularly preferable. The coenzyme may be fixed to the enzyme electrode together with the enzyme, or may be added to the fuel solution.

  In addition to the fuel cell, the enzyme electrode of the present invention can be used as an electrode for a biosensor, for example. Since the high output is obtained when the enzyme electrode of the present invention is used, it is advantageous in such applications.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not limited to these Examples.
1. Preparation of enzyme-immobilized electrode (1) Preparation of carbon fine particle slurry A carbon fine particle slurry was prepared using an ultrasonic crusher. Teflon (registered trademark) was used as the binder. Each component was weighed into a glass vial in the following amounts, and sonicated until it was sufficiently dispersed. Table 1 shows the slurry composition subjected to ultrasonic treatment.

The metal oxides used in Examples and Comparative Examples are as follows: zirconium oxide (ZrO ₂ , Example 1), molybdenum oxide (MoO ₃ , Example 2), manganese oxide (MnO ₂ , Comparative Example 2) ), Yttrium oxide (Y ₂ O ₃ , comparative example 3), titanium oxide (TiO ₂ , comparative example 4), iron oxide (Fe ₂ O ₃ , comparative example 5). In Comparative Example 1, no metal oxide was used.

(2) Preparation of enzyme immobilization substrate electrode Appropriate amount of each carbon slurry prepared in (1) above was applied to both sides of a carbon cloth (manufactured by Nippon Carbon Co., Ltd.) cut out with a cork borer so as to have an area of 1 cm ^2. Then, the solvent was removed by drying with a dryer at 60 ° C.

(3) Immobilization of enzyme catalyst electrode The bilirubin oxidase (BOD: Amano Enzyme Co., Ltd.) was prepared in advance so that each electrode prepared in (2) was 50 mg / ml with 0.1 M sodium phosphate buffer (pH 7). (Made) It was immersed in the solution. It left still at 4 degreeC overnight, and BOD was fix | immobilized on the electrode by the physical adsorption method. In the case of an electrode using a metal oxide, in particular in the case of an electrode using zirconium oxide and molybdenum oxide, it was observed that the enzyme solution rapidly penetrated.

2. Evaluation of Electrode Fuel cell for evaluation (schematic diagram is shown in FIG. 1) in which an enzyme-immobilized electrode prepared in accordance with the procedure of 1 (1) to (3) above was made of an acrylic plate together with a titanium mesh as a current collector. The output at a single pole was evaluated. For the measurement, an electrochemical analyzer LS / CH 708c (manufactured by ALS) was used, and constant potential current measurement (chronoamperometry method) at 0.1 V was performed. In addition, the amount of BOD immobilized on the electrode by the BOD immobilization performed in 1 (3) above was measured, and the relationship with the output current value of each electrode was examined. In addition, conditions were examined for the amount of metal oxide mixed, and the optimum value was investigated.

(1) Chronoamperometry measurement Using 0.1M sodium phosphate buffer (pH 7) as an electrolytic solution, measurement was performed using an electrochemical analyzer LS / CH 708c (manufactured by ALS). The measurement was performed with a three-electrode system including a working electrode, a reference electrode, and a counter electrode. Here, the enzyme electrode produced this time is used as the working electrode, the silver-silver chloride electrode (manufactured by ALS: RE-1B reference electrode) is used as the reference electrode, and the platinum electrode (manufactured by ALS: VC-2) is used as the counter electrode. Pt counter electrode) was used. All measurements were performed at room temperature, and a steady current value of BOD catalyst current at 0.1 V (500 seconds after the start of measurement as a guide) was measured.

  The result of examining the mixing effect of each metal oxide is shown in FIG. Compared with the case where the metal oxide was not mixed (Comparative Example 1), in the case where the metal oxide was mixed, the output was improved and the mixing effect on the electrode could be confirmed. In particular, when zirconium oxide is mixed (Example 1) and when molybdenum oxide is mixed (Example 2), other metal oxides such as manganese oxide, which are frequently used as electrode materials, are mixed (Comparative Example 2). A higher effect was obtained for ˜5). In particular, when molybdenum oxide was mixed (Example 2), a dramatic output improvement effect was obtained.

(2) Evaluation of enzyme affinity of electrode The amount of enzyme protein immobilized on each electrode by the enzyme immobilization treatment performed in 1 (3) above was measured and compared. For measurement, a protein content measurement kit manufactured by BioRad was used, and the protein amount was calculated by the Bradford method. A graph showing the relationship between the amount of enzyme immobilized on the electrode and the current value obtained by chronoamperometry measurement is shown in FIG.

  It was found that the electrode mixed with the metal oxide had a larger amount of protein adsorbed than the electrode not mixed, and that the affinity of the enzyme for the electrode was improved by adding the metal oxide. In particular, when zirconium oxide is mixed (Example 1), compared with the case where manganese oxide, which is known as a good electrode material, is mixed (Comparative Example 2), the output of protein is relatively small. Improved by about 40% or more. Further, when molybdenum oxide was mixed (Example 1), the enzyme adsorption to the electrode was the largest, and a dramatic output improvement effect was recognized as compared with the case where other metal oxides were mixed.

  In order to confirm the mixing effect of molybdenum oxide, an electrode containing molybdenum oxide but not immobilizing an enzyme was prepared, and chronoamperometry measurement was performed in the same manner as 2 (1) above. The result is shown in FIG. It was found that even if the electrodes were mixed with the same molybdenum oxide, no output could be obtained unless the enzyme was immobilized. Although it was considered that molybdenum oxide itself has a catalytic action, since an output cannot be obtained when an enzyme is not included, the output improvement in an electrode mixed with molybdenum oxide is between the catalytic activity of molybdenum oxide and the enzyme. It is thought to be due to the interaction.

(3) Optimization of Molybdenum Oxide Mixing Amount According to the above procedure 1, enzyme electrodes (4 conditions of 0 mmol, 0.49 mmol, 0.98 mmol, 1.95 mmol, 3.9 mmol) with different molybdenum oxide mixing amounts were prepared. The output current was evaluated. The result is shown in FIG. As for molybdenum oxide, it was found that an optimum value of the mixing amount exists around 1.95 mmol.

  Biofuel cells using enzyme electrodes may be used as low-cost and low environmental load batteries as an alternative to current lithium ion batteries. Applications include medical devices and mobile devices, as well as power supplies for small mobility, and the market size in that case is extremely large.

Claims (5)

  An enzyme electrode in which a mixture containing carbon fine particles, an enzyme, and a metal oxide containing at least one of zirconium oxide and molybdenum oxide is fixed to a conductive substrate.   The enzyme electrode according to claim 1, comprising 15 to 130 mmol of metal oxide with respect to 1 g of carbon fine particles.   The enzyme electrode according to claim 1, wherein the enzyme electrode is produced by preparing a slurry in which carbon fine particles and a metal oxide are dispersed in a solvent, applying the slurry to a conductive substrate, and then immobilizing an enzyme. .   A fuel cell comprising the enzyme electrode according to any one of claims 1 to 3 on at least one of an anode electrode and a cathode electrode.   Preparing a slurry in which carbon fine particles and a metal oxide containing at least one of zirconium oxide and molybdenum oxide are dispersed in a solvent, applying the slurry to a conductive substrate, and then immobilizing an enzyme. A method for producing an enzyme electrode.

Images