JP2010267537A - Enzyme electrode - Google Patents

Abstract

In an enzyme-immobilized electrode used as an electrode of an electrochemical reaction device such as an enzyme fuel cell or a biosensor, an electrode capable of increasing the amount of gas substrate supplied to the enzyme and exhibiting high output is provided.
The other part of the conductive substrate 2 is in contact with the substrate-containing gas 8 so that a part of the conductive substrate 2 constituting the enzyme-immobilized electrode is in contact with the electrolyte solution 9. By immobilizing the enzyme 6 on at least a part of the part in contact with the substrate-containing gas 8, the output power is improved. The enzyme 6 is immobilized on the carrier 5 by fixing the carrier 5 on the binder 7 and indirectly immobilizing it on the carrier 5 or directly immobilizing on the carrier 5. Moreover, an enzyme can also be immobilized on the electrolyte contact portion side.
[Selection] Figure 1

Description

      The present invention relates to an enzyme electrode, and more particularly to an enzyme electrode that can be used as an electrode of an electrochemical reaction device such as an enzyme fuel cell or a biosensor.

      Enzymes have a high substrate specificity and generally act strongly under a certain reaction of a certain substrate. In addition, enzymes have the characteristic that the reaction proceeds under relatively mild conditions at room temperature and neutrality.

  The catalytic action of such an enzyme has been used for a long time, but in recent years, its application to the electronic engineering sector such as a biosensor using an enzyme and a biofuel cell has been studied and put into practical use. Among them, an enzyme electrode using an electric current generated in accordance with an oxidation-reduction reaction between an enzyme and a substrate is expected to be used and is being developed.

      In the enzyme electrode, generally, in the anode electrode, sugar, amine, organic acid, hydrogen and the like are used as a substrate. On the other hand, oxygen is often used as a substrate in the cathode electrode. Gases may be used as enzyme substrates, such as hydrogen for the anode electrode and oxygen for the cathode electrode.

In Patent Document 1, there are an anode electrode and a cathode electrode in an electrolytic solution, and in order to supply oxygen to the cathode electrode, air or oxygen is sent into the electrolytic solution through a gas supply pipe and bubbling is performed. Is disclosed. However, since the solubility of oxygen in the liquid is low and the diffusion coefficient is small, there is a limit to the supply of oxygen to the cathode electrode, and the output improvement effect is not sufficient.
Therefore, in Non-Patent Document 1, in order to make it easy to supply oxygen to the cathode electrode, one surface of the conductive substrate is disposed in the air and the other surface is in contact with the electrolyte solution, and the enzyme is disposed in the conductive substrate. An enzyme electrode immobilized only on the surface in contact with the electrolyte is disclosed.

JP2007-121280

Academic Meeting 2008 214th Meeting of the Electrochemical Society October 12-17 (Honolulu)

      An object of the present invention is to provide an electrode capable of increasing the supply amount of a gaseous substrate to the enzyme and exhibiting high output in the enzyme electrode.

      In order to achieve the above-described object, the present inventors have intensively studied. As a result, the other part of the conductive substrate is in contact with the substrate-containing gas so that the part of the conductive substrate constituting the electrode is in contact with the electrolytic solution. Thus, the inventors have found a new finding that the output power can be improved by immobilizing an enzyme on at least a part of the conductive substrate in contact with the substrate-containing gas, and the present invention has been completed.

That is, the enzyme electrode according to the present invention is an enzyme electrode having a conductive substrate and an enzyme immobilized on the conductive substrate, wherein the conductive substrate includes an electrolyte contact portion that contacts the electrolyte, and a substrate. And a gas contact portion that contacts the contained gas, wherein the enzyme is immobilized on at least a part of the gas contact portion.
The enzyme electrode according to the present invention is characterized in that a first enzyme is immobilized on the gas contact portion and a second enzyme is immobilized on the electrolyte contact portion. The first enzyme and the second enzyme may be the same or different.
The enzyme electrode according to the present invention is characterized in that a carrier is fixed to the conductive substrate with a binder, and the enzyme is supported on the carrier.
Furthermore, the binder of the gas contact part is hydrophobic, and the binder of the electrolyte solution contact part is hydrophilic.
Biofuel cells and biosensors can be used as targets for utilizing the enzyme electrode of the present invention.

      In the enzyme electrode of the present invention, the gas substrate supplied to the enzyme can be increased by the enzyme directly contacting the substrate-containing gas. In addition, by separately applying a binder for immobilizing the carrier carrying the enzyme depending on hydrophobicity and hydrophilicity, the supply of the electrolyte solution in contact with the enzyme can be controlled, and a high enzyme function can be exhibited. Therefore, output improvement can be aimed at by using the enzyme electrode of the present invention for a biofuel cell or a biosensor.

It is a schematic cross section which shows the structure of the enzyme electrode which shows an example of 1st embodiment of this invention. It is a schematic cross section which shows the structure of the enzyme electrode which shows an example of 2nd embodiment of this invention. It is a graph which shows the chronoamperometry measurement result of the enzyme electrode of Examples 1, 2 and Comparative Example 1. It is a graph which shows the chronoamperometry measurement result of the enzyme electrode of Example 3, 4 and the comparative example 2. FIG. It is a schematic cross section which shows an example of the enzyme fuel cell using the enzyme electrode of this invention.

      Hereinafter, specific embodiments of the present invention will be described with reference to the drawings. However, the present invention is not limited to the following examples, and conventionally known configurations are added or materials are changed. Various applications are possible without departing from the scope of the present invention.

      As shown in FIG. 1, the enzyme electrode 1 which is 1st embodiment of this invention has the electroconductive base | substrate 2, the enzyme 6, the support | carrier 5, and the binder 7. As shown in FIG. The conductive substrate 2 has a gas contact portion 3 that comes into contact with the substrate-containing gas 8 and an electrolyte solution contact portion 4 that comes into contact with the electrolyte solution 9. 7 is fixed.

      As shown in FIG. 2, the enzyme electrode 10 according to the second embodiment of the present invention includes a conductive substrate 2, a first enzyme 6 a, a second enzyme 6 b, a carrier 5, and a binder 7. The conductive substrate 2 has a gas contact portion 3 that contacts the substrate-containing gas 8 and an electrolyte solution contact portion 4 that contacts the electrolyte solution 9. In the gas contact portion 3, the first enzyme 6 a is electrolyzed. A second enzyme 6 b is fixed to the liquid contact portion 4 together with the carrier 5 with a binder 7.

      In FIG. 1 and FIG. 2, the carrier 5 is fixed to the conductive substrate 2 with the binder 7 and the enzyme 6, 6a, 6b is supported on the carrier 5 so that the enzyme is fixed to the conductive substrate. Immobilization of the enzyme to the conductive substrate is not particularly limited to indirect immobilization, and the enzyme may be directly immobilized to the conductive substrate.

      An electron mediator may be used to efficiently transfer electrons between the enzyme and the conductive member. For example, ABTS (2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)), diammonium salt, vitamin K3, 2-amino-3carboxy-1,4-naphthoquinone is used as the electron mediator can do. Further, these electron mediators can be used after being derivatized.

  The conductive substrate 2 is preferably an aggregate of carbon fibers having a high surface area, such as carbon felt, carbon paper, carbon cloth, and carbon sponge, in that more enzyme, carrier and mediator can be immobilized. For example, a conductive resin can be used instead of the carbon fiber material as long as it is a material capable of carrying the enzyme and the mediator solution and transferring electrons.

The carrier 5 is necessary for supporting the enzyme and the mediator,
A porous fine particle material or the like is preferably used in terms of supporting more enzymes and mediators. Specific examples include ketjen black, carbon cryogel, activated carbon, graphite, carbon nanotube, and carbon nanohorn.

The enzymes 6, 6a and 6b only need to be able to oxidize or reduce a gaseous substrate. In the anode electrode, when hydrogen is used as the substrate, it is sufficient that hydrogen can be oxidized. Specifically, hydrogen oxidase (hydrogenase, nitrogenase) or the like is used. In the cathode electrode, when oxygen is used as a substrate, it is sufficient that oxygen can be reduced. Specifically, bilirubin oxidase, laccase, peroxidase, multi-copper oxidase (CueO) or the like is used.
Enzyme immobilization is not particularly limited to the surface of the conductive substrate, and the enzyme may be immobilized inside the conductive substrate.

      The binder 7 is used for fixing the carrier 5 to the conductive substrate 2. Specifically, examples of the hydrophobic binder include polytetrafluoroethylene (Teflon (registered trademark)) and polyvinylidene fluoride (PVDF). Examples of the hydrophilic binder include polyvinyl pyridine and polyhydroxyethyl methacrylate. , Calcium alginate, κ-carrageenan and the like. In addition, a bipolar compound such as lecithin may be used, or the hydrophobic binder and the hydrophilic binder may be mixed and used. When a hydrophobic binder is used for the gas contact portion 3 and a hydrophilic binder is used for the electrolyte solution contact portion 4, the amount of electrolyte supplied to the gas contact portion can be controlled and fixed to the gas contact portion. This is preferable because the supply balance of the gas substrate and the electrolyte to the enzyme can be balanced, and the output of electric power can be increased.

  The substrate-containing gas 8 is not particularly limited as long as it is a gas substrate alone or a mixed gas containing a gas substrate. When oxygen is used as the substrate of the cathode electrode, air is preferably used because it can be easily carried out without preparing other devices and materials.

      Electrolyte 9 includes sodium phosphate buffer, potassium phosphate buffer, Tris hydrochloride buffer, MOPS (3- (N-morpholino) propanesulfonic acid) buffer solution, MacIlvine solution, etc. Depending on the use. The electrolytic solution 9 may be mixed with a substrate substance that acts on the enzyme of the anode electrode or the cathode electrode. For example, alcohols such as methanol, sugars such as glucose, fats, proteins, and intermediate products of sugar metabolism Organic acids (glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, triosephosphate isomerase, 1,3-bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerin Acid, phosphoenolpyruvate, pyruvate, acetyl-CoA, citric acid, cis-aconitic acid, isocitric acid, oxalosuccinic acid, 2-oxoglutaric acid, succinyl-CoA, succinic acid, fumaric acid, L-malic acid, oxalo Acetic acid, etc.), and mixtures thereof. Moreover, in order to raise the ionic strength in a solution, you may mix suitable salts as a supporting electrolyte.

      The enzyme electrode shown in the first embodiment and the second embodiment is preferably used as an electrode of an enzyme fuel cell or a biosensor because the amount of gas substrate supplied to the enzyme is large. An example of an enzyme fuel cell in which the enzyme electrode is assembled as a cathode electrode 11 and oxygen is used as a gas substrate is shown in FIG. A separator may be provided between the cathode electrode 11 and the anode electrode 12. The separator is configured to have proton conductivity so that the electrolyte solution 9 oozes out, and the electrolyte solution 9 oozed out of the separator touches the cathode electrode 11. Specifically, any material may be used as the separator, such as cellophane, Nafion, PVDF membrane, filter paper, etc., as long as the above purpose is achieved.

          Specific samples of the enzyme electrode of the present invention were evaluated by chronoamperometry measurement.

<Production of enzyme electrode>
Carbon paper (manufactured by Toray Industries, Inc.) having a thickness of 370 μm cut out to a diameter of 1.2 cm was prepared, and enzyme electrodes A to D were prepared according to the following procedure.
Ketjen Black (manufactured by Lion) 10 mg, 2-propanol 4 ml, and Teflon 6.67 mg were weighed, and components were sufficiently dispersed using an ultrasonic crusher to prepare a carbon slurry. An appropriate amount of the carbon slurry was applied to one side of the carbon paper, the solvent was removed by drying at 60 ° C. to form a carrier surface, and an electrode material A having the carrier surface only on one side was produced. Immerse electrode material A in a bilirubin oxidase solution (Amano Enzyme) adjusted to a concentration of 10 mg / ml, leave it at 4 ° C for 12 hours to immobilize the enzyme, and place the enzyme-supported surface on one side Enzyme electrode A having only was prepared.
An enzyme electrode B having an enzyme carrying surface on both sides was prepared in the same manner as the enzyme electrode A, except that an appropriate amount of the carbon slurry was applied to both sides of the carbon paper.
An enzyme electrode C having an enzyme-supporting surface only on one surface was prepared in the same manner as the enzyme electrode A except that ketjen black was changed to carbon cryogel.
An enzyme electrode D having an enzyme carrying surface on both sides was produced in the same manner as the enzyme electrode B except that ketjen black was changed to carbon cryogel.

<How to assemble electrodes>
Table 1 shows the structure and assembly method of each enzyme electrode.
(Example 1) Air containing oxygen serving as a substrate on the enzyme-carrying surface of enzyme electrode A was assembled in a housing for electrode evaluation so that the electrolyte solution was in contact with the non-enzyme-carrying surface.
(Example 2) Air containing oxygen serving as a substrate on one surface of the enzyme-carrying surface of enzyme electrode B was assembled in a housing for electrode evaluation so that the electrolyte solution was in contact with the other surface.
(Example 3) Except that the enzyme electrode A was changed to the enzyme electrode C, it was assembled in a housing for electrode evaluation in the same manner as in Example 1.
(Example 4) Except that the enzyme electrode B was changed to the enzyme electrode D, it was assembled in a housing for electrode evaluation in the same manner as in Example 2.
(Comparative Example 1) The electrode was assembled in an electrode evaluation casing so that the electrolyte solution was in contact with the enzyme-carrying surface of the enzyme electrode A and the oxygen-containing surface was in contact with air containing oxygen as a substrate.
(Comparative Example 2) Same as Comparative Example 1 except that the electrode material A is changed to the electrode material C
And it assembled | attached to the housing | casing for electrode evaluation.



Enzyme electrode
Carrier
Electrolyte contact part
Gas contact part

Example 1
A
Ketjen Black Enzyme not supported Enzyme supported

Example 2
B
Ketjen Black Enzyme-supported Enzyme-supported

Example 3
C
Carbon cryogel Enzyme not supported Enzyme supported

Example 4
D
Carbon cryogel Enzyme support Enzyme support

Comparative Example 1
A
Ketjen Black Enzyme-supported Enzyme non-supported

Comparative Example 2
B
Carbon cryogel Enzyme supported Not enzyme supported

<Chronoamperometry measurement>
As an electrolytic solution, a sodium phosphate buffer of pH 7 was used, and an electrochemical analyzer LS / CH 708c was used to measure with a three-electrode apparatus of a working electrode, a reference electrode, and a counter electrode. The working electrode is the enzyme electrode, the reference electrode is a silver-silver chloride electrode (product name: RE-1B reference electrode), and the counter electrode is a platinum electrode (product name: Pt counter electrode for VC-2). Using. At room temperature, the measurement potential was 0.1 V, and the measurement was performed until 600 seconds as a guide until the obtained current became stable, and a stable current value was recorded.

The results of the output values of Examples 1 and 2 and Comparative Example 1 using ketjen black as a carrier are shown in FIG.
It can be seen that the electrode of Example 1 in which the enzyme is immobilized in the gas contact portion has higher output than the electrode of Comparative Example 1 in which the enzyme is immobilized in the electrolyte contact portion. Moreover, it turns out that the electrode of Example 2 by which the enzyme was fix | immobilized in both the electrolyte solution contact part and the gas contact part has the highest output, and is preferable.
The results of the output values of Examples 3 and 4 and Comparative Example 2 using carbon cryogel as the carrier are shown in FIG. FIG. 4 also has the same tendency as FIG. 3, and it can be seen that the type of carbon that is the carrier of the enzyme is not particularly limited.

Thus, the conductive substrate constituting the electrode has a gas contact portion that contacts the substrate-containing gas and an electrolyte solution contact portion that contacts the electrolyte, and an enzyme is immobilized on at least a part of the gas contact portion. By using the enzyme electrode characterized in that the gas substrate was sufficiently supplied to the enzyme, the output could be improved.
The enzyme electrode of the present invention having a high output can be used for an enzyme fuel cell and an enzyme sensor.

1, 10 Enzyme electrode 2 Conductive substrate 3 Gas contact part 4 Electrolyte contact part 5 Carrier
6, 6a, 6b Enzyme 7 Binder 8 Substrate-containing gas 9 Electrolyte
11 Cathode electrode 12 Anode electrode

Claims (6)

In an enzyme electrode having a conductive substrate and an enzyme immobilized on the conductive substrate,
The conductive substrate has a gas contact portion that contacts a substrate-containing gas, and an electrolyte solution contact portion that contacts an electrolyte solution,
The enzyme electrode, wherein the enzyme is immobilized on at least a part of the gas contact portion.         The enzyme electrode according to claim 1, wherein a first enzyme is immobilized on the gas contact portion, and a second enzyme is immobilized on the electrolyte contact portion.               The enzyme electrode according to claim 1 or 2, wherein a carrier is fixed to a conductive substrate with a binder, and an enzyme is supported on the carrier.       The enzyme electrode according to claim 3, wherein the binder in the gas contact portion is hydrophobic and the binder in the electrolyte solution contact portion is hydrophilic.               A fuel cell comprising the enzyme electrode according to claim 1. An enzyme sensor comprising the enzyme electrode according to claim 1.