JPH01182747A - Enzyme electrode - Google Patents

Abstract

PURPOSE:To obtain an electrode characterized by excellent current dependency for enzyme concentration, stable performance for a long period and a low cost, by using a reduction type redox compound, which is insoluble in water and soluble in organic solvent, as an electron transfer material. CONSTITUTION:As an enzyme electrode, a reduction type redox compound such as phenothiazine, phenoxazine and the like, which is insoluble in water and soluble in organic solvent, is used as mediator. Electrons are made to trans fer between oxidation-reduction enzyme and a current collector with the media tor, and an electric signal is detected. Thus the substrate is determined. The stable redox compound in the reduced state is used as the mediator. A potential, which is higher than the oxidation-reduction potential of the redox compound, is applied, and electrolytic oxidation is performed. Therefore, the reaction speed of the entire system does not become a rate determining state owing to the reducing reaction of the mediator. Thus the electron acceptance reaction from the enzyme is efficiently performed, and the large current value is obtained. Furthermore, the reduction type redox compound can be used in a pattern, in which it is incorporated in a macromolecule compound such as poly tetrafluoroethylene.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an enzyme electrode suitable for use in fuel cells, bioreactor electrode materials, biosensor electrodes, and the like.

- Research has been actively conducted on sensors that detect chemical substances using enzymatic reactions and fuel cells that convert chemical energy into electrical energy to generate electricity. Among them, various systems have been proposed for glucose sensors and sensors using glucose oxidase. in particular.

There is a method of quantifying H and O□ produced by the reaction using a hydrogen peroxide electrode, and a method of quantifying consumed O2 using an oxygen electrode. However, these methods have the disadvantage that glucose cannot be accurately quantified unless there is sufficient dissolved oxygen. Therefore, even if there is not enough dissolved oxygen, an electron transfer substance (hereinafter referred to as a mediator) is used to transfer electrons between the oxidoreductase and the current collector, and an electrical signal is detected to quantify glucose. A method has been proposed. In this method, oxidized redox compounds such as ferrocene, benzoquinone, chloranil, and thionine are mainly used as mediators, but these low-molecular mediator substances are not sufficiently insoluble in water and therefore elute from the electrode. However, there is a problem that the sensitivity of the electrode gradually decreases. Furthermore, when constructing an enzyme electrode using an enzyme that oxidizes a substrate, an oxidized redox compound is used as a mediator of the enzyme.

Since the oxidized state of oxidized redox compounds is thermodynamically stable, the reaction rate of the entire system depends on the reduction reaction of the mediator,
That is, the rate is determined by the electron acceptance reaction from the enzyme, and the electron acceptance reaction from the enzyme is not performed efficiently, so that a large current value may not be obtained.

The present invention was developed in view of the above circumstances, and allows accurate quantitative determination of the substrate even when sufficient oxygen is not present, and the mediator substance is eluted from the electrode, reducing the sensitivity of the electrode. It is an object of the present invention to provide a highly accurate and inexpensive enzyme electrode that does not cause such problems and allows electron acceptance reactions from enzymes to be carried out efficiently to obtain a large current value.

In order to achieve the above object, the present inventors conducted intensive studies and found that reduced redox compounds such as phenothiazines and phenoxazines are good mediators of oxidoreductases. Reduced redox compounds have very low solubility in water and are soluble in organic solvents. Electrodes are made by molding a composition consisting of a compound, a polymer compound, and a conductor such as carbon.

By immobilizing enzymes such as glucose oxidase on this, it has excellent current dependence on oxygen concentration, shows good correspondence between substrate concentration such as glucose and current value, and is stable for a long time without elution of mediator. The inventors have discovered that an enzyme electrode can be obtained that exhibits excellent performance, is inexpensive, and can be easily produced, and has led to the present invention.

Therefore, the present invention provides an enzyme electrode in which an electron transfer substance mediates electron transfer between an enzyme and a current collector, in which the electron transfer substance is a reduced redox compound that is insoluble in water and soluble in an organic solvent. Provides an enzyme electrode with characteristics.

The present invention will be explained in more detail below.

The enzyme electrode according to the present invention uses, as a mediator, a reduced redox compound such as phenothiazines and phenoxazines that is insoluble in water and soluble in organic solvents, and the mediator is combined with an oxidoreductase. It allows electron transfer between an electric body and detects an electric signal to quantify the substrate.

Here, how the substrate reacts in the presence of the enzyme and the mediator (M) is as follows when glucose is quantified using glucose oxidase (GOD) as the enzyme.

Glucose + (GOD) ox” Gluconic acid + (COD)
RED (1) Mox+ (GOD)RED=
MRED+ (COD)OX (2) Electrode+MRHD=ne-(electronic tin) +Mox
(3) (However, .X indicates an oxidized state and RED indicates a reduced state.

Note that the description of H+ movement has been omitted. ) Therefore, oxidized redox compounds have been mainly used as mediators for enzymes that oxidize substrates, such as glucose oxidase, but as mentioned above, oxidized redox compounds have thermodynamically stable oxidation states. Therefore, the reaction rate of the entire system is rate-limited by the reduction reaction of the mediator, that is, the electron acceptance reaction from the enzyme, and the equation (2) above.As a result, the electron acceptance reaction from the enzyme is not performed efficiently, and a large current value is not obtained. In contrast, the enzyme electrode of the present invention uses redox compounds that are stable in a reduced state as the mediator, and electrolytically oxidizes them by applying a potential higher than the redox potential of these redox compounds. Since the reaction rate is not rate-limited by the reduction reaction of the mediator, the electron acceptance reaction from the enzyme is carried out efficiently and a larger current value can be obtained.

The reduced redox compound used in the enzyme electrode of the present invention is not particularly limited as long as it is insoluble in water and soluble in organic solvents. or an oxygen atom, R1-R1 are each a hydrogen atom, an alkyl group, a halogen atom, a nitro group,
Atoms or groups selected from alkoxy groups and aryl groups, R
9 represents a hydrogen atom or an alkyl group. ) Phenothiazines or phenoxazines shown in the following are preferably used. In addition, when glucose oxidase is used as the enzyme, phenothiazine is particularly preferred since it has a good mediator function with glucose oxidase.

The above-mentioned reduced redox compound can be used in the form of being included in a polymer compound. In this case, the polymer compound may include polytetrafluoroethylene, polychlorotrifluoroethylene, polyvinylidene fluoride, tetrafluoroethylene-hexafluoroethylene, etc. Fluoropolymer electrolytes such as fluorinated propylene copolymers and tetrafluorinated ethylene-ethylene copolymers, fluoropolymer electrolytes such as perfluorosulfonic acid polymers and perfluorocarboxylic acid polymers, and highly hydrophilic materials such as polyvinyl alcohol and polyacrylamide. Molecular compounds or crosslinked products thereof, thermoplastic resins such as polyester, polyethylene terephthalate, polystyrene, vinyl chloride, curable resins such as silicone, epoxy, urethane, or biologically derived polymeric substances such as albumin, cellulose acetate, nitrocellulose, etc. One type or a mixture of two or more types of derivatives can be used. In addition, in order to improve the wettability with the measurement solution, surfactants or fine powders such as silica and titanium oxide may be added to the composition consisting of a reduced redox compound and a polymer compound, and carbon black, graphite, etc.
There is no problem in adding a conductor such as carbon fiber.

Further, the mixing ratio of the reduced redox compound and the polymer compound is not particularly limited, but 1 to 100 parts by weight, particularly 5 to 20 parts by weight of the polymer compound to 1 part by weight of the reduced redox compound. It is preferable to do so.

The enzyme contained in the enzyme electrode of the present invention is appropriately selected depending on the substrate specificity and reaction specificity depending on the type of chemical reaction desired to proceed, and is not particularly limited; for example,
Examples include glucose oxidase, alcohol dehydrogenase, urease, glucokinase, peroxidase, cholesterol esterase, lipase, phospholipase, catalase, lactate dehydrogenase, glucoamylase, galactose oxidase, penicillinase, and tyrosinase.

Next, a method for producing the enzyme electrode of the present invention will be explained.

The electrode base used in the electrode of the present invention is a carbon electrode formed by screen printing.

Preferably used are plates of noble metals such as platinum, gold, and palladium, or vapor-deposited films thereof, and stainless steel.

The method for producing the electrode is not particularly limited, but the reduced redox compound is dissolved in a small amount of an organic solvent, mixed with the polymer compound in a solution state or water dispersion state, and the above i. ! ! A method is used in which the composition on the top plate of the electrode is molded by coating or printing, and after drying and solidification, the enzyme is immobilized, or a method in which the composition mixed with the enzyme is coated, printed, or crimped on the electrode. . Note that a conductor such as carbon may be added to these compositions.

Furthermore, it is also possible to use a method in which a reduced redox compound is mixed with the above-mentioned polymer compound together with a conductor such as carbon, and an electrode is prepared by printing or the like, and the enzyme is immobilized. As a printing method, a screen printing method is suitable because it is simple and allows a relatively large amount of coating.

There are no particular restrictions on the method for immobilizing enzymes on the molded article formed from the composition made of the above-mentioned reduced redox compound, but examples include entrapment method, carrier bonding method, covalent bonding method, ionic bonding method, adsorption method, and crosslinking method. Among them, an entrapment method in which the enzyme is wrapped in a polymer network and a method in which a Schiff base is formed with glutaraldehyde are preferably used. Here, the amount of enzyme relative to the reduced redox compound is not necessarily limited, but may range from 1 to 50.
000 units/mg, the treatment temperature is preferably 0 to 30°C, and the pH is preferably 3 to 10.

As explained above, a composition consisting of a reduced redox compound and a polymer compound can be coated, printed, or crimped onto an electrode, or a reduced redox compound can be mixed with a polymer compound together with a conductor such as carbon. The resulting composition is molded by printing to create an electrode, and an enzyme such as glucose oxidase is immobilized thereon, or by pre-mixing the enzyme into the composition, it has excellent current dependence on enzyme concentration and An enzyme electrode can be obtained that exhibits good correspondence with the current value, exhibits stable performance over a long period of time without elution of the mediator, is inexpensive, and can be easily produced. This enzyme electrode is suitably used as a sensor for measuring, for example, blood sugar level or urine sugar level.

EXAMPLES Hereinafter, the present invention will be specifically explained by showing examples and comparative examples, but the present invention is not limited to the following examples.

[Example 1] 1 part by weight of phenothiazine (Seiko Chemical Division) was dissolved in a small amount of acetone, mixed with 10 parts by weight of water-dispersible Teflon dispersion (solid content 60%), and thoroughly mixed in an agate mortar. . The paste-like composition thus obtained was applied to a screen-printed carbon electrode and dried.

To this electrode, 500 units of glucose oxidase (manufactured by Sigma) was added in 1% glutaraldehyde phosphate buffer solution.
It was air-dried and fixed using μQ to obtain an enzyme electrode.

Next, using this enzyme electrode, glucose (manufactured by Wako Pure Chemical Industries, Ltd.) was added in a pH 7.4 phosphate buffer solution, and the current at 6 V compared to the silver-silver chloride electrode was measured, as shown by the solid line A in the drawing. The same results were obtained, and it was confirmed that the enzyme electrode according to this example showed good correspondence with the current value up to a glucose concentration of 450 μ/d.

[Example 2] 1 part by weight of phenothiazine and 50 parts by weight of glucose oxidase
0 unit was dissolved in a small amount of acetone, mixed with 10 parts by weight of water-dispersible Teflon dispersion (solid content 60%), and thoroughly mixed in an agate mortar. The paste-like composition thus obtained was applied to a screen-printed carbon electrode and dried.

Glucose oxidase was immobilized on this electrode in the same manner as in Example 1, and the response current to glucose of the obtained enzyme electrode was measured in the same manner as in Example 1. Results as shown by the solid line B in the drawing were obtained. It was found that the enzyme electrode according to this example showed good correspondence with the current value up to a glucose concentration of 450 mg/a.

[Comparative example]

10 parts by weight of a water-dispersible Teflon dispersion (solid content 60%) was mixed with 1 part by weight of chloranil (Wako Pure Chemical Industries, Ltd.) and thoroughly mixed in an agate mortar.

The paste-like composition thus obtained was applied to a screen-printed carbon electrode and dried.

An enzyme was immobilized on this electrode in the same manner as in Example 1, an enzyme electrode was prepared, and the response current to glucose was measured. The results are as shown by the broken line C in the drawing, and 300■/dl
The enzyme electrode of this comparative example reached saturation at a glucose concentration of 1 liter. Furthermore, some elution of chloranil was observed.

[Brief explanation of the drawing]

The drawing is a graph showing the relationship between glucose concentration and current value when the enzyme electrodes of Example 1, Example 2, and Comparative Example are used. Applicant Brideston Co., Ltd. Agent Patent Attorney Takashi Kojima

Claims (1)

[Claims] 1. An enzyme electrode in which an electron transfer substance mediates electron transfer between an enzyme and a current collector, wherein the electron transfer substance is a reduced redox compound that is insoluble in water and soluble in an organic solvent. electrode.