JPS5853745B2 - enzyme electrode - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention has electrochemical activity toward a substrate that is subject to specific catalytic action of an enzyme, can quickly and easily measure the concentration of the substrate, and can be used continuously and repeatedly. The aim is to obtain a usable enzyme electrode.

The present invention also relates to an enzyme electrode used in a battery that converts the chemical energy of a substrate into electrical energy by combining it with an oxygen electrode or the like.

In recent years, with the advancement of techniques for utilizing various enzymes, attempts have been made to industrially utilize the specific catalytic reactions possessed by these enzymes.

As one example, attempts have been made to detect the concentration of a substrate, which is a substance that specifically reacts with an enzyme, by linking an enzyme reaction system and an electrochemical reaction system.

To treat an enzymatic reaction as an electrochemical reaction, for example,
A method is used in which an appropriate redox compound conjugated with the enzyme reaction system is interposed, and the redox reaction of this redox compound is electrochemically detected.

As an example, when glucose is used as a substrate, glucose oxidase is used as an oxidoreductase, and benzoquinone is used as a redox compound, the reactions shown by the first-order equations (1) and (2) are carried out in a mixed solution in which an electrode is inserted. can be done.

Gluco-Z + Penzoquinone Glucose Oxidase Gluconolactone + Hydroquinone ・・・・・・・・・・・・
... (1) Hydroquinone → benzoquinone + 2H + ...
(2) Hydroquinone, which is the reduction product in formula (1), is electrochemically oxidized to benzoquinone in formula (2), and the glucose concentration can be detected as the oxidation current at this time.

This method can be applied to many oxidoreductases,
This is a powerful method for measuring substrate concentration.

However, from a practical standpoint, it is desirable to immobilize enzymes and redox compounds in order to avoid having to use expensive enzymes and redox compounds for each measurement and to solve the complexity of measurement operations.

Conventionally, there is an example of an enzyme electrode in which an immobilized enzyme and a redox compound are integrated, as disclosed in US Pat. No. 3,838,033.

In this example, the outside of the current collector is covered with a semipermeable membrane, and the enzyme and redox compound are interposed in the space surrounded by the current collector and the semipermeable membrane, and the redox compound is placed in a buffer solution. We have adopted a method in which the compound is involved in the reaction while dissolved in the compound.

For this reason, if the semipermeable membrane is removed, it will be of no use at all.

Furthermore, the substrate diffuses into the interior through this semipermeable membrane and undergoes a catalytic reaction by the enzyme, after which the reaction product diffuses out of the membrane again, and it is thought that this is caused by the diffusion of redox compounds inside the electrode. This may include delays in response speed.

For these reasons, it takes a long time to obtain a steady current based on the substrate.

In addition, the redox compound, which is a molecule with the same size as the substrate, will naturally be gradually lost to the outside of the membrane.
The electrode life is also very short and cannot withstand continuous or repeated use for long periods of time.

The present invention relates to a new enzyme electrode that eliminates the drawbacks of these conventional examples, and eliminates the need for a semipermeable membrane etc. by constructing the electrode from an immobilized oxidoreductase, an insoluble redox compound, and a current collector. First, the present invention provides a practical enzyme electrode capable of rapid measurement, continuous use, and repeated use.

The feature of the present invention is that an immobilized enzyme, a redox compound, and an electron conductive substance as a current collector are integrated in an appropriate state. In particular, an insoluble compound having a quinone skeleton is used as the redox compound, It is configured so that it is brought into contact with the current collector and conjugated with the enzyme in a state that is not dissolved in the solution that is in contact with the electrode.

Therefore, the decrease in electrode response speed that is thought to be caused by the diffusion of redox compounds and the like is significantly improved.

Additionally, since there is no need for a semipermeable membrane to prevent enzymes and redox compounds from being lost from the electrode, substrates and reaction products can diffuse smoothly into and out of the electrode, allowing for rapid measurement. is possible.

Naturally, enzymes and redox compounds will not be lost from the electrode during measurement or due to operations such as cleaning the electrode, and the response performance of the electrode will not deteriorate.

The present invention will be explained below with reference to Examples.

Example 1 The enzyme electrode is produced by the method described below.

A phosphate buffer is added to glucose oxidase as an oxidoreductase and carbon powder such as acetylene black, and the mixture is thoroughly mixed. Next, after vacuum drying, the enzyme is immobilized on the carbon using gluculaldehyde.

Chloranil as a redox compound is added to this mixture, graphite powder is further added thereto, and after thorough mixing, press molding is performed.

An enlarged cross-sectional view of the electrode thus obtained is shown in FIG.

In the figure, a indicates acetylene black particles, b indicates glucose oxidase immobilized on the surface of the acetylene black particles,
C is a chloranil particle which is a redox compound, and d is a graphite particle.

This electrode is mounted on an electrode support as shown in FIG.

In the figure, reference numeral 1 denotes a cylindrical resin electrode support, which has a circular recess 2 at its lower end, and a platinum plate 3 is attached to the bottom of this recess.

Reference numeral 4 denotes a platinum lead connected to the platinum plate 3, which passes through the support and is led out to the top.

Reference numeral 5 denotes the above electrode, which is mounted in the recess 2 in close contact with the platinum plate 3 and fixed with a resin bag nut 6 screwed onto the lower end of the support 1.

7 is the backing.

FIG. 3 shows a measurement system for measuring substrate concentration using the enzyme electrode 8 having the above configuration.

In the figure, 9 is a recorder, 10 is a potentioscuit, 11 is a reference electrode, 12 is a salt bridge, 13 is a counter electrode, and 14 is a phosphate buffer containing glucose as a substrate.

FIG. 4 shows the response characteristics of the enzyme electrode 8 to glucose at 25°C.

If the potential of the enzyme electrode is set to +0.4■ with respect to the saturated calomel electrode potential of the reference electrode, and a glucose solution is added at A in the figure to make the concentration 1X10-3 mol/l, the oxidation current will be approximately 30 After a second, it increased by 35 μA/- and reached a steady value.

Such an increase in current is not observed in enzyme electrodes that do not contain redox compounds.

The relationship between the glucose concentration measured in this manner and the amount of increase in current is shown by curve B in FIG.

As is clear from the figure, the glucose concentration is approximately 8 x 10-3
A good linear relationship is obtained up to mol/l.

In addition, C in the figure shows the case where the enzyme electrode used in the measurement in B above was thoroughly washed, stored in a phosphate buffer for one week, and then subjected to the measurement again.

It can be seen from this that there is hardly any decrease in responsiveness and excellent reproducibility is observed.

As the redox compound, in addition to the above-mentioned chloranil, compounds having a quinone skeleton as shown in D-G below can be used.

D) Parabenzoquinone derivatives such as fromanil, iodoanil, tetramethyl parabenzoquinone.

E) a-naphthoquinone, β-naphthoquinone, 2゜3
- Naphthoquinones such as dichloro-a-naphthoquinone.

F) 1,4-anthraquinone, 1,2-anthraquinone, 9,10-anthraquinone, 2,3-dichloro-1,
Anthraquinones such as 4-anthraquinone.

G) 4,4'-diphenoquinone, 4,4'-stilbenequinone.

FIG. 6 shows the response characteristics when these water-insoluble redox compounds are used.

DG in the figure corresponds to the redox compound groups listed above, respectively.

Although there are differences in response characteristics between the groups DG, which are considered to be based on differences in reaction reversibility, any redox compound can be used for the purpose of the present invention.

Example 2 Acetylene black is used as a current collector, and chloranil is thoroughly mixed with the acetylene black.

In this case, more uniform mixing can be achieved by dissolving chloranil in chloroform or the like, mixing it with acetylene black, and drying.

After adding graphite to the mixture thus obtained and mixing,
Press mold.

The obtained molded body is immersed in a glucose oxidase buffer solution and impregnated under reduced pressure.

After sufficient impregnation and drying, the enzyme is immobilized with glutaraldehyde.

The obtained electrode is subjected to measurement in the same manner as in Example 1.

The response performance of this electrode to glucose was as good as in Example 1.

In addition to carbon, the electron conductive material used for the current collector may include noble metals such as platinum and gold, corrosion-resistant metals such as titanium and quantal, and conductive materials such as tin oxide and ruthenium oxide. Can be done.

When used as a powder, it can be made into a pressed electrode by adding graphite or a binder mainly composed of a synthetic resin such as a fluororesin.

In addition, when using it as a plate-shaped current collector, first apply a redox compound dissolved in an organic solvent to the surface of the current collector, dry it, then apply an enzyme buffer solution, dry it again,
Enzymes can be immobilized with glutaraldehyde and used as enzyme electrodes.

In the enzyme electrode of the present invention, by fixing and using oxidoreductases such as xanthine oxidase and amino acid oxidase in addition to glucose oxidase, it becomes possible to quantify each enzyme substrate.

As described above, according to the present invention, it is possible to obtain an enzyme electrode that has rapid response performance to an enzyme substrate and is simple and can be used repeatedly.

[Brief explanation of drawings]

FIG. 1 is an enlarged sectional view of an enzyme electrode in an embodiment of the present invention, FIG. 2 is a longitudinal sectional view showing the structure of the enzyme electrode, FIG. 3 is a diagram showing the configuration of the substrate concentration measurement system, and FIG. 4 Figure 5 shows the relationship between glucose concentration and current increase, and Figure 6 shows the relationship between glucose concentration and current increase when various redox compounds are used. Show relationships. a, cl...electronic conductive substance, b...
Enzyme, C... Redox compound.

Claims (1)

[Scope of Claims] 1 Consists of an immobilized oxidoreductase, a redox compound conjugated thereto, and an electron conductive substance, wherein the redox compound is an insoluble compound having a quinone skeleton and the electron conductive substance is an insoluble compound having a quinone skeleton. An enzyme electrode configured to be conjugated with the enzyme while in contact with the enzyme. 2. The enzyme electrode according to claim 1, wherein the redox compound is a compound selected from the group consisting of rosebenzoquinone derivatives, naphthoquinones, anthraquinones, 4,4'-diphenoquinone, and 4,4'-stilbenequinone. 3. The enzyme electrode according to claim 1 or 2, which is made of a molded body of a mixture of an electron conductive substance on which an enzyme is immobilized and a redox compound. 4. The enzyme electrode according to claim 1 or 2, wherein the enzyme is immobilized on a molded body of a mixture of a redox compound and an electron conductive substance.