JPH021261B2 - - 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, enables rapid and simple measurement of substrate concentration, and can be used repeatedly. The present invention relates to an enzyme electrode that can be used for various purposes.

As an example of an attempt to industrially utilize the specific catalytic action of enzymes, an attempt was made to measure the concentration of a substrate, a substance that specifically reacts with enzymes, by linking an enzyme reaction system and an electrochemical reaction system. It is being As an example, as shown in the following formulas (1) and (2), an oxidoreductase that uses oxygen as a hydrogen acceptor,
For example, due to the action of glucose oxidase, a substrate such as glucose is oxidized to produce H ₂ O ₂ ,
Next, this H ₂ O ₂ is oxidized using a platinum electrode etc.
The concentration of glucose can be determined from the oxidation current value obtained at this time.

Glucose + O ₂ Glucose Oxidase――――――――――→ Gluconolactone + H ₂ O ₂ …(1) H ₂ O ₂ →2H ⁺ +2e+O ₂ …(2) Repeat by applying this principle In order to construct a usable enzyme electrode for measuring substrate concentration, it is necessary to immobilize a water-soluble enzyme on or near a platinum electrode. On the other hand, when measuring the substrate concentration using such an enzyme electrode, interfering substances contained in the sample become a problem. For example, when measuring glucose in blood, various coexisting substances contained therein, such as uric acid and ascorbic acid, are directly electrochemically oxidized on a platinum electrode. In other words, when H ₂ O ₂ is oxidized on the electrode as shown in equation (2) above, these coexisting substances are oxidized at the same time.
This will give an error to the obtained current value.

Conventional examples of countermeasures against such interfering substances include the following.

(1) Two platinum electrodes are used, the enzyme is immobilized on only one electrode, and the influence of interfering substances is compensated for by subtracting the current values obtained from both electrodes.

(2) By placing a membrane made of silicone rubber, cellulose acetate, etc. to block interfering substances in front of the platinum electrode (test liquid side),
Prevents uric acid and ascorbic acid from diffusing into the platinum electrode.

In the above method, (1) has the drawback that it is very difficult to balance the responsivity of the two platinum electrodes. In addition, method (2) is simple, but because it uses the membrane itself made of cellulose acetate, it avoids a decrease in response current (decrease in sensitivity) and a decrease in response speed compared to when no membrane is used. It was something I couldn't do.

Therefore, the present inventors conducted various studies to improve the above-mentioned points, and as a result, they discovered an enzyme electrode with excellent characteristics. The enzyme electrode of the present invention is characterized by using a porous membrane, forming an acetyl cellulose layer in the pores of this membrane or on one membrane surface, and directly coating platinum etc. on the other membrane surface. The point is that an electrode for detecting hydrogen peroxide is formed and the enzyme is immobilized on the other membrane surface.
That is, a platinum electrode, an immobilized enzyme layer, and an acetyl cellulose layer for blocking uric acid and ascorbic acid are each formed on one porous membrane.

FIG. 1 shows a schematic cross-sectional view of an example of the structure of the enzyme electrode of the present invention. In the figure, 1 is a porous membrane that serves as a carrier.
An acetylcellulose layer 2 is formed in the pores of the membrane and on the membrane surface. An immobilized enzyme layer 3 is formed on the acetyl cellulose layer on the membrane surface, and a thin layer 4 of, for example, platinum is formed on the opposite membrane surface by vapor deposition, sputtering, etc., so that the whole is in the form of one thin film. .

When using this enzyme electrode, it is arranged so that the immobilized enzyme layer 3 is on the test liquid side with respect to the hydrogen peroxide detection electrode 4. The substrate in the test solution generates H ₂ O ₂ by the action of the enzyme, and this H ₂ O ₂ diffuses through the membrane and is detected as an anode current by the hydrogen peroxide detection electrode 4 on the opposite side. On the other hand, when uric acid, ascorbic acid, etc. coexist, the diffusion of these interfering substances is blocked by the acetyl cellulose layer, making it difficult for them to reach the hydrogen peroxide detection electrode.

In this way, the enzyme electrode of the present invention can effectively reduce the influence of interfering substances, and since the hydrogen peroxide detection electrode is formed directly on the membrane and the entire membrane is thin, the response speed is , it has excellent response sensitivity, and the response characteristics are hardly affected by expansion, contraction, deformation, etc. of the membrane during use, and a stable response can be obtained.

The enzyme used is not limited to one type, and may be a complex enzyme system as long as it generates H ₂ O ₂ during the oxygen reaction. Further, as the hydrogen peroxide detection electrode formed on the membrane surface, any metal or metal oxide other than platinum, such as ruthenium or oxides thereof, may be used as long as it meets the purpose described above.

The porous membrane used as a carrier is preferably made of a material on which an electrode for detecting hydrogen peroxide can be formed and which does not impair adhesion to platinum or the like when used in an aqueous solution. Such porous membranes include polycarbonate, polyethylene,
Porous membranes that do not swell with water, such as polypropylene, are optimal.

Examples of the present invention will be described below.

A polycarbonate porous membrane with a pore diameter of 2000 Å, a membrane thickness of 10 μm, and a pore density of 3×10 ⁸ /cm ² was used as the carrier membrane. One side of the membrane is immersed in an acetone solution of cellulose acetate and then dried. By repeating this operation several times, an acetylcellulose layer can be formed in the pores and on one side of the membrane.
Platinum was sputtered onto the surface of the obtained membrane on the side where the acetyl cellulose layer was not formed, to form a hydrogen peroxide detection electrode having a thickness of several hundred to several thousand angstroms. Next, an aqueous solution of glucose oxidase (concentration 200 mg/ml) was spread as an enzyme on the membrane surface opposite to the platinum-forming surface, dried, and immobilized in glutaraldehyde vapor, followed by sufficient Washed with water. The enzyme electrode according to the present invention thus obtained is designated as A.

For comparison, an enzyme electrode B was prepared under the same conditions as A above without acetylcellulose treatment.

The enzyme electrode described above was attached to a cylindrical electrode holder shown in FIG. 2 and subjected to measurement. In the figure, 5 is a reference electrode, 6 is a counter electrode, and 7 is a platinum lead. Reference numeral 8 denotes an enzyme electrode, the platinum electrode side of the membrane is in contact with the lead 7, and is held in a resin cylindrical body 11 by a cap 10 via a packing 9. Further, the inside of the electrode holder is filled with an electrolytic solution 12.

This electrode holder is immersed in a pH5.6 buffer solution,
After setting the potential of the enzyme electrode to a sufficient oxidation potential of H ₂ O ₂ with respect to the reference electrode, glucose or ascorbic acid was added, and changes in current (steady value) due to changes in concentration were measured. Figure 3 shows the amount of current increase for each of the enzyme electrodes A and B. In the figure, the solid line shows the response characteristics to changes in glucose concentration, and the broken line shows the response characteristics to changes in ascorbic acid concentration. As is clear from the figure, enzyme electrode A according to the present invention is enzyme electrode B without acetylcellulose treatment.
It can be seen that compared to the above, it has the property that it almost maintains its responsiveness to glucose and is almost unaffected by ascorbic acid. These excellent properties against ascorbic acid were also observed against interfering substances such as uric acid and even glutathione. Furthermore, the enzyme electrode of the present invention had extremely excellent characteristics in terms of response speed, with the oxidation current of H ₂ O ₂ reaching a steady value in about 10 seconds after the addition of glucose.

As described above, the enzyme electrode of the present invention has excellent performance.

[Brief explanation of drawings]

FIG. 1 is a schematic cross-sectional view showing an example of the structure of the enzyme electrode of the present invention, FIG. 2 is a vertical cross-sectional view showing an electrode system using an electrode holder equipped with an enzyme electrode, and FIG. 3 is a cross-sectional view showing the concentration of glucose and ascorbic acid. FIG. 3 is a diagram showing the relationship between the current increase amount and the current increase amount. 1... Porous membrane, 2... Acetyl cellulose layer, 3... Enzyme, 4... Hydrogen peroxide detection electrode.

Claims (1)

[Scope of Claims] 1. A porous membrane, an acetylcellulose membrane formed in the pores of the porous membrane or on one membrane surface, and an enzyme immobilized on the one membrane surface of the porous membrane. An enzyme electrode comprising: a hydrogen peroxide detection electrode formed on the other membrane surface of the porous membrane; 2. The enzyme electrode according to claim 1, wherein the porous membrane has no swelling property with respect to water.