JPS585643A - Enzyme electrode - Google Patents

Abstract

PURPOSE:To check an impeding material and to prevent deaceleration of responding rate and reduction in sensitivity, by a method wherein an acetyl-cellulose layer is formed in an aperture in a porous film or on one film surface, an enzyme is fixed to the one film surface, and H2O2 detecting electrode is formed on the other film surface. CONSTITUTION:One surface side of a porous film 1 comprising polycarbonate having no swelling property against water is immersed in an aceton solution consisting of acetylcellulose and is dried. The operation is repeated several times to form an acetylcellulose layer 2 in an aperture in the film 1 or on one side surface. A H2O2 detecting electrode 4 is formed on other side surface of the film 1 by spattering Pt and the like. A water thick solution of a glucol oxidase 3 is spread on the film 1 surface located opposite to an electrode 4, and is dried to fix said enzyme 3 in glutaraldehyde vapor to obtain an enzyme electrode 8. The electrode 8 is held to a cylindrical body 11 with a cap 10 through the medium of a packing 9. This causes a reliable measurement of concentration of a substrate, i.e., glucose, in a sample without being affected by uric acid, ascorbic acid and without reduction in a responding rate and sensitivity, and enables the enzyme electrode to be repeatedly used.

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 as an enzyme electrode.

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, (1) (as shown in formula 21), a substrate such as glucose is oxidized to produce H2O2 by the action of an oxidoreductase that uses oxygen as a hydrogen acceptor, such as glucose oxidase. Next, this H2O) is oxidized using a platinum electrode or the like, and the concentration of glucose can be determined from the oxidation current value obtained at this time.

Glucose + O2 Swelling and L Niotoshihiki 2 meyo ± 4 gluconolactone + H2O
2・・・・・・・・・・・・(1) )1202 →2H” + 2e + 02e * 1
1 @ @ e a * * * (2) In order to apply this principle and construct an enzyme electrode for measuring substrate concentration that can be used repeatedly, a water-soluble enzyme is immobilized on or near the platinum electrode. There is a need. 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. That is, during the oxidation on the electrode 02 shown in equation (2) above, these coexisting substances are oxidized at the same time, giving an error to the obtained current value.

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

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

(2) Placing a membrane made of silicone rubber, cellulose acetate, etc. in front of the platinum electrode (test liquid side) to prevent uric acid and ascorbic acid from diffusing into the platinum electrode .

In the above method, (1) has the disadvantage 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 impossible.

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 acetylcellulose layer in the pores of the membrane or on the membrane surface, and directly coating one side of the resulting membrane with platinum or the like. In this method, an electrode for detecting hydrogen peroxide was formed, and the enzyme was immobilized on the other membrane surface. That is, a platinum dillinate, 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 serving as a carrier, and 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,
A thin layer 4 of, for example, platinum is formed on the opposite film surface by vapor deposition, sputtering, etc., so that the entire film 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 H2O2 by the action of the enzyme, and this H2O2 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 inhibited by the action of the acetyl cellulose layer, making it difficult for them to reach the peroxide rate 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 enzyme electrode is in the form of a thin film as a whole, it responds well. It has excellent speed and response sensitivity, and the response characteristics are hardly affected by expansion, contraction, deformation, etc. of the membrane during use, and stable responses can be obtained.The enzyme used is not limited to one type. Instead, a complex enzyme system may be used as long as it generates H2O2 during the enzymatic reaction. Further, the hydrogen peroxide detection electrode formed on the membrane surface may be made of any metal or metal oxide other than platinum, such as ruthenium or oxides of these metals, as long as they meet the above-mentioned purpose.

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. Examples of such porous membranes include polycarbonate and polyethylene.

Porous membranes that do not swell with water, such as polypropylene, are optimal.

Examples of the present invention will be described below.

As a carrier membrane, the pore diameter is 200 degrees and the membrane thickness is 10 μm.

A polycarbonate porous membrane with a pore density of 3 × 10 pores/tri was used. 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 acetylcellulose layer was not formed, to form a hydrogen peroxide detection electrode having a thickness of several hundred to several thousand angstroms. Next, on the membrane surface opposite to the platinum-forming surface, an aqueous solution of glucose oxidizer (concentration 20 orng/rILe) was spread as an enzyme, dried, and an immobilization reaction was carried out in glutaraldehyde vapor. After that, it was thoroughly 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, 6 is the reference electrode, 6 is the counter electrode, and 7
is a platinum lead. 8 is an enzyme electrode, which is in contact with the beam 7 on the platinum electrode side of the membrane; : It is held in the resin cylindrical main body 11 by the cap 1o. Further, the inside of the electrode holder is filled with an electrolytic solution 12.

After immersing this electrode holder in a buffer solution of pH 6.6 and setting the potential of the enzyme electrode to a sufficient oxidation potential of H2O2 with respect to the reference electrode, glucose or ascorbic acid is added, and the current changes as the concentration changes. (steady value) was measured. For each enzyme electrode A and B, the amount of current increase is
As shown in the figure. 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, the enzyme electrode A according to the present invention has the characteristics that it almost maintains the response characteristics to glucose and is almost unaffected by ascorbic acid compared to the enzyme electrode B without acetylcellulose treatment. As can be seen, these excellent properties against ascorbic acid were also observed against interfering substances such as uric acid and even glutathione. In addition, in the enzyme electrode of the present invention, after adding glucose, the oxidized form of H2O2 is added.

The response speed was also extremely excellent, with the flow reaching a steady value in about 10 seconds.

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

[Brief explanation of the drawing]

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 longitudinal sectional view showing an electrode system using an electrode holder equipped with an enzyme electrode, and FIG. 3 is a diagram showing the relationship between concentration and current increase amount for glucose and ascorbic acid. 1. ■ Porous membrane, 2. Acetylcellulose layer, 3. Enzyme, 4. Hydrogen peroxide detection electrode. Name of agent: Patent attorney Toshio Nakao and 1 other person No. 1
figure

Claims (2)

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