JPS58139059A - enzyme electrode - Google Patents

Abstract

PURPOSE:To provide an enzyme electrode which is stabilized for a long period of time, fast in responsiveness, and is capable of preventing diffusion of an obstacle material to an H2O2 detecting electrode, by a method wherein the H2O2 detecting electrode is formed on one surface of a porous film, and an enzyme is secured in the hole of the porous film and to said electrode side. CONSTITUTION:A thin layer such as Pt is formed as an H2O2 detecting electrode 2 on one surface of a porous film 1 such as polycarbonate. After, for example, a glucose oxidase aqueous solution is dripped and developed on the film 1 formed on the electrode 2, it is dried. Then, after an enzyme is cured in steam of a crosslinking agent such as glutalaldehyde, it is washed by water, and an enzyme electrode 9 is obtained such that the enzyme is, like a 3a, secured in a hole 4 in the film 1, and it is, like a 3b, secured to the surface of the electrode 2 on the film 1. The enzyme electrode 9 is held to a cylindrical holder body 12 through the medium of a packing 10 by a cap 11 in a manner to bring the Pt electrode 2 side into contact with an electrolyte 13 to obtain an enzyme electrode which is suitable for quantitative determination of glucose and the like in blood.

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, particularly an enzyme electrode that includes an immobilized enzyme and an electrode for detecting hydrogen peroxide.

Glucose, cholesterol, etc. are also important as analytical items for blood components, and various methods have been attempted using enzyme electrodes as analysis means. In other words, the following (1)
As in the glucose example shown in equation (2), the substrate (glucose) is oxidized by the action of an enzyme (glucose oxidase) to generate hydrogen peroxide (H2O2), and then this H2O2 is oxidized using, for example, a platinum electrode. The substrate concentration can be determined from the oxidation current value.

Glucose +02. -1. Knee◆ Gluconolactone +
H2o2・・・・−・−・−・−・・0) H2O2+ 2H++2e+02. ,-,, (2) However, since halogen is water-soluble, in order to enable repeated use of expensive enzymes and to quickly and easily measure the substrate concentration, it is necessary to immobilize the enzyme and use peroxidized It is desirable to have an enzyme electrode integrated with a hydrogen electrode.

Conventionally, this type of enzyme electrode has been used by immobilizing an enzyme using an organic polymer membrane made of cellulose or the like as an immobilization carrier, and overlapping this enzyme immobilization membrane with a platinum plate as an electrode for detecting hydrogen peroxide.・There is a way to be there. This enzyme electrode has the drawbacks that the enzyme-immobilized membrane must be brought into close contact with the electrode for detecting arsenic, which complicates the operation when replacing the membrane, and leads to a decrease in reproducibility. Furthermore, in order to obtain a rapid response to changes in substrate concentration, it is necessary to minimize the gap between the hydrogen peroxide detection electrode and the enzyme-immobilized membrane, and improvements are desired from this perspective as well. Met.

Therefore, the present inventors conducted various studies regarding the points mentioned above, and as a result, they discovered an enzyme electrode with excellent characteristics. The enzyme electrode of the present invention is characterized by a membrane on one side of the porous membrane. The hydrogen peroxide detection electrode is formed on the surface, and the enzyme is immobilized in the pores of the porous membrane and on the hydrogen peroxide detection electrode side. Furthermore, the pores of the porous membrane are filled with an immobilized enzyme.

Figure 1 shows a schematic cross-sectional view of one embodiment of the enzyme electrode of the present invention. In the figure, 1 is a porous membrane that serves as a carrier. Layer 2 was formed to serve as an electrode for detecting hydrogen peroxide.

3a and 3b are enzymes immobilized in the pores 4 of the membrane and on the hydrogen peroxide detection electrode side 2, respectively.

When using this enzyme electrode, it is arranged so that the membrane surface opposite to the hydrogen peroxide detection electrode 2, that is, the membrane surface 6 on which no enzyme is immobilized, faces the test liquid side. The substrate in the test solution mainly generates H2O2 by the action of immobilized enzyme 3a,
O2 diffuses and is detected as an anode current by the hydrogen peroxide detection electrode 2.

The immobilized enzyme 3b has the role of sufficiently firmly immobilizing the enzyme on the membrane together with the immobilized enzyme 3a in addition to its catalytic effect on the substrate. In addition, no enzyme is immobilized on the membrane surface 5, but this is because if the enzyme is immobilized on the membrane surface that comes into direct contact with the test liquid, the enzyme on this part may fall off depending on the conditions of use. This is because changes in characteristics are also expected. In this respect, □ In the enzyme electrode according to the present invention, the enzyme is immobilized in the pores, and a stable response can be maintained even during long-term use.

In addition, if substances that are susceptible to direct electrolytic oxidation, such as ascorbic acid and uric acid, coexist as interfering substances in the test solution, these interfering substances can be removed by hydrogen peroxide by filling the pores with immobilized enzyme. Diffusion to the detection electrode can be effectively prevented.

As described above, the enzyme electrode of the present invention can effectively reduce the influence of interfering substances, and the hydrogen peroxide detection electrode is formed directly on the porous membrane, so the enzyme electrode has a thin film shape as a whole. It has excellent response sensitivity and response speed, and stable response characteristics can be obtained over a long period of time even when used continuously or repeatedly.

The enzyme used is not limited to one type, and may be a complex enzyme system as long as it generates H2O2 in the enzymatic reaction. For the hydrogen peroxide detection electrode formed on the porous membrane, in addition to the above-mentioned platinum, metals and metal oxides suitable for the purposes already mentioned, such as ruthenium, can also be used.

The porous membrane used as a carrier should be made of a material on which an electrode for detecting hydrogen peroxide can be formed, and which does not impair adhesion to platinum, etc. formed on the membrane when used in an aqueous solution. The one is good.

An example of such a porous membrane is polycarbonate.

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

Hereinafter, the present invention will be explained with reference to examples thereof.

Example 1 A polycarbonate porous membrane (pore size 2oo) was used as a carrier membrane.
A platinum layer with a thickness of several hundred to several thousand angstroms was formed on one side of this membrane by sputtering, and was used as an electrode for detecting hydrogen peroxide. did. Next, a glucose oxidase aqueous solution (200 my
/IRl) was added dropwise at a rate of 10 pl/crrt, spread uniformly, and dried. Next, an immobilization reaction was performed at 26°C for 60 minutes in glutaraldehyde vapor as a cross-linking reagent. After that, it was thoroughly washed with water. In this way, an enzyme electrode as shown in FIG. 1 was obtained. Enzymes 3b and 3a are integrally immobilized on the platinum layer and on the inner wall surface of the pores, respectively, as shown in the figure.

The enzyme electrode described above was cut into a circular shape with a diameter of 10 mm, and was mounted on a cylindrical electrode holder shown in FIG. 2 for measurement. In the figure, 6 is an Aq/Aq C1l reference electrode, 7 is a counter electrode, and 8 is a platinum lead, which is in contact with the platinum layer of the membrane. Reference numeral 9 denotes an enzyme electrode, and the cylindrical body 1 made of resin is attached to the cap 11 via a gasket 10 so that the platinum layer of the membrane is on the inside of the holder.
It is held at 2.

The inside of the electrode holder is filled with electrolyte 13. After immersing this electrode holder in a phosphate buffer solution of pH 5.6 and setting the potential of the enzyme electrode to +0.6 V with respect to the reference electrode, to increase the concentration to 2 x 10-5 mol/
1, the current rapidly increased as shown in FIG. 3, and a rapid response was obtained, with the current reaching a steady μ value after about 6 seconds. In addition,
In the figure, A indicates the time point of glucose addition. In addition, a good linear relationship was obtained between the amount of current increase up to the steady-state value and the glucose concentration, as shown in Figure 4B, and this characteristic shows that there is almost no decline in deer usage after approximately 1000 uses. It had excellent life characteristics.

Example 2 An enzyme electrode prepared in exactly the same manner as in Example 1 was washed with water.

After drying, add glucose oxidase aqueous solution (20
01rIg//) was dropped onto the membrane surface on the platinum layer side at a rate of 1 apl/cni, spread uniformly, dried, and immobilized in the same manner as in Example 1 above. In the enzyme electrode obtained by repeating such enzyme immobilization process six times in total, the porous pores 4 shown in FIG. 1 are almost filled with the immobilized enzyme. The response characteristics of this enzyme electrode to glucose were good as shown in FIG. 4C, and the response speed and lifespan characteristics were also excellent as in Example 1.

In addition, in the same manner as in the case of glucose, Example 1,
FIG. 6 shows the response characteristics of the enzyme electrodes obtained in 2 and 2 to ascorbic acid.

In the case of the enzyme electrode of Example 1, some oxidation current is observed as shown in B, but in the case of the enzyme electrode of Example 2, C
As shown, the oxidation current of ascorbic acid can be sufficiently suppressed. This is thought to be because the pores of the porous membrane are filled with the immobilized enzyme, which inhibits the permeation of ascorbic acid.

Enzyme immobilization on porous membranes is not limited to enzymes alone; by developing and immobilizing a mixed solution with proteins such as albumin, the strength of the immobilized enzyme layer and suppression of interfering substances can be improved. The effect can be further improved.

[Brief explanation of the drawing]

FIG. 1 is a schematic cross-sectional view showing an embodiment of an enzyme electrode according to the present invention, FIG. 2 is a schematic view showing an electrode holder equipped with an enzyme electrode and an electrode system, and FIG. 3 is a schematic cross-sectional view showing an example of an enzyme electrode according to the present invention. FIG. 4 is a diagram showing the relationship between glucose concentration and current increase amount, and FIG. 6 is a diagram showing the relationship between ascorbic acid concentration and current increase amount. 1...Porous membrane, 2...Hydrogen peroxide detection electrode. 0 3a, 3b... Enzyme, 4... Pore. Name of agent: Patent attorney Toshio Nakao and 1 other person11
111 Time (Figure 4 Groups' own skin (7/Ju〆)

Claims (2)

[Claims] (1) It has a porous membrane, a hydrogen peroxide detection electrode formed on one side of the membrane surface, and an enzyme immobilized on the pores of the porous membrane and the hydrogen peroxide detection electrode side. Enzyme electrode. (2) The enzyme electrode 0 according to claim 1, wherein the pores of the porous membrane are filled with an immobilized enzyme.