JPS6319818B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention aims to effectively utilize enzymes in enzyme-based reactions, and particularly to obtain an enzyme electrode equipped with an enzyme-immobilized membrane that can be used continuously and repeatedly.

In recent years, with advances in enzyme immobilization technology, various attempts have been made to detect the concentration of a substrate, which is a substance that specifically reacts with an enzyme, by combining an enzymatic reaction and an electrochemical reaction. As an example, a method is known in which hydrogen peroxide (H ₂ O ₂ ) produced by an enzymatic reaction is electrochemically detected. That is, as shown in the following formulas (1) and (2), a substrate such as glucose is oxidized to produce H ₂ O ₂ by the action of an oxidoreductase such as glucose oxidase that uses oxygen as a hydrogen acceptor. Next, the generated H ₂ O ₂ is oxidized using, for example, a platinum electrode, and the concentration of the substrate can be determined from the oxidation current value obtained at this time.

H ₂ O ₂ →2H ⁺ +2e+O ₂ ...(2) However, since the enzyme is water-soluble, in order to enable repeated use of the expensive enzyme, the enzyme must be placed near the hydrogen peroxide detection electrode using an appropriate method. It is necessary to immobilize (insolubilize). On the other hand, in this hydrogen peroxide detection method, dissolved oxygen in the liquid is used as the hydrogen acceptor, so when the substrate concentration becomes high, it is not possible to supply the sufficient amount of oxygen required for the reaction of equation (1). The linear relationship between the resulting H ₂ O ₂ oxidation current and substrate concentration is lost. In principle, the oxygen consumed in equation (1) is electrochemically regenerated in equation (2), but in reality, H ₂ O ₂ generated in equation (1) also diffuses into the liquid. , linearity is lost more quickly.

Furthermore, if substances that are easily oxidized directly on the electrode, such as uric acid or ascorbic acid, coexist in the test solution, the oxidation current of these coexisting substances will be generated in addition to the oxidation current of H ₂ O ₂ generated by the enzyme-substrate reaction. This will cause errors.

As a solution to these various problems,
The following methods are possible. That is, as shown in equation (3), H ₂ O ₂ that diffuses into the liquid can be decomposed into oxygen using catalase. In this case Kata As for lase, it is necessary to immobilize it so that it is located on the test liquid side.

In addition, for uric acid, ascorbic acid, etc.
A known method is to use a film of silicone rubber or cellulose acetate to prevent these interfering substances from diffusing into the electrode. However, when such a membrane is used, the substrate in the test liquid will diffuse through the membrane, resulting in a delay in response. Such a delay in response becomes a problem, especially when a large number of analytes are analyzed continuously. In addition, uricase,
There is also a method of oxidizing uric acid and ascorbic acid using ascorbic acid oxidase, but at the same time
Since H ₂ O ₂ is also generated, an error will occur.

Practically speaking, an enzyme electrode is desired that has high selectivity even for the above-mentioned coexisting substances, has excellent linearity with respect to substrate concentration, and exhibits a rapid response. As a result of various studies on these points, we discovered an enzyme electrode with excellent properties.

An example of the structure of an enzyme electrode according to the present invention is shown in FIG. In the figure, 1 is a layer formed by immobilizing catalase or, in addition, uricase and ascorbic acid oxidase, 2 is a porous membrane for enzyme immobilization that has pores made of polycarbonate, etc., and 3 is selected according to the target substrate. An immobilized layer of an oxidoreductase that acts on the membrane to produce H ₂ O ₂ , and 4 is a hydrogen peroxide detection electrode made of platinum or the like.

The present invention is characterized by using a porous membrane as an immobilization carrier, immobilizing necessary enzymes by supplying immobilization reagents from the gas phase to both sides of the membrane, and using hydrogen peroxide detection electrodes and The point is that it is placed in an appropriate positional relationship with respect to the test liquid. In other words, an enzyme that acts on the target substrate to generate H ₂ O ₂ is immobilized on the hydrogen peroxide detection electrode side of the membrane.
Catalase is immobilized on the other side, that is, on the test liquid side. By arranging it in this way, it is possible to decompose H ₂ O ₂ that diffuses into the test liquid into oxygen in advance and suppress a decrease in response linearity in the high concentration region of the substrate. Furthermore, for interfering substances such as ascorbic acid and uric acid, by immobilizing ascorbate oxidase and uricase together with catalase, these interfering substances that diffuse from the test liquid to the hydrogen peroxide detection electrode are pre-empted by the enzymatic reaction. The H ₂ O ₂ produced at this time is decomposed into oxygen by the action of catalase.

As described above, the enzyme electrode of the present invention has excellent response linearity and also has high selectivity against the above-mentioned interfering substances.

Furthermore, using a membrane to immobilize enzymes has advantages in terms of ease of immobilization and strength, but as already mentioned, the problem is the delay in response. However, in the present invention, a porous membrane is used as a carrier for immobilization, and by supplying the immobilization reagent from the gas phase, both sides of one membrane are used to transfer multiple enzymes. are properly placed and fixed. Therefore, it is possible to obtain an enzyme electrode that has excellent strength and quick response.

Examples of the present invention will be described below.

An electrode for detecting hydrogen peroxide was fabricated by coating the surface of Nesa glass with platinum using a pyrolysis method and electrically connecting it with silver paste.

This electrode was used to construct enzyme electrodes A and B below.

(A) Polycarbonate porous membrane (film thickness 8 μm, pore diameter 10 μm, pore density 1×
10 ⁵ pieces/cm ² ) was used. On one side of this membrane,
A mixed solution of catalase and ascorbic acid oxidase supplemented with albumin was applied, slightly dried, and then soaked in glutaraldehyde vapor for 25 minutes.
The mixture was reacted at ℃ for about 1 hour to achieve crosslinking and fixation, and then thoroughly washed with water to remove unreacted substances. next,
A glucose oxidase aqueous solution was applied to the other side of the membrane, and after slightly drying, crosslinking and immobilization was carried out in the same manner as above. In this way, different types of enzymes can be immobilized on both sides of the porous membrane by supplying glutaraldehyde as an immobilization reagent not from the liquid phase (solution) but from the gas phase and allowing the immobilization reaction to proceed. Can be done. This is because since the reagent is not in a solution state, the enzyme previously retained on the porous membrane is not eluted and is immobilized in almost the same retained state.

The glucose oxidase-immobilized side of the enzyme-immobilized membrane obtained above was closely fixed to the hydrogen peroxide detection electrode to form an enzyme electrode.

(B) Glucose oxidase alone was immobilized on one side of the membrane by the same method as above, and this immobilized surface was tightly immobilized on a hydrogen peroxide detection electrode to serve as an enzyme electrode for comparison.

Using the enzyme electrodes A and B obtained above, response characteristics to changes in concentration of glucose and ascorbic acid were measured using the measurement system shown in FIG.

In Figure 2, 5 is a recorder, 6 is a potentiostat, 7 is a saturated calomel reference electrode, and 8 is a resin electrode holder with an enzyme electrode attached to its lower end, which is connected to the potentiostat via a lead. ing. 9 is a phosphate buffer containing a substrate, 10 is a salt bridge, and 11 is a counter electrode. After immersing _the enzyme electrode in the solution and setting the potential to be sufficient to oxidize _H2O2 ,
Glucose or ascorbic acid was added while stirring to obtain a predetermined concentration, and the change in current flowing at this time was measured.

FIG. 3 shows the response characteristics of enzyme electrodes A and B to glucose. It can be seen that electrode A, which has catalase immobilized thereon, has good properties such as excellent linearity in the high concentration range. It was also found that the response speed was fast and the system had sufficient promptness.

FIG. 4 shows the response characteristics of enzyme electrodes A and B to ascorbic acid. In electrode A, in which ascorbic acid oxidase is immobilized, direct oxidation of ascorbic acid is reduced, and selectivity is significantly improved.

Furthermore, for uric acid, by immobilizing uricase together with catalase, the same effect as in the case of ascorbic acid was obtained.

As substrate enzymes to be analyzed, in addition to glucose oxidase, oxidoreductases that generate H ₂ O ₂ through enzymatic reactions such as xanthine oxidase, amino acid oxidase, cholesterol oxidase, and alcohol oxidase can be used. Furthermore, it can also be applied to complex enzyme systems containing these enzymes. Further, when immobilizing an enzyme, as shown in the Examples, immobilization can be facilitated by using a protein other than the enzyme, such as albumin, in combination. In addition, as an electrode for hydrogen peroxide detection,
In addition to platinum, various noble metals and metal oxides can be used.

As described above, the enzyme electrode of the present invention has great utility value as it has excellent response linearity and selectivity and can be used repeatedly.

[Brief explanation of the drawing]

Figure 1 is a schematic cross-sectional view showing an example of the configuration of the enzyme electrode of the present invention, Figure 2 is a diagram showing the configuration of the measurement system, and Figure 3 is a diagram showing the configuration of the measurement system.
Figure 4 shows the response characteristics to glucose.
The figure also shows the response characteristics to ascorbic acid. 1... Catalase immobilization layer, 2... Porous membrane,
3... Immobilized layer of oxidoreductase, 4... Electrode for detecting hydrogen peroxide.

Claims (1)

[Scope of Claims] 1. Consisting of a porous membrane in which an enzyme is immobilized by supplying an immobilized reagent from a gas phase and a hydrogen peroxide detection electrode, the hydrogen peroxide detection electrode side of the porous membrane has a An enzyme electrode characterized in that an oxidoreductase that uses oxygen as a hydrogen acceptor is immobilized, and calatase is immobilized on the other side. 2. The enzyme electrode according to claim 1, wherein at least one of uricase and ascorbate oxidase is immobilized together with the catalase.