JPH023944B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention has electrochemical activity toward a substrate that undergoes a specific catalytic action of the substrate, enables rapid and simple measurement of the concentration of the substrate, and can be used repeatedly. This invention relates to enzyme electrodes that can be used. More specifically, when measuring the substrate concentration in a test solution using the oxidation current of hydrogen peroxide generated in an enzymatic reaction, there are various interfering substances contained in the test solution that interfere with electrochemical detection. This invention relates to an enzyme electrode that can eliminate the effects of

As an example of measuring the substrate concentration by anodic oxidation of hydrogen peroxide (H ₂ O ₃ ) produced by an enzymatic reaction, the case of glucose will be described below.

As shown in equation (1), glucose is oxidized by the action of glucose oxidase and H ₂ O ₂ is generated,
Next, this H ₂ O ₂ is oxidized using a platinum electrode as shown in equation (2), and the concentration of the substrate 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 Platinum electrode――――→ 2H ⁺ +2e＋O ₂ ………(2 ) To construct an enzyme electrode for substrate concentration measurement that can be used repeatedly by applying this principle, for example, in the above example, water-soluble glucose oxidase is immobilized on or near a current collector such as a platinum electrode. There is a need to. Various methods are known for fixing oxygen, such as using an organic polymer membrane such as acetyl cellulose as an immobilization carrier. An enzyme electrode is constructed.

On the other hand, when measuring the substrate concentration using the enzyme electrode as described above, there is a problem of interfering substances contained in the test liquid. For example, when measuring glucose in blood, various coexisting substances contained therein, such as uric acid and ascorbic acid, are electrolytically oxidized directly on the electrode. In other words, as shown in equation (2) above,
When H ₂ O ₂ is oxidized on the electrode, these coexisting substances are oxidized at the same time, giving an error to the obtained current value. A conventional example of an enzyme electrode that takes measures against these interfering substances is a method that uses two platinum anodes, immobilizes enzyme on only one, and compensates for the influence of interfering substances by subtracting the current values of both. (U.S. Patent No.
No. 3539455). However, this method has the disadvantage that it is very difficult to balance the responsivity of the two electrodes (platinum anode).

On the other hand, as an example of attempting to prevent diffusion of uric acid, ascorbic acid, etc. to the platinum electrode by placing a membrane made of cellulose acetate, silicone rubber, etc. on the test liquid side of the platinum anode, US Pat. No. 3,979,274, There is No. 4240889. The effect of the method described in this example on interfering substances is
It depends on the selectivity for H ₂ O ₂ and interfering substances in the above example, and it is difficult to completely block interfering substances. It is thought that increasing the film thickness to a certain extent will increase the effect, but this will conversely lead to a decrease in reaction current (decrease in sensitivity) and a decrease in response speed.

The present invention provides an enzyme electrode having excellent characteristics that are improved in the various points mentioned above.

The enzyme electrode of the present invention is composed of two electrodes, a first electrode and a second electrode. That is, the first electrode is for detecting H ₂ O ₂ generated in connection with the enzyme reaction, and the second electrode is for detecting a substance that interferes with the detection of H ₂ O ₂ at the first electrode. For example, it is for electrochemically oxidizing uric acid and ascorbic acid in advance. These two electrodes are each formed directly on both sides of the porous membrane.

FIG. 1 shows an enlarged schematic cross-sectional view of an embodiment of the enzyme electrode of the present invention. In the figure, 1 is a porous membrane that serves as a carrier, and a first electrode 2 for detecting H ₂ O ₂ is formed on one side of the membrane, and a membrane for electrolytically removing interfering substances is formed on the other side. A second electrode 3 is formed. Further, the enzyme is integrally immobilized in the pores 4 of the porous membrane and on the first electrode side to form an immobilized enzyme layer 5, thereby forming a thin film-like enzyme electrode as a whole.
Regarding the first electrode and the second electrode, an electrode material such as platinum is formed on the porous membrane by a method such as vapor deposition or sputtering. Regarding the positional relationship between the two electrodes, in order to prevent interfering substances in the test liquid from being oxidized by the first electrode, the second electrode is on the test liquid side with respect to the first electrode. Place it like this. For example, in measuring glucose concentration, if ascorbic acid coexists in the test solution, the second
Electrolytic oxidation can be performed in advance by setting the electrode potential at a sufficient oxidation potential of ascorbic acid. Since glucose is difficult to undergo immediate electrolysis, it passes through the second electrode and reaches the immobilized enzyme layer 5 (glucose oxidase in this case), where an enzymatic reaction generates H ₂ O ₂ . The generated H ₂ O ₂ is
is oxidized on the electrode, and a current depending on the glucose concentration in the test liquid is finally obtained. Like this,
In the enzyme electrode of the present invention, substances that interfere with electrochemical detection can be removed by electrochemical means, which is rational and highly effective.

As is clear from the above, the enzyme immobilization position is a place other than the second electrode side, that is,
It is most desirable to immobilize it in the pores of the porous membrane and on the first electrode side.

In the enzyme electrode of the present invention, two electrodes are formed directly on both sides of the porous membrane to form an independent electrode system, so it is necessary to configure the electrodes so that they do not short-circuit. From this point of view, it is preferable that at least one of the first electrode and the second electrode be formed inside the peripheral portion of the porous membrane. As an example, FIGS. 2 and 3 show a case where two electrodes are formed on a circular porous membrane. In the figure, 1 is a porous membrane, 2 is a first electrode, and 3 is a second electrode. In this case, the first electrode is formed inside the periphery of the porous membrane. Of course, contrary to this example, the second electrode may be formed inside the periphery of the porous membrane, or both electrodes may be formed inside the periphery.
In either case, the purpose of providing a structure that prevents short-circuiting between the first electrode and the second electrode is met.

Further, the shape of the porous membrane and the electrodes formed on the membrane is not limited to the circular shape shown in the above example, but can be any shape that suits the purpose of use. Furthermore, the distance between the periphery of the porous membrane and the outer periphery of the electrode formed inside the porous membrane is not particularly limited as long as it meets the objective of not short-circuiting the two electrodes.

Examples of the present invention will be described below.

As a carrier, pore diameter is 2000 Å, film thickness is 10 μm, and pore density is 3.
A polycarbonate porous membrane with ×10 ⁸ pieces/cm ² was used. A platinum layer having a surface resistance of 10 to 20 Ω was formed on one side of this film by sputtering, and this was used as a second electrode. Next, for the opposite surface, a platinum layer with a surface resistance of 10 to 20 Ω was formed by overlapping masks with holes with a diameter of 9 mm and sputtering in the same manner as above, and this was used as the first electrode. . The obtained membrane was cut into a diameter of 11 mm concentrically with respect to the platinum surface serving as the first electrode. The structure of the film thus obtained was as shown in FIGS. 2 and 3 above, and the resistance between the two electrodes was 10 MΩ or more.

Next, 10μ of a 100mg/ml aqueous solution of glucose oxidase as an enzyme was applied to the first electrode side of the membrane.
/ ^cm2 . Through this operation, the enzyme solution permeates into the pores of the membrane. Next, after drying this membrane, a crosslinking reaction was carried out at 25° C. for 1 hour in glutaraldehyde vapor as a bifunctional crosslinking reagent to immobilize it. After the reaction was completed, it was thoroughly washed with water.
This oxygen electrode is designated as A.

For comparison, a platinum layer was formed in the same manner as above, and the membrane obtained by cutting was immersed in an aqueous glucose oxidase solution (100 mg/ml), pulled up, dried, and then immobilized in the same manner as above. , washed. The enzyme electrode thus obtained has a layer of immobilized glucose oxidase on both electrode surfaces and in the pores. Let this be B.

FIG. 4 shows the cross section of the cylindrical electrode holder equipped with the enzyme electrode described above and the electrode system. In the figure, 6 is an enzyme electrode, which is attached to a resin main body 8 through a resin outer tube 7 so that the platinum layer of the first electrode is inside the electrode holder. is in contact with the platinum lead 9, and the platinum layer of the second electrode is in contact with the platinum lead 10, respectively. In addition, Ag/AgCl reference electrode 11 and counter electrode 1 for the first electrode
2 inside the electrode holder and against the second electrode.
An Ag/AgCl reference electrode 13 and a counter electrode 14 are arranged outside the electrode holder to constitute an electrode system. Further, the inside of the electrode holder is filled with a phosphate buffer solution 15 having a pH of 5.6.

The above electrode system was immersed in a phosphate buffer solution of pH 5.6, glucose or ascorbic acid was added, and changes in current for each of the enzyme electrodes A and B as a result of changes in concentration were measured. Glucose concentration and the first
FIG. 5 shows the relationship between the amount of increase in current of the electrode and the amount of increase in current of the electrode. In the figure, for each of A and B, the potential of the first electrode and the potential of the second electrode are both +0.60V (vs.Ag/
The solid line shows the case where the potential of the first electrode is +0.60V (vs.Ag/AgCl) and the circuit is cut off with no potential applied to the second electrode. Ta. As is clear from the figure, in enzyme electrode A according to the present invention, a good linear relationship was obtained regardless of whether the second electrode was on or off. On the other hand, in case B, when the second electrode was turned off, it showed almost the same performance as A, but when it was on, almost no response was obtained. this is,
Since glucose oxidase is also immobilized on the second electrode, glucose is reacted and consumed at this location, and the generated H ₂ O ₂ is also oxidized at the second electrode. In A, since the enzyme is immobilized in the pores of the membrane and on the first electrode side, B
There is no drop in response as in

Similarly to the case of glucose, the relationship between the ascorbic acid concentration and the amount of increase in current of the first electrode is
As shown in the figure. The solid line and the broken line each mean the same measurement conditions as in FIG. 5. As is clear from the figure, the second
It can be seen that the influence of ascorbic acid can be effectively removed by operating the electrode. The set potential of the second electrode is +0.60V (vs.Ag/
AgCl) or higher, sufficient effects were obtained.

In the examples, the case of ascorbic acid is shown, but other than this, uric acid, glutathione,
Good removal effects similar to those described above were also obtained for various reducing substances including bilirubin and the like.

In the oxygen electrode of the present invention, two electrodes and an immobilized enzyme layer are integrated on a porous membrane, and the whole has a thin film shape. It demonstrated excellent high-speed response. Furthermore, regarding the various response characteristics described above, stable performance was obtained over a long period of time even in repeated and continuous use.

Porous membranes that can be used are not limited to those shown in the examples, and any porous membrane may be used as long as it meets the gist of the present invention.

In the examples, the case where glucose oxidase was used as the enzyme was shown, but the present invention is not limited thereto. Any method that meets the gist of the present invention can be similarly applied. Moreover, in this case, a plurality of enzyme reactions may be involved.

The materials for the first electrode and the second electrode include:
It is not limited to the platinum shown in the examples, and any platinum may be used as long as it meets the gist of the present invention.

[Brief explanation of drawings]

Fig. 1 is a schematic cross-sectional view showing one embodiment of the enzyme electrode of the present invention, Fig. 2 is a plan view showing the positional relationship between the porous membrane and the electrode formed on this membrane, and Fig. 3 is a side view of the same. , Figure 4 is a schematic diagram showing the electrode holder and electrode system equipped with the enzyme electrode, Figure 5 is a diagram showing the relationship between concentration and current increase amount for glucose, and Figure 6 is a diagram showing the relationship between concentration and current increase amount for glucose.
The figure is a diagram showing the relationship between concentration and current increase amount for ascorbic acid. DESCRIPTION OF SYMBOLS 1... Porous membrane, 2... First electrode, 3... Second electrode, 4... Pore, 5... Immobilized enzyme layer.

Claims (1)

[Claims] 1 Consisting of at least a first electrode for detecting oxygen, a porous membrane, and hydrogen peroxide, and a second electrode for electrolytically removing substances that interfere with detection by the first electrode, a first electrode on one side of the porous membrane and a second electrode on the other side, and at least one electrode is formed inside the peripheral part of the porous membrane, and the enzyme is An enzyme electrode characterized in that the enzyme electrode is immobilized in the pores of a plasma membrane and on the first electrode side.