JPH0643130A - Composite type fixing enzyme film - Google Patents

Abstract

PURPOSE:To greatly reduce the deterioration of selection transmissive film under a high pH value so as to realize a long life thereof by forming the selection transmissive film integrally on one face of a fixing enzyme film and further forming a specific auxiliary film integrally on the surface of the selection transmissive film. CONSTITUTION:A hydrogen peroxide selection transmissive film 2 made of acetylcellulose is integrally formed under a fixing enzyme film 1 that is formed by fixing uricase on a polyacrylnitrile film and an auxiliary film 3 made of polymer perfluorosulfonate is integrally formed under the film 2 to make a composite type fixing enzyme film A. The film A is attached on the surface of hydrogen peroxide electrode B, and it is immersed in a buffer solution of high pH during non-measurement, while, when a test solution is dropped thereon for measurement, a high pH solution (for example, urine) is neutralized to some extent by sulfonic acid containing in the film 3 between the surface of the electrode B and the film 2 where the high pH solution may tend to remain, thereby reducing the deterioration of the film 2 to the utmost. As a result, the service life of entire film A can be greatly improved.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composite-type immobilized enzyme membrane, and more specifically, a composite-type immobilized enzyme membrane including a permselective membrane and an immobilized enzyme membrane, which are arranged on an electrode in a laminated state. Regarding

[0002]

2. Description of the Related Art An enzyme obtained by immobilizing an enzyme on the surface of a base electrode has been focused on the characteristic that a complex organic compound represented by a protein can be detected with extremely high sensitivity. Research and development have been conducted to measure the above organic compounds by electrodes. Further, in recent years, in performing measurements in clinical trials in the fields of medical care, industrial processes, environment, etc., the diversification of measurement items and the increase in the amount of measurement have become remarkable.
There is a strong demand for an enzyme sensor that can measure easily and continuously.

This enzyme sensor is generally constructed by immobilizing an enzyme on the surface of a base electrode having a measuring electrode made of platinum or the like and a counter electrode made of silver or the like. It is possible to cause the oxidation reaction or reduction reaction of only the measurement target substance by simply contacting the test solution containing the measurement target substance with, and to determine the amount of the substance that is generated or lost as a result of the oxidation reaction or reduction reaction. A corresponding electric signal can be output from the base electrode. Therefore, the concentration of the substance to be measured in the test solution can be easily measured by performing a predetermined process based on the output electric signal. Further, if a base electrode having a thermistor or the like embedded therein is used, an electric signal corresponding to the amount of heat generated by the oxidation reaction or reduction reaction carried out in the presence of an enzyme can be output,
Similarly, the concentration of the substance to be measured in the test solution can be easily measured.

By the way, in order to immobilize the enzyme on the surface of the base electrode, an enzyme-immobilized film having the enzyme immobilized thereon is obtained in advance, and the enzyme-immobilized film is attached to the surface of the base electrode. In order to improve the workability of wearing, for example, a complex-type immobilized enzyme membrane in which an immobilized enzyme membrane obtained by immobilizing an enzyme on a polyacrylonitrile membrane and a hydrogen peroxide selective permeation membrane composed of an acetylcellulose membrane are laminated. Has been adopted.

[0005]

The above-mentioned complex-type immobilized enzyme membrane is most strongly required to maintain high enzyme activity. Depending on the type of enzyme, low pH may be the optimum condition, High pH may be the optimum condition. For example, uricase, which is one of the enzymes suitable for measuring uric acid, has a pH of 8 to 9 under optimum conditions. Therefore, a buffer solution of pH 8 to 9 must be used in order to maintain the activity of uricase during non-measurement. In this case, the activity of uricase can be maintained high, but the acetylcellulose membrane, which is a hydrogen peroxide selective permeable membrane, is significantly deteriorated due to hydrolysis under high pH conditions, and the desired hydrogen peroxide selection There is an inconvenience that the permeation performance cannot be exhibited (for example, the ascorbic acid inhibiting ability is significantly reduced when used as a uric acid tester). In addition, since the high pH condition is set at the time of measurement, there is a disadvantage that the acetyl cellulose membrane is similarly deteriorated. And if acetyl cellulose deteriorates, even if the activity of uricase itself is sufficient,
The complex-type immobilized enzyme membrane itself has to be replaced with a new complex-type immobilized enzyme membrane, which is a disadvantage that uricase is discarded more than necessary.

Although the case where uricase is used as the enzyme and the acetylcellulose membrane is used as the hydrogen peroxide selective permeation membrane have been described above, the enzyme having a high pH as an optimum condition and an excessive tolerance to the high pH. The same inconvenience also occurs when a hydrogen oxide selective permeable membrane is used. Also,
Similar disadvantages occur when an oxygen electrode is used instead of the hydrogen peroxide electrode.

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and an enzyme and a high p for which high pH is the optimum condition.
An object of the present invention is to provide a novel complex-type immobilized enzyme membrane capable of significantly reducing the deterioration of the permselective membrane and achieving a long life even when the permselective membrane having little resistance to H is used. .

[0008]

In order to achieve the above object, the composite immobilized enzyme membrane according to claim 1 has a selective permeation membrane integrally formed on one surface of the immobilized enzyme membrane. An auxiliary membrane made of a perfluorosulfonic acid polymer is integrally formed on the surface of the permeable membrane. Here, when the composite immobilized enzyme membrane is attached to the hydrogen peroxide electrode, the hydrogen peroxide selective permeable membrane is adopted as the selective permeable membrane, and when it is attached to the oxygen electrode, the oxygen selective permeable membrane is used. Should be adopted.

In the composite immobilized enzyme membrane of claim 2, an auxiliary membrane made of a perfluorosulfonic acid polymer is integrally formed on the other surface of the immobilized enzyme membrane.

[0010]

According to the complex type immobilized enzyme membrane of claim 1, the selective permeation membrane is integrally formed on one surface of the immobilized enzyme membrane, and the auxiliary permeation membrane made of a perfluorosulfonic acid polymer is formed on the surface of the selective permeation membrane. Since the membrane is integrally formed, when the complex type immobilized enzyme membrane is attached to the surface of the electrode, it is immersed in a high pH buffer solution during non-measurement and the test solution is spotted during measurement. The high pH solution is most likely to remain. Since the auxiliary membrane made of a perfluorosulfonic acid polymer is interposed between the electrode surface and the selectively permeable membrane, the sulfonic acid neutralizes the high pH solution to some extent. And then
The deterioration of the selectively permeable membrane can be significantly reduced. As a result, the life of the composite-type immobilized enzyme membrane as a whole can be significantly lengthened.

According to the complex type immobilized enzyme membrane of claim 2,
Since the auxiliary membrane made of perfluorosulfonic acid polymer is integrally formed on the other surface of the immobilized enzyme membrane,
Even when a high pH solution is supplied from the immobilized enzyme membrane side, the sulfonic acid will neutralize the high pH solution to some extent,
The deterioration of the selectively permeable membrane can be further reduced significantly. As a result,
The life of the complex-type immobilized enzyme membrane as a whole can be further extended.

[0012]

Embodiments will be described in detail below with reference to the accompanying drawings showing embodiments. FIG. 1 is an exploded perspective view showing an embodiment of a composite-type immobilized enzyme membrane of the present invention together with a hydrogen peroxide electrode. Acetylcellulose is formed on the lower surface of an immobilized enzyme membrane 1 obtained by immobilizing uricase on a polyacrylonitrile membrane. The composite immobilization enzyme membrane A is formed by integrally providing the hydrogen peroxide selective permeation membrane 2 made of A and the auxiliary membrane 3 made of a perfluorosulfonic acid polymer integrally on the lower surface of the hydrogen peroxide selective permeation membrane 2. The composite immobilized enzyme membrane A is attached to the surface of the hydrogen peroxide electrode B.

As a method of forming the above-mentioned auxiliary membrane 3, for example, on the hydrogen peroxide selective permeable membrane side of the already formed composite membrane consisting of the immobilized enzyme membrane 1 and the hydrogen peroxide selective permeable membrane 2, Nafion (registered trademark) 1 manufactured by Aldrich
5% aqueous solution of 17 (perfluoro-3,6-di
oxa-4-methyl-7-octensulfo
nic acid

[0014]

[Chemical 1]

) 0.5 ml was dropped, and after 5 seconds, the composite membrane was removed at 1000 r. p. m. Rotate for 5 seconds, then 3
000r. p. m. By rotating for 45 seconds,
It is possible to obtain the composite immobilized enzyme membrane A in which the auxiliary membrane 3 is integrally formed on the hydrogen peroxide selective permeable membrane side. FIG. 2 is a diagram showing a change in permeation rate of ascorbic acid measured using the complex-type immobilized enzyme membrane of the present invention and the complex-type immobilized enzyme membrane that does not simply include the auxiliary membrane 3. In addition, Na ₂ HPO ₄ 50 mM and KCl 100 mM were used as a buffer solution, heated to 60 ° C. in an oven with a fan, and 100 mg / dl of ascorbic acid was used to output the output current (n
The change with time of A) was measured. The white circles represent the output current when the composite immobilized enzyme membrane of the present invention is used, and the open squares represent the output current when the composite immobilized enzyme membrane that does not include the auxiliary membrane 3 is simply adopted. .

As is apparent from FIG. 2, when the composite immobilized enzyme membrane of the present invention was adopted, the output current was at a significantly low level and hardly increased, whereas the auxiliary membrane 3 was simply used. When a complex-type immobilized enzyme membrane containing no is used, the output current rises sharply and is almost saturated after about 1 hour. That is, when the complex-type immobilized enzyme membrane of the present invention was adopted, the ascorbic acid permeation-inhibiting ability was hardly reduced, whereas the complex-type immobilized enzyme membrane not containing the auxiliary membrane 3 was simply adopted. In case of about 1
It can be seen that the ascorbic acid permeation-inhibiting ability rapidly decreased until the time elapsed, and the ascorbic acid permeation-inhibiting ability almost disappeared after about 1 hour. Further, comparing based on the increase rate of the output current up to the time of 30 minutes, it is found that the ascorbic acid permeation inhibiting ability can be increased to about 16.6 times by simply providing the auxiliary membrane 3 integrally. As is clear from the above, the hydrogen peroxide selective permeable membrane 2
The life of the composite immobilized enzyme membrane can be remarkably extended by simply forming the auxiliary membrane 3 integrally on the surface of the.

[0017]

[Embodiment 2] FIG. 3 is an exploded perspective view showing another embodiment of the composite-type immobilized enzyme membrane of the present invention together with the hydrogen peroxide electrode. The difference from FIG. The only difference is that the auxiliary film 4 made of perfluorosulfonic acid polymer is integrally formed. Therefore, in the case of this embodiment, when the high pH solution (for example, urine) is spotted, the sulfonic acid of the auxiliary membrane 4 neutralizes the high pH solution to a certain extent, and thus the hydrogen peroxide selective permeable membrane 2 of Deterioration can be further reduced,
As a result, the life of the complex-type immobilized enzyme membrane can be further extended.

The present invention is not limited to the above-mentioned embodiment. For example, instead of forming the auxiliary membrane 4 on the upper surface of the immobilized enzyme membrane 1, the immobilized enzyme membrane 1 and hydrogen peroxide are selected. It is possible to form an auxiliary membrane between the membrane and the permeable membrane 2, and it is also possible to apply it to a complex-type immobilized enzyme membrane mounted on an oxygen electrode. It is possible to make various design changes within the range that does not.

[0019]

As described above, the invention of claim 1 has a high pH.
Since the solution is most likely to remain, since the auxiliary membrane made of perfluorosulfonic acid polymer is interposed between the electrode surface and the selectively permeable membrane, the sulfonic acid neutralizes the high pH solution to some extent. In addition, it has a unique effect that the deterioration of the permselective membrane can be significantly reduced and the life of the entire composite-type immobilized enzyme membrane can be significantly lengthened.

According to the second aspect of the present invention, even when the high pH solution is supplied from the immobilized enzyme membrane side, the sulfonic acid neutralizes the high pH solution to some extent, thereby further significantly degrading the permselective membrane. It has a unique effect that it can be reduced and the life of the composite-type immobilized enzyme membrane as a whole can be further extended.

[Brief description of drawings]

FIG. 1 is an exploded perspective view showing an embodiment of a composite-type immobilized enzyme membrane of the present invention together with a hydrogen peroxide electrode.

FIG. 2 is a diagram showing changes in the rate of permeation of ascorbic acid measured using the complex-type immobilized enzyme membrane of the present invention and the complex-type immobilized enzyme membrane that does not simply include the auxiliary membrane.

FIG. 3 is an exploded perspective view showing another embodiment of the composite-type immobilized enzyme membrane of the present invention together with a hydrogen peroxide electrode.

[Explanation of symbols]

1 Immobilized enzyme membrane 2 Hydrogen peroxide selective permeation membrane 3,4 Auxiliary membrane composed of perfluorosulfonic acid polymer A Composite immobilized enzyme membrane B Hydrogen peroxide electrode

Claims (2)

[Claims] 1. Arranged in a laminated state on the electrode (B),
A composite immobilized enzyme membrane (A) comprising a permselective membrane (2) and an immobilized enzyme membrane (1), wherein the permselective membrane (2) is integrally formed on one surface of the immobilized enzyme membrane (1). A composite type immobilized enzyme membrane, wherein an auxiliary membrane (3) made of a perfluorosulfonic acid polymer is integrally formed on the surface of the permselective membrane (2). 2. The composite immobilized enzyme membrane according to claim 1, wherein an auxiliary membrane (4) made of a perfluorosulfonic acid polymer is integrally formed on the other surface of the immobilized enzyme membrane (1). .