JPS60117143A - Enzyme electrode - Google Patents

Abstract

PURPOSE:To maintain enzyme activity and sensitivity by arranging a storage section for storing replenishing enzyme to an enzyme section and a means of introducing it in an oxygen electrode equipped with an immobilized enzyme section having selectivity for a specified substrate. CONSTITUTION:An immobilized enzyme membrane 6 functions as immobilized enzyme section. The immobilized enzyme membrane 6 is connected to an enzyme storage section 1 provided outside with a fine tube 2 and a fine tube 3. Then, enzyme is replenished only by a required amount through these tubes 2 and 3. An O ring 4 for putting a plastic protective cover 10 tight on an electrode, an oxygen transmitting film 5, a platinum electrode 7 connected to the immobilized enzyme membrane 6 and a lead electrode 8 as a reference electrode are also arranged. Thus, the enzyme electrode can be replenished with enzyme to improve the activity, sensitivity and long-time stability of enzyme substantially.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field of the Invention] The present invention relates to an enzyme electrode equipped with an immobilized enzyme portion having a constant selectivity for a substrate of 1%.

[Technical background of the invention and its problems]

For example, an enzyme electrode equipped with an immobilized enzyme part utilizes the function of an enzyme to selectively react only with respect to its substrate. Because it has a catalytic effect and can be used repeatedly, its future development is attracting attention. In particular, it can play an important role in fields such as biochemical analysis and clinical chemical analysis.

For example, uricase can be used to analyze uric acid, which plays an important role in gout diagnosis, and glucose oxidase can be used to analyze glucose, which is extremely important in diabetes diagnosis and treatment.
Since analysis can be performed easily and in a short time, it is useful for early diagnosis and bedside treatment. For example, the gelcol sensor, which immobilizes this glucose oxidase on the immobilized enzyme portion of an enzyme electrode and determines the blood sugar level from the amount of decrease in dissolved oxygen or increase in dissolved hydrogen peroxide caused by the enzyme reaction, is the most general-purpose sensor. This is an example of an enzyme electrode, but it has been pointed out that a problem with such an enzyme electrode is that the activity of the enzyme used decreases over time, and after a certain period of time, it must be verified after each measurement. has been done. This is thought to be because the immobilized enzyme is deactivated by repeated use. In response, various measures have been proposed to stabilize the activity of immobilized enzymes for a long period of time. In the case of glucose oxidase, hydrogen peroxide produced as a result of the reaction is thought to be a factor in enzyme deactivation, so camerase, which decomposes fugitive hydrogen, is immobilized together with gelcol oxidase in the immobilized enzyme part. Attempts have been made to reduce the rate of deactivation of glucose oxidase. However, when monitoring the reaction with an oxygen electrode, the decrease in dissolved oxygen due to the oxidative decomposition of glucose is offset by the oxygen generated from the decomposition of hydrogen peroxide, and the apparent measurement sensitivity is halved. turn into. Also.

In the case of proteases such as xotrypsin, it is known that the stability can be increased several times by covalently immobilizing them on carriers, but in this case, immobilization significantly reduces the activity of the enzyme. A problem has arisen.

[Purpose of the invention]

In view of the above points, the present inventors aimed to provide an enzyme electrode that maintains enzyme activity and sensitivity and allows stable measurement over a long period of time.

[Summary of the invention]

The present inventors provided an enzyme storage part in the enzyme electrode, and by having a structure that allows enzyme to be supplied from the enzyme storage part to the immobilized enzyme part, the present inventors maintained sensitivity for long-term water retention and achieved stability. It has been found that an enzyme electrode having the following properties can be obtained. Specifically, as an example, as shown in FIG. The structure is such that the required amount of enzyme is supplied through the microtubule 2 and microvalve 3. Alternatively, as illustrated in FIG. 2, a structure in which the enzyme storage part 1 integrated with the enzyme electrode is not provided and the required amount of the enzyme separated by the microspheres 12 is supplied to the immobilized enzyme membrane 6 is also possible. be.

In addition, in FIGS. 1 and 2, O-rings 4, which are used to bring the plastic protective cover 10 into close contact with the electrodes,
A platinum cathode 7, which is connected to an oxygen permeable membrane 5 and also to an immobilized enzyme membrane 6.23, and a lead anode 8 as a reference electrode are shown. 9 and 9' constitute ultrafiltration membranes, which have a porous nature and are impermeable to substances, and 9 has finer pores. 9 and 9' may be separated into two layers or may be made in one piece. Small hand valve 3
Alternatively, the enzyme supplied from the microspheres 12 enters the immobilized enzyme membrane 6, but may also enter the 9' part.The pores 9 are large enough to prevent the enzyme from passing through. Shi, I am visiting the outflow of #replenishment. Further, the oxygen permeable membrane 5 and the electrode part 11 are closely attached by a method such as adhesion or pressure bonding, and have a structure that prevents supplementary enzyme from entering between the oxygen permeable JIi 5 and the electrode part 11. Furthermore, since the enzyme cannot pass through the oxygen permeable membrane 5, it is retained between the oxygen permeable membrane 5 and the ultraviolet membrane 9.

Any enzyme applicable to the present invention may be used as long as it is compatible with the substrate to be measured, but when considering the stability of the stored enzyme, a relatively stable enzyme is desirable. For example, preferred enzymes include glucose oxidase, camerase,
Peroxidase chamber alkaline phosphatase, pyruvate oxidase, lactate oxidase, urease, uricase.

Malate dehydrogenase, glucose-6-phosphate dehydrogenase, lipase, lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, glutamate decarboxylase.

Examples include cholesterol oxidase, cholesterol esterase, amylase, and acetylcholinesterase. Further, as the electrodes used in the present invention, commonly used electrodes such as oxygen, hydrogen peroxide, and PH1 ions can be used. Furthermore, in the enzyme electrode consisting of the enzyme and electrode mentioned above, when the enzyme is immobilized, the method of immobilizing the enzyme to the immobilized enzyme part is to consider replenishment of the enzyme, and to enclose the enzyme in the membrane or gel. A method of immobilization is preferred. For example, entrapment of enzymes in a thin film of fibrous protein a such as collagen is used.

When replenishing enzymes using the microtubule 2 shown in Figure 1, the material of the microtubule 2 may be glass, metal, etc.; It is preferable that there be. If the inner diameter is smaller than this, the flow of the enzyme-containing solution to be replenished will be hindered due to surface tension. Furthermore, if the inner diameter of the tube is too large, the amount of enzyme to be replenished will exceed the amount to be replenished, so an appropriate size is determined depending on the nature of the solution containing the enzyme to be replenished. Therefore, the inner diameter is 100μ
It is preferable to use a plastic tube with a diameter of about 1.5 m.

In addition, the microvalve 3, microsphere 12, etc. that adjust the supply of enzymes and prevent the outflow of unnecessary enzymes when the enzyme electrode is not in use are made of water-resistant plastic membranes or beads. etc. apply. The adjustment mechanism using these valves, balls, etc. is appropriately selected depending on the form of the enzyme electrode, etc.

The enzyme to be supplied to the identified enzyme section is stored in the enzyme storage section in the form of a concentrated aqueous solution with an enzyme concentration of 0.1 W% or more, or in the form of a solution to which coenzymes or stabilizers are roughly added if necessary. Storage type. This is because if the concentration is less than 0.1% IW, the stability of the enzyme is impaired and storage is not possible. In addition, it is also effective to encapsulate enzymes in microcapsules such as liposomes in order to stabilize the enzymes. Alternatively, instead of using an aqueous solution of the enzyme, a method of dispersing the enzyme in a water-soluble polymer may be used. As the water-soluble polymer, a polymer having appropriate solubility in relation to microporosity must be selected. Examples include agar and pullulan.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

[Embodiments of the invention]

Example 1 An enzyme electrode was prepared, which was equipped with an external enzyme storage section 1 as shown in FIG. 1, and an electrode including immobilized pyruvate oxidase as the identified enzyme section (hereinafter referred to as an "immobilized POP electrode"). As for the reagents used, pyruvate oxidase (hereinafter referred to as PoP) was manufactured by Toyo Jozo@), and thiamine pyrophosphate and 7-rabin adenine dinucleotide (hereinafter referred to as F'AJ) were manufactured by Tokyo Kasei Co., Ltd. I used the one from

Other reagents were commercially available products (special grade) and were used as they were without purification. In addition, collagen was prepared from cowhide, stored frozen, and thawed before use. Note that ion-converted water was used throughout the entire operation. In addition, as a buffer solution,
0.05M phosphate buffer (pH 7.5).
A solution containing 01mM FAD, 0.045mM manganese chloride and 0.1mM thiamine pyrophosphate was used.

Using these, For immobilization; Lagen membrane was prepared as follows. 0.6% collagen suspension (pH 4,
o) After stirring the xog well, POP (21 units
s/mg) were mixed, stirred lightly, and then defoamed for 1 minute using a vacuum pump. Next, the obtained suspension was spread on a Teflon plate (4 cm x 5 cm) and air-dried at 28 υ for 3 hours. Next, the film peeled off from the Teflon plate was cut into 1 cm x I cm pieces, and this was soaked in a gas phase using a 0.1% glutaraldehyde aqueous solution (pH 8,0).
A POP-immobilized collagen membrane was prepared by stretching at 8°C for 10 minutes. Then, the sensitive surface formed by covering the platinum cathode with an oxygen permeable membrane was covered with the POP-immobilized collagen membrane (thickness: approximately 60 μm + elemental activity: approximately x001 U/cI).
If) Teea L was further covered with a dense layer of cellulose-acetate membrane (thickness: about 50 μm) to produce an electrode part in an enzyme electrode. According to the present invention, which is an immobilized POP electrode, this electrode part is covered with a plastic protective cover which has a micro/J% tube and a microvalves (or beads) and is connected to an enzyme reservoir. An enzyme electrode was created. The sample solution used was the above buffer solution to which 0.5 mM sodium pyruvate was added.

Then, using this enzyme electrode, a solution containing 0.1 mM FAD in the buffer solution was added to the enzyme reservoir at a concentration of 5 mg/r.
Disperse and store 9 POPs in nll, at least 10 per day
When the sample was repeatedly immersed in the sample solution more than once, almost no decrease in the output current value was observed during the 6-dip period.

Moreover, the deviation of response values was within 5%.

Example 2 An enzyme electrode according to the present invention, consisting of an identified glucose oxidase electrode with an integrated enzyme storage portion as shown in FIG. 2, was prepared as follows.

The reagent used was glucose oxidase (hereinafter GO
D) was from Sigma, and the other reagents were commercially available products (% grade) and were used as they were without purification. Then, GOD was fixed to the collagen membrane in the same manner as in Example 1. The structure of the enzyme electrode is basically the same as in Example 1, but differs from Example 1 in that an enzyme storage section is provided and integrated within a plastic protective cover as shown in FIG.

As the sample solution, a solution obtained by diluting raw urine 10 times with water was used.

Using the enzyme electrode fabricated by the method described above, (K)D dispersed at a concentration of 1 mg/ml in 0.1 M IJ acid buffer (pH 6,5) was stored in the enzyme reservoir. Urine sugar was measured repeatedly at least 10 times a day.

That is, the electrode was immersed in the sample solution to measure the glucose concentration, and then immersed in the Mk solution up to the enzyme reservoir. Through this operation, a small amount of GOD is transferred to the enzyme reservoir.
It is supplied through the small hole of the OD fixation machine. As a result, it was confirmed that the product can be used stably for at least one year at different temperatures.

Furthermore, similar results were obtained when GOD dispersed in 1% agar was stored in the enzyme storage section, confirming long-term stability.

〔Effect of the invention〕

The enzyme electrode according to the present invention, which can be supplemented with enzymes, has greatly improved enzyme activity, sensitivity, long-term stability, etc. compared to conventional examples, and can be applied to a wider range of usage conditions. Ta.

[Brief explanation of the drawing]

FIG. 1 is a partial cross-sectional view showing an example of an enzyme electrode according to the present invention in which an enzyme storage portion is provided outside the enzyme electrode, and FIG. 2 is a partial cross-sectional view showing an example of an enzyme electrode in which an enzyme storage portion is integrated with the enzyme electrode. FIG. 1. Enzyme reservoir 2. Microtube 3. Microvalve 40 ring 5. Oxygen permeable membrane 6. Immobilized enzyme membrane 7. Platinum cathode 8. Lead anode filter 9.9'. Ultra-N membrane 10. Plastic protective cover. 11. Electrode part 12. Microsphere

Claims (1)

[Scope of Claims] (1) An enzyme electrode equipped with an immobilized enzyme portion having selectivity for a specific substrate, comprising: an enzyme storage portion storing enzyme to be supplied to the immobilized enzyme portion; An enzyme electrode characterized by comprising means for guiding enzyme to an immobilized enzyme part.