JPS5843797A - Enzyme electrode - Google Patents

Abstract

PURPOSE:To obtain an enzyme electrode having high safety and stable for a long period, consisting of an immobilized enzyme membrane having high activity, and a permselective membrane attached to the surface of said immobilized enzyme membrane. CONSTITUTION:The objective enzyme electrode is composed of an electrode to pick up the electrical signals, an immobilized enzyme membrane 1 attached to the surface of said electrode and consisting of a hydrophilic gel dispersed with an emzyme activating agent such as cystine and enzymes such as urease, catalase, peroxidase, etc. or enzymes immobilized to granular carrier, and a permselective membrane 2 attached to the surface of said immobilized enzyme membrane. The properties of the enzyme electrode can be further improved by using an asymmetric porous membrane having non-uniform pore size distribution along the direction of thickness, as the permselective membrane 2. In the figure, 3 is ammonium gas electrode cylinder, 4 is ammonium gas permeable membrane, 5 is spacer, and 6 is conductive liquid in the electrode.

Description

[Detailed description of the invention] The present invention relates to a II elementary electrode equipped with an immobilized enzyme.

The enzyme electrode, which is equipped with an immobilized enzyme membrane, utilizes the ability of enzymes to selectively react only with respect to their substrates. Because it is an eggplant, it can be used repeatedly for a long time, and even expensive enzymes can be analyzed at low cost, so its future development is attracting attention.
In particular, it can play an overlapping role in fields such as biochemical analysis and clinical chemical analysis. For example, when using uricase, it is possible to analyze uric acid, which plays an overlapping role in gout diagnosis, and glucose oxidase can be used to analyze glucose, which plays an extremely important role in diabetes diagnosis and treatment. Since analysis can be performed quickly and easily, it is useful for early diagnosis, bedside treatment, etc.

However, in the past, directly immobilized enzyme membranes came into contact with test liquids such as blood and urine, so the proteins contained in these
Due to decomposition of the enzyme by proteolytic enzymes, etc., the sensitivity response of the enzyme electrode is significantly reduced due to the immobilized enzyme lacking stability. There was the inconvenience and inconvenience of repeatedly having to replace the immobilized enzyme membrane within a short period of time. Also.

When immobilizing enzymes, the enzyme activity generally decreased, depending on the immobilization method, and lI1 particles fell off the membrane, resulting in an unstable immobilized enzyme membrane.

In addition, the stability of immobilized enzyme membranes during long-term storage, such as storing them in a dry state or in a buffer solution, is not necessarily sufficient, and the practical performance is limited to high-sensitivity membranes equipped with an enzyme layer. The purpose is to provide an enzyme electrode that is stable for a long period of time.

The present invention comprises an electrode for extracting an electrical signal, an enzyme activator and an enzyme or an enzyme immobilized on a granular carrier dispersed in a gel hydrophilic substance, and an immobilized enzyme layer provided on the surface of the electrode; This is an enzyme electrode equipped with a permselective layer provided on the surface of the enzyme membrane, and an asymmetric porous membrane with non-uniform pore diameters in the thickness direction is used as the permselective layer, resulting in an enzyme electrode with even better properties. is obtained.

In the present invention, as an immobilized enzyme membrane, an enzyme activator and an enzyme or an enzyme immobilized on a granular carrier are mixed with a gel hydrophilic substance (by using a dispersed immobilized enzyme).
In addition to increasing the enzyme activity and making it highly sensitive, it also increases the stability of the enzyme and significantly improves its lifespan.

In addition, the selectively permeable membrane provided on the surface of the immobilized enzyme membrane in the present invention prevents blood cells and bacteria from the sample from adhering to and contaminating the immobilized enzyme layer, and prevents enzyme reactions in the immobilized enzyme layer. The influence of harmful components can be reduced. In particular, when an asymmetric porous membrane with non-uniform pore diameters in the thickness direction is used as a selectively permeable membrane, the gel hydrophilic substance on which the enzyme is immobilized is embedded in the side with larger pores of the asymmetric selectively permeable membrane. This results in an immobilized enzyme membrane that does not fall off from the small pore diameter on the side that is in contact with the sample body fluid, resulting in a constant voltage enzyme electrode that maintains high sensitivity for a long period of time.

The selectively permeable membrane in the present invention refers to, for example, chemical compounds (mainly molecules with a molecular weight of several tens to thousands) that are substrates for elementary reactions such as glucose, uric acid, ``elevation,'' and cholesterol.
The material can be selected as appropriate, as long as it does not substantially impede the permeation of 1° and does not allow the passage of large molecules (most of the proteins in the blood are over 20,000), which play a major role in contamination. , cellulose conductor, polyamide, polyacrylonitrile, polystyrene/etc. It is preferable to decide on the solvent. The thinner the selectively permeable layer is, the faster the response speed is, but in practice it is preferably about 10 λ to several μm.

In addition, examples of carriers for binding enzymes that may be used as needed include polyvinyl alcohol, porous glass, PV
You can use A fi, super fine fiber, glass pieces, po, rhino onomer beads, etc. As the gel hydrophilic substance, synthetic substances such as polyvinylpyrrolidone, polyacrylamide trout conductor, polyvinyl alcohol, sodium polyacrylate gel, polyethylene oxide, and natural polymer substances such as gelatin starch and konjac powder can be used.

In addition, enzyme activators that promote or protect enzyme activity include cysteine and mercaptoethanol for enzymes with 8H groups such as urease, and trypsin for trypsinogen. EDTA to remove metal ions that inhibit
It is thought that catalase, which is present in the reeds and uricases, suppresses the activity, so sodium azide (N), which inhibits the activity of catalase, is
aN5) can be used. others,! Appropriate addition of complement, metal ions, etc. bound to element II is also effective in improving stability and longevity.

As the enzyme used in the present invention, many other enzymes such as urease uricase, trypsinogen, glucose oxidase, cholesterol oxidase, catalase, peroxidase, creatinase, and L-amino acid I oxidase can be selected as appropriate. can be immobilized on the above-mentioned carrier by chemical bonding through a covalent bond reaction such as the sialyte method, adzation method, alkylation method, or a polyfunctional reagent (e.g., glutaraldehyde, hexamethylene diisocyanate, etc.). I can do it.

Furthermore, the electrodes used in the present invention include oxygen electrodes, hydrogen peroxide electrodes, carbon dioxide electrodes, ammonium gas electrodes,
Ammonium ion electrodes, hydrogen ion electrodes, cation electrodes, conductivity electrodes, etc. can be used.

・Hereinafter, the present invention will be explained in detail using examples〇Example 1 Particles having carboxyl groups (average ff 12 μm) 3
25ml of ion-exchanged water whose pH was adjusted to slightly acidic with hydrochloric acid
, and then 1.5 m7 of 1,8-dianno-4-aminomethyloctane was added and kept at a stirring state of 11 ml. After the particles are dispersed in the liquid, a water-soluble calcium compound for peptide synthesis is added.
Diimide trout conductor (1-cyclohexyl-3-2-morpholinoethylcarbosiodo-meso-P-)luenesulfonate;]E) . continue.

The particles were mixed with 5s glutaraldehyde solution (pH 7, 6).
) to form a granular carrier for enzyme immobilization.
2 degrees. Then, urease was immobilized by adding to the solution (Ocal Co., Ltd.) to obtain a predetermined immobilized enzyme.

The amount of enzyme immobilized on the granular carrier for enzyme immobilization was calculated based on the decrease in absorbance of the urease solution, and was approximately 5 mg/
Immobilized urease particles of g wet particles were obtained.

On the other hand, polyvinyl alcohol t''lnM pH 7 with a degree of saponification of 98.5% and a degree of polymerization of 500 is used as a gel hydrophilic material.
, 5 in phosphate buffer,
After heating to 50'0, it was rapidly cooled and degassed. This solution contains β-
1 mM of mercaptoethanol was added, and the immobilized enzyme with the urease immobilized above was added, stirred, and stored at 5°C.

In use, as shown in Figure 1, a selectively permeable membrane (1) consisting of an asymmetric porous membrane is immersed in a 5% by weight boric acid solution.
After opening (Fig. 1 cA)) the solution in which the immobilized enzyme is dispersed is poured onto the larger pore size side (IJI) of the asymmetric permselective membrane (Fig. 1 (B)). Gel the alcohol.

The immobilized urease particles were thus:

A dispersed and immobilized enzyme membrane (2) was provided. First, wash the selectively permeable membrane (1) with a 0.5 weight percent boric acid solution.
Diagram (0>J consisting of an asymmetric porous membrane excluding water-soluble substances) Inside 11 (la) K in contact with ammonia ft A
An enzyme electrode was prepared by installing a lj x electrode (Orion Co., Ltd. 95-1o) and having the outside (IJI) in contact with the test liquid.90 Using the enzyme electrode thus obtained (Fig. 2) While continuously pumping 0.1M phosphate buffer pH 7.5 at a rate of 1 minute, a sample of urea (μ) was injected into the buffer flow at the inlet of the enzyme electrode using a microsyringe. In Figure 2, (1) is the immobilized enzyme membrane, (2) is the selectively permeable membrane, (3) is the ammonium electrode cylinder, (4) is the ammonium gas permeable membrane, (5) is the spacer, and (6) is the ammonium gas permeable membrane. ) indicates the electrode internal solution, respectively.

Since urea is decomposed within the immobilized enzyme membrane as shown in the following equation, the amount of urea was measured by measuring the amount of ammonium ions produced by voltage changes.

JR element + HzO force G top - <2NH4" + HCO
When human serum was used as a sample using the enzyme electrode according to the present invention obtained in this manner, it exhibited a high level of friendliness and could withstand repeated use more than 9 times. In addition, 30 g of acryl acid monomer obtained using the conventional method and 0.58 g of N-N methylenebisacrylic acid were mixed in 25 rnl O0, IM) of Lith buffer pH.
The immobilized urease membrane combed with 7.0 became unusable after about 400 cycles. Furthermore, no decrease in urease activity was observed in the solution containing the immobilized urease particles dispersed at 5° C. for 3 months or more.

As detailed above, according to the present invention, it is possible to prevent contamination of the enzyme electrode with the body fluid to be examined, and to provide a highly sensitive and long-term stable enzyme electrode.
It can be used as a part of clinical chemistry analysis.

[Brief explanation of the drawing]

1st (50■(B)) (0 is a diagram showing the manufacturing process of the immobilized enzyme membrane in the present invention, and FIG. 2 is a cross-sectional view of the enzyme electrode in the present invention. 1... Immobilized enzyme layer, 2 ... selectively permeable membrane (hole diameter 2 a > 2 b ), 3...
- Ammonium gas electrode cylinder, 4... Ammonium gas permeation 111. ．． 5... Spacer, 6... Electrode internal liquid agent Patent attorney Noriyuki Chika (1 person) No.
1 Figure 2

Claims (1)

[Claims] (1) An electrode for extracting an electric signal, an enzyme activator and an enzyme, or an enzyme immobilized on a granular carrier, dispersed in a gel aqueous substance, and nine immobilized enzymes Jl provided on the electrode surface! - an enzyme electrode comprising: a selectively permeable membrane provided on the surface of the immobilized elementary membrane; (2. An enzyme electrode according to claim 1, characterized in that an elongated symmetrical porous membrane with non-uniform pore diameters in the thickness direction is used as the permselective membrane.