JPH0328752A - Enzyme immobilized electrode and its production - Google Patents

Abstract

PURPOSE:To prevent a film from peeling off from an electrode main body by causing the interfering substance removing film of an enzyme immobilized electrode which is obtained by providing an enzyme immobilized film and the interfering substance removing film on an electrode made of conductive material to contain moisture holding agent. CONSTITUTION:In the case of using the enzyme immobilized electrode, it is necessary to protect the electrode from the interference of various components other than a substance to be measured in sample solution. Then, the electrode has the interfering substance removing film for removing an interfering substance between the surface of the electrode main body and the enzyme immobilized film but the interfering substance removing film has such a defect that its detection sensitivity is lowered by the change of humidity because the film is easily dried, so that the corresponding moisture holding agent is set to be used. As an example, the interfering substance removing film 4 is formed by applying interfering substance removing film producing liquid 6 having specified composition on the surface of the electrode main body 1 provided with a lead part 2 on a substance 3 and causing a cross-linking reaction. Liquid which keeps moisture holding ability and has a little effect in removing the interference is used as the producing liquid. Thus, the interfering substance removing film is prevented from peeilng interfering substance removing film is prevented from peeling off from the electrode main body caused by the dryness. The sensitivity becomes excellent as the enzyme immobilized electrode, so that stable measurement is performed.

Description

[Detailed Description of the Invention] [Industrial Application Field] This invention relates to an enzyme-immobilized electrode (also referred to as an "enzyme electrode") used as an electrode for detecting a substrate such as glucose.
and its manufacturing method.

[Conventional technology]

Place the intoxicant on the electrode body (or electrode material; the same applies hereinafter) made of platinum, etc.
As an electrode with an enzyme immobilized on the surface, a dilute enzyme solution containing gelatin and glutaraldehyde as a crosslinking agent is applied to a platinum electrode, a film is formed on the surface of the electrode body, and the enzyme is covalently bonded to the platinum electrode. Some enzymes are immobilized to form an enzyme-immobilized film on the surface of the electrode body. Here, gelatin serves as the matrix for the enzyme-immobilized membrane, and is dilute. Since the strength of the enzyme-immobilized membrane obtained by the crosslinking reaction is weak when only the enzyme solution and glutaraldehyde as a crosslinking agent are used, the enzyme solution is used to increase the strength of the membrane. For example, in a glucose oxidase immobilized membrane,
The membrane is formed by adding gelatin in an amount of 5 to 10 times the amount of enzyme and crosslinking with glutaraldehyde.

When using such an enzyme-immobilized electrode, it is necessary to protect the enzyme-immobilized electrode from interference from a wide variety of substances other than the substance to be measured in the sample solution. Therefore, an interference-insensitive enzyme-immobilized electrode has been proposed that has an interference-removing membrane (hereinafter referred to as "interference-removal membrane") for removing interfering substances between the surface of the electrode body and the enzyme-immobilized membrane.

For example, in Japanese Patent Application Laid-Open No. 62-88953, an electrode body with a glucose oxidase immobilized membrane provided on the surface thereof is immersed in an electrolytic polymerization solution containing virol and Na Cff to undergo electrolytic polymerization treatment, and the immobilized membrane is An enzyme-immobilized electrode with an electropolymerized membrane on top of the membrane is described. This electrolytically polymerized membrane is an interference removal membrane. Also, JP-A-63-30
Publication No. 4150 Tsubasa describes an enzyme-immobilized electrode in which an inner membrane, an intermediate membrane, and an outer membrane are provided in this order on the surface of the electrode body. The intermediate membrane is a fixed membrane for glucose oxidase. The outer membrane is a polycarbonate 1 film and provides removal of macromolecular substances relative to glucose molecules in undiluted blood or serum. The inner membrane is a membrane made of cellulose acetate and removes interfering substances (eg, ascorbic acid and uric acid) not removed by the polycarbo-1-film.

[Problem to be solved by the invention]

In interference-insensitive enzyme-immobilized electrodes that have an interference removal membrane formed on the surface of the electrode body, the interference removal membrane is susceptible to drying, so the membrane shrinks due to changes in humidity, making measurements in aqueous solutions and If drying is repeated, the interference removal film and the enzyme immobilization film will peel off from the surface of the electrode body, resulting in a decrease in detection sensitivity and the like, making it unusable.

Furthermore, as shown in FIG. 3(a), if the humidity is too low, the interference removal film 5 on which the enzyme is not immobilized will peel off from the electrode body l.
% or more must be carried out in a humidifier.

This invention was made in view of the above circumstances, and has a structure that prevents the interference removal film from peeling off from the electrode body.
The first objective is to provide an enzyme-immobilized electrode with small initial fluctuations in output sensitivity.

Furthermore, a second object of the present invention is to provide a method for manufacturing an enzyme-immobilized electrode that does not require the humidity of the atmosphere when an enzyme-immobilized membrane is formed on an interference removal membrane. do.

[Means to solve the problem]

The inventors investigated means for preventing the interference removal membrane from peeling off from the electrode body and for forming an enzyme-immobilized membrane on the interference removal membrane without controlling the humidity of the atmosphere. As a result, they discovered that the interference removal film itself should have moisture retention properties, and completed this invention.

Therefore, in order to solve the first problem described above, an enzyme-immobilized electrode according to the present invention is provided with an enzyme-immobilized membrane and an interference removal membrane on an electrode body made of a conductive material. Features a child whose crotch contains moisturizer.

In order to solve the above second problem, the method for manufacturing an enzyme-immobilized electrode according to the present invention includes forming an enzyme-immobilized film on an interference removal film formed on the surface of an electrode body made of a conductive material. The enzyme-immobilized electrode of the present invention is characterized in that the interference-removing film contains a humectant.The enzyme-immobilized electrode of the present invention is provided with at least an interference-removing film and an enzyme-immobilizing film on the surface of the electrode body. Preferably, a base film is provided on the surface of the electrode body,
It has at least three layers: an interference removal membrane and an enzyme immobilization membrane.

Here, the base film is used as necessary to improve the adhesion of the interference removal film to the electrode body. The base film is a film formed by, for example, applying a crosslinking agent to an aqueous biopolymer solution such as an aqueous gelatin solution to the surface of the electrode body and performing a crosslinking reaction. The matrix content of the base film is not limited to gelatin. As the crosslinking agent, for example, a dialdehyde such as glutaraldehyde is used.

At least a part of the mal-resox component is a humectant, and the interference removal film is a solution containing a protein such as albumin, a water-soluble polymer such as polyallylamine, a humectant, and a crosslinking agent, on the electrode body or under the electrode body. A predetermined amount of the solution is applied onto a geomembrane or an enzyme-immobilized membrane, a crosslinking reaction is performed, and a membrane is formed on the electrode surface. The moisturizer is not particularly limited as long as it retains or increases the moisture content of the interference removal membrane, but it may include collagen, surfactants such as polyethylene glycol alkyl compounds, and water-soluble polymers such as polyvinylpyrrolidone and polyvinyl alcohol. However, it is preferable to use collagen as a humectant in order to maintain the moisturizing properties of the interference removal film and to reduce the decrease in interference removal effect. As the collagen, any of acid-treated collagen, alkali-treated collagen, enzyme-fusible collagen, etc. can be used, and the type of collagen is not particularly limited. Melt solution (hereinafter referred to as “
In the "interference removal membrane preparation solution"), the concentration of collagen is not particularly limited, but the concentration of collagen is o. A range of oi to 0.1% by weight is preferred. If it exceeds 0.1% by weight, the permeability of the membrane will deteriorate and the response speed will deteriorate. The responsiveness of the enzyme-immobilized electrode according to the present invention is, for example, 20 to 30 seconds. Further, it is also possible to appropriately change the type of substance that can be removed depending on the degree of crosslinking of the interference removal membrane. The matrix components of the interference removal film are not limited to those mentioned above, as long as they include a humectant. As the crosslinking agent, for example, a dialdehyde such as glutaraldehyde is used.

The enzyme-immobilized membrane is made by applying a predetermined amount of a solution (e.g., an aqueous solution) containing enzymes such as glucose oxidase and cholesterol oxidase, biopolymers such as gelatin, and a crosslinking agent onto the electrode body or onto the interference removal membrane, and then crosslinking the membrane. It is produced by conducting a reaction to form a film and immobilizing it.

Only one type of enzyme may be used, or two or more types may be used as long as no adverse effects such as deactivation occur. The matrix material for the enzyme-immobilized membrane is not limited to those mentioned above, as long as it does not impair the function of the enzyme. As the crosslinking agent, for example, a dialdehyde such as glutaraldehyde is used.

The enzyme-immobilized electrode according to the present invention obtained in this manner can maintain moisture retention because the moisture content of the interference removal film is retained by the humectant, and the separation of the interference removal film from the electrode body due to drying can be maintained. It can be prevented. In addition, the swelling rate in the measurement solution and the shrinkage rate when dried are reduced.
There is no fluctuation in sensitivity when used as an enzyme-immobilized electrode, and stable measurements can be performed.

In addition, the applicability of the aqueous solution for preparing the interference removal membrane is improved,
The film can be formed to have a uniform thickness on the electrode surface.

With conventional interference removal films that do not contain humectants, after applying the interference removal film to the electrode body, if you leave it outside a humidifier for 5 to 10 minutes in an atmosphere with a humidity of about 40 to 60%, As shown in FIG. 3 (al), the interference removal film 5 peels off from the electrode body 1.

For this reason, it was necessary to maintain the atmosphere at a humidity of 80% in a humidity conditioner and to manufacture the interference removal filter 4 as shown in FIG. 3(b). On the other hand, in the method for manufacturing an narcotic acid-immobilized electrode according to the present invention, before forming the enzyme-immobilized film on the second part of the interference-removing membrane, the interference-removing membrane is pre-impregnated with a humectant. Outside the humidifier, for example, humidity 40~
Even if left in an atmosphere of about 60% for 5 to IO minutes, the third
As shown in Figure tb+, the interference removal film 4 is formed and peeling is less likely to occur. In Figures 1 to 3, 3 is an insulating substrate made of ceramic material, Hei resin, or the like. The electrode body 1 is formed, for example, on this substrate 3, but is not limited thereto.

Note that the enzyme-immobilized electrode according to the present invention is capable of continuous measurement of 1000 times or more.

[For production]

Since the hair removal crotch contains a humectant, changes in shrinkage and swelling due to dryness and moisture are reduced, making it difficult to peel off.

In addition, since the interference removal film contains a humectant, there is no need to adjust the humidity of the atmosphere when forming the enzyme immobilization membrane on the interference removal film, and the interference removal film peels off from the electrode body. You also have the advantage of being prevented from doing so.

〔Example〕

Specific examples and comparative examples of the present invention are shown below, but the present invention is not limited to the following examples.

Examples 1 to 5 and Comparative Examples A base membrane preparation solution, an interference removing membrane preparation solution, and an enzyme-immobilized F6 wave of Kumihi as shown in Table 1 were used.

9 10 Polyallyl and albumin were used as matrix components of the interference removal membrane, glutaraldehyde was used as a crosslinking agent, and collagen was used as a humectant. The matrix components of the enzyme-immobilized membrane are gelatin, polyallylamine, and albumin, and the crosslinking agent is glutaraldehyde. As shown in Fig. 1, a predetermined amount of the base+1Q preparation solution is applied onto the surface of the electrode body (platinum electrode) 1, which has a lead part 2 and is provided on the substrate 3, and then a -W was used to form a fi1 base film. The first layer is applied to the surface of this base film.
The interference removal film preparation solution 6 having the composition shown in the table was applied (see Fig. 2) to cause a crosslinking reaction to form the interference removal film 4 (see Fig. 3 (bl). An enzyme immobilization solution prepared by Kumiui (not shown in Table 1) was applied to the surface to carry out a crosslinking reaction to form an enzyme immobilization membrane.

In addition, in the comparative example, after applying the interference removal film production liquid as shown in Figure 2, it was applied for 5 to 1 hour in an atmosphere with a humidity of 40 to 60%.
When left for 0 minutes, as shown in Figure 3 (=1),
The interference removal film 5 has peeled off from the electrode body 1. For this purpose, the interference removal film was prepared by applying the interference removal film production liquid and placing it in a humidity controller in an atmosphere with a humidity of 80% or more (see Figure 3).
bl) and an enzyme-immobilized membrane was prepared.

The polyallylamine used in the above Examples and Comparative Examples has an average molecular weight of about 60,000, and the collagen used in the Examples is Bovine Dermal/Flusible Type I Collagen Solution (pH 3.0, concentration 0.3%).

The detection sensitivity (measurement sensitivity), moisture retention property, and sensitivity fluctuation were investigated for each enzyme-immobilized electrode of Examples 1 to 5 and Comparative Example, and the results are shown in Table 1. Sensitivity is determined by connecting the enzyme-immobilized electrode to a measuring device such as a polar analyzer, and applying a voltage of 0.6 V (
(Ag electrode), the sample amount was 20 μC.

The sample was a 150 nw/d1 glucose solution, 10
A cholesterol solution of 0 .mu./d1, ascorbic acid (concentration of 20 .mu./d1) and NaF (concentration of 50 .mu./d!), which is used to prevent coagulation of blood vessels, were used as interfering substances.

Moisture retention is determined at a humidity of 40 to 60 after applying the interference removal film preparation solution.
A case in which the l1 12 interference removal film did not peel off within about 5 to 10 minutes in an atmosphere of 10% was indicated by ◯, and a case in which it did peel off was indicated by ×. Sensitivity fluctuation was determined by calculating the change between the first sensitivity measurement value and the 20th sensitivity measurement using the following formula when measuring 20 times with a standard solution using the obtained enzyme-immobilized electrode. .

13 As can be seen from Table 1, the enzyme-immobilized electrodes of Examples 1 to 3 can all detect glucose with high sensitivity, and the enzyme-immobilized electrodes of Examples 4 and 5 can both detect cholesterol with high sensitivity. We were able to detect it well. Also,
In Examples 1-5, little or no interfering substances were detected. On the other hand, in the enzyme-immobilized electrode of the comparative example, the sensitivity fluctuation was larger (±4 to 5%) than that of the example (within ±2%). In addition, the comparative example has a humidity of 4
In an atmosphere of 0 to 60% RH, the interference removal film 5 peeled off from the electrode body 1 as shown in FIG. 3 (al), but in the example, it did not peel off under the same conditions.

In the above example, glucose oxidase and cholesteryl oxidase were used as the enzymes to be immobilized, but the enzymes that can be used in the present invention are not limited to these two. Furthermore, the precepts for measurement are not limited to blood smear.

[Effect of First Effect] The enzyme-immobilized electrode according to the present invention has an enzyme-immobilized film and an interference removal crotch provided on an electrode body made of a conductive material, and the interference removal film does not contain a humectant. It is characterized by the fact that there is no peeling of the interference removal film,
Able to perform sensitive and stable measurements.

In the method for producing an enzyme-immobilized electrode according to the present invention, when forming an enzyme-immobilized film on an interference-removing film formed on the surface of an electrode body made of a conductive material, a humectant is included in the interference-removing film. Therefore, there is no need to adjust the humidity when forming the enzyme-immobilized membrane on the interference removal membrane.

[Brief explanation of drawings]

FIG. 1 is a perspective view showing a female platinum electrode used in Examples and Comparative Examples, FIG. 2 is a schematic cross-sectional view showing the same platinum electrode coated with an interference removal film preparation solution, and FIG. +8+ is
Schematic cross-sectional view showing the state before formation of the enzyme-immobilized film after interference removal film formation in the enzyme-immobilized electrode of the comparative example, FIG. 3 fb
FIG. 1 is a schematic cross-sectional view showing the state in which the enzyme-immobilized 15 16 membrane is formed after the interference removal membrane is formed in the enzyme-immobilized electrode of the example. 1... Electrode body 4... Interference removal diagram Figure 1

Claims (1)

[Scope of Claims] 1. An enzyme-immobilized electrode in which an enzyme-immobilized film and an interfering substance removing film are provided on an electrode body made of a conductive material, characterized in that the interfering substance removing film contains a humectant. Enzyme-immobilized electrode. 2. In the method for producing an enzyme-immobilized electrode, in which an enzyme-immobilized film is formed on an interfering substance-removing film formed on the surface of an electrode body made of a conductive material, the interfering substance-removing film contains a humectant. A method for producing an enzyme-immobilized electrode characterized by: