JPH0777509A - Electrochemical measurement electrode - Google Patents

Abstract

(57) [Summary] [Objective] To provide an electrode for use in amperometric measurement, which is widely used among electrochemical measurement methods, with a particularly simple structure and excellent in durability and stability. The purpose is to A film-shaped conductor (2) serving as a working electrode and a film-shaped conductor (3) serving as a counter electrode are provided on an insulating substrate (1), and a hydrogen peroxide selective permeable film is formed on the conductor (2) serving as a working electrode. 7 and an immobilized enzyme membrane 8 are formed, an underlying polymer membrane 6 is formed between the hydrogen peroxide selective membrane 7 and the working electrode, the insulating substrate 1 adjacent thereto and the working electrode. Electrode for electrochemical measurement.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrode for electrochemical measurement, and more particularly to an electrode for electrochemical measurement which has high sensitivity and has small variation in response value between electrodes in the manufacturing process.

[0002]

2. Description of the Related Art Electrochemical measurement has been attracting attention as an analytical method capable of high-sensitivity measurement with a relatively simple device configuration that is less affected by a sample matrix than conventional analytical methods such as optical measurement. It is well known as a technique. Among them, the amperometric measurement method of observing the amount of current flowing through the working electrode is widely used for detecting an electrochemically active substance in a sample solution. For example, it is widely known that the concentration of an electrochemically active substance such as hydrogen peroxide or oxygen in a sample solution is determined from the amount of current flowing through a working electrode that is kept at a constant potential with respect to a reference electrode such as a saturated calomel electrode. It is done. The characteristic of this measuring method is that the electron-accepting reaction involving the substance to be detected is directly observed as the amount of current at the working electrode, so that highly sensitive measurement can be performed.

On the other hand, the conventional electrochemical measuring method has a drawback that the electrode reaction is poor in selectivity, but in recent years, the progress of utilization technology of physiologically active substances such as enzymes, antibodies and microbial cells has been achieved. As a result, it has become possible to combine these biochemical reactions and electrochemical analysis to achieve highly selective and highly sensitive analysis. Sensors using such electrodes are about to be applied to a wide range of fields such as clinical analysis and fermentation control. For this reason, it has been desired to establish a technique for mass-producing small-sized and inexpensive functional electrodes such as immobilized enzyme electrodes with good reproducibility.

In recent years, studies have been conducted on the use of electrodes having a simple structure as a source of such functional electrodes suitable for mass production. For example, JP-A-61-26
As disclosed in Japanese Patent No. 2652, there has been devised a method of forming a film-like electrode on an insulating substrate that can be easily mass-produced and then forming an enzyme film thereon. By simplifying and downsizing the electrodes with such a configuration, it becomes easy to integrally configure a plurality of sensors of different systems as disclosed in, for example, JP-A-61-133853.

In these conventional techniques, processing techniques such as a lift-off method, a photo-etching method, a mask vapor deposition method, a screen printing method and a plating method, which are usually used at the time of manufacturing a semiconductor with a conductive material on an insulating substrate, are applied. A functional film such as an enzyme film is provided on the plate-shaped or film-shaped electrode so as to cover at least the working electrode portion (refers to the portion of the conductor that functions as the working electrode that contacts the sample). There is. When a functional film such as an enzyme film is formed on the surface of such a plate-like electrode, a method of applying a film material prepared in a solution form to form a film is often used because it is simple. That is, to give an example of the case of producing an immobilized enzyme membrane, a membrane material solution in which an enzyme and, if necessary, a protective protein and the like coexist is applied, and a cross-linking agent such as a polyfunctional aldehyde or a stilbazolium group. It is a method of forming an immobilized enzyme membrane by reacting with a crosslinking agent such as a photocrosslinkable polymer having a functional group such as a diazo group.

However, in these methods, since the electrode shape is formed in a flat plate shape, even if a membrane material solution containing an enzyme is dropped on the surface of the working electrode, the solution spreads in an indefinite shape, resulting in a thickness per unit area. It is difficult to form a reproducible immobilized enzyme membrane that regulates the enzyme activity contained in and. Then, in order to form the immobilized enzyme membrane with good reproducibility on the surface of the working electrode, which is the detection portion, and to reduce the variation in the electrode response value at the time of preparation between electrodes, complicated steps and devices were required. .

For example, JP-A-61-262652 discloses a method of spin coating an aqueous solution of an enzyme containing a photocrosslinkable polymer,
A method of uniformly applying by a spray method or the like is described. However, the spin coating method and the spray method are effective means for applying a thin solution on a flat surface, but since it is not possible to apply a highly viscous solution with good reproducibility, an attempt is made to obtain a highly active immobilized enzyme membrane. However, it is difficult to increase the enzyme concentration of the applied film material solution. Further, if the solution viscosity changes during the work, reproducible results cannot be obtained. Therefore, it is necessary to prevent the change in the solution viscosity during the work by using an expensive crosslinking agent such as photocrosslinking polymerization.

That is, in the conventional method, it is indispensable to uniformly apply the film material solution to almost the entire flat electrode such as the spin coating method and the spray method. It was necessary to apply a large amount of the enzyme solution as compared with the required amount of the enzyme to be immobilized on the above, or a dedicated device was required. Therefore, there is a limit even when trying to obtain a highly active immobilized enzyme membrane, and it is difficult to reduce the production cost because the amount of expensive enzyme used is large and the production apparatus becomes large even when mass production is performed. .

[0009]

As described above, a film-like electrode (including a plate-like film) made of a conductive material is provided on an insulating substrate and fixed by spin coating, spraying or dip coating. Although the method of forming the immobilized enzyme membrane is a simple method suitable for mass production, it is necessary to sufficiently examine the preparation of the highly sensitive immobilized enzyme electrode and the reproducibility of the response value between the electrodes. It hasn't arrived. In other words, it cannot be said that the conventional method is excellent in high sensitivity and reproducibility, which is required for constructing an originally stable measuring device.

The present invention relates to an immobilized enzyme electrode used for amperometric measurement, which is widely used among electrochemical measurement methods, and has a particularly simple structure, high sensitivity and excellent reproducibility of response values between electrodes. Another object of the present invention is to provide a measuring electrode.

[0011]

Embodiments of the present invention will be described below.

The present invention has at least a film-shaped conductor serving as a working electrode and a film-shaped conductor serving as a counter electrode on an insulating substrate, and a hydrogen peroxide selective permeation film on the conductor serving as a working electrode. An electrochemical measurement electrode formed by forming an immobilized enzyme membrane on the working electrode and an insulating substrate adjacent to the working electrode, and an underlayer polymer membrane formed between the hydrogen peroxide selective permeable membrane. It is a chemical measurement electrode.

The hydrogen peroxide selective permeable membrane is formed by applying a membrane material solution onto the base polymer film, and the base polymer membrane is a hydrogen peroxide selective permeable membrane film material solution. The above-mentioned electrode for electrochemical measurement which is insoluble.

The above-mentioned electrode for electrochemical measurement in which the underlying polymer film contains a protein.

The electrode for electrochemical measurement as described above, wherein the underlying polymer film is formed by crosslinking a protein with a polyfunctional aldehyde.

The above-mentioned electrode for electrochemical measurement in which the base polymer film contains perfluoropolyethylene sulfonic acid.

The electrode for electrochemical measurement according to the above or above, wherein the hydrogen peroxide selective permeable membrane contains a protein.

An electrochemical measurement electrode having at least two film-shaped conductors on an insulating substrate, wherein the surface of the insulating substrate including the conductors is uniformly coated with a polymer to form a base polymer film. And a hydrogen peroxide selective permeable membrane covering at least the working electrode portion on a part of the base polymer membrane, and an immobilized enzyme membrane formed on the base polymer membrane and the selective permeable membrane. Electrode for electrochemical measurement.

[0019]

The insulating substrate that can be used in the present invention is a material such as acrylic resin, fluororesin, vinyl chloride or other plastic, ceramic, glass or the like as long as it is a material having an insulating property required when immersed in a solution used for measurement. A general insulator can be used. As a material used for at least two conductors formed in a film shape (including a plate shape), it is possible to use a noble metal such as gold, silver or platinum, or carbon.

These conductors are formed into a film on an insulating substrate to form an electrode body, or in the case of a firing insulator such as ceramics, the insulating substrate and the film conductor are simultaneously formed. Is also possible. The at least two film conductors provided on the insulating substrate do not necessarily have to be the same material, and different materials such as a combination of platinum and silver can be used. In addition, the formation of two or more film-shaped (including plate-shaped) conductors does not have to be performed simultaneously, and can be performed individually.

As a method for forming a conductor on an insulating substrate using a conductive material, vapor deposition, screen printing,
Various methods such as a method combining the above method and electroplating can be used. The electrode having at least two conductors is a two-electrode system composed of a working electrode / counter electrode, or a three-electrode composed of a working electrode / reference electrode / counter electrode.
An electrode system can be illustrated.

If necessary, an insulating layer such as an epoxy resin may be formed on the conductor to prevent a short circuit due to the carrier liquid during measurement.

In the electrode having at least two film-like (including plate-like) conductive portions provided on the insulating substrate as described above, at least one surface of the insulating substrate having the film-like conductor is raised. Uniformly coat with a molecular film. The type of polymer membrane to be used is not particularly limited, but non-water-soluble polymer substances such as acetyl cellulose, spherical proteins such as albumin and globulin, water-soluble polymer substances such as fibrous proteins such as collagen, perfluoropolyethylene sulfonic acid, etc. Can be used. Preferred is protein or perfluoropolyethylene sulfonic acid. Perfluoropolyethylene sulfonic acid may be used in the form of sulfonate.

Unlike the hydrogen peroxide selective permeable membrane, the underlying polymer membrane does not need to have selective permeability, and it is generally desirable that it is thinner than the thickness of the selective permeable membrane described later. This is because, in the case of a thick film, the interfering substance for measurement may permeate from the part not covered with the permselective film, which may affect the measured value.

For example, when acetyl cellulose or the like is used, it is dissolved in a soluble solvent such as a mixed solution of dichloromethane and acetone and at least one side of the insulating substrate having the film-shaped conductor is uniformly coated, and then the solvent is evaporated to form a base. Form a polymer film. When a water-soluble polymer substance such as protein is used, an aqueous solution containing at least one of these proteins is applied and dried to form a film. Further, a solution containing at least one of these proteins is applied together with a cross-linking agent such as a polyhydric aldehyde such as glutaraldehyde or glyoxal and dried to form a film, or a solution of the underlying protein is applied and then reacted with the cross-linking agent. It is also possible to form a film. As the cross-linking agent, glutaraldehyde, which allows the reaction to proceed relatively mildly, is preferable.

When the membrane material solution of the permselective membrane is a non-aqueous solvent, the protein of the underlying polymer membrane does not dissolve in the membrane material solution of the permselective membrane, but as an electrode for electrochemical measurement in an aqueous solvent. When an electrode for the purpose of measurement is prepared, it is preferable to use a crosslinking agent to make it insoluble in water so that the underlying polymer film is not solubilized. Further, when the membrane material solution of the permselective membrane is an aqueous solvent, it is preferable to insolubilize it with a crosslinking agent so that the protein of the underlying polymer membrane is not dissolved in the membrane material solution of the permselective membrane.

When a protein is used as the water-soluble polymer substance, the protein concentration in the membrane material solution is 0.5 to 10
%, The concentration range is preferable, and within this concentration range, the viscosity is relatively low and the handling is easy. It is preferable that a cross-linking agent for polyvalent aldehydes such as glutaraldehyde coexist in this protein solution. In this case, the concentration of the cross-linking agent is not particularly limited, but it is desirable that the concentration is such that the cross-linking reaction does not abruptly occur during handling, for example, in the case of glutaraldehyde, the concentration range is about 0.05-5%. The amount of coating per unit area of the film electrode is not particularly limited, but if the electrodes are substantially equal to each other, the accuracy may be such that it can be performed by a simple method such as a dip coating method, a spin coating method or a spraying method. . The protein solution applied to the membranous electrode is dried to complete the crosslinking reaction and form a base polymer film.

When covering the base polymer film, a physical or chemical bond strengthening treatment may be performed if necessary in order to strengthen the adhesive force between the insulating substrate and the base polymer film. Examples of such a bond strengthening treatment include chemical bond strengthening treatment such as hydrophilization treatment by metal sodium treatment on the resin surface, which is performed when fluororesin, polyethylene, polypropylene or the like is used for the insulating substrate, and glass or the like. There is a physical bond strengthening treatment such as a treatment for increasing an adhesion area by a roughening treatment performed when used for an insulating substrate.

It is sufficient for the portion covered with the base polymer film to include at least the working electrode portion (the portion that works as the working electrode and is in contact with the sample) and the insulating substrate around the working electrode portion. For covering, the method of covering the entire surface of the insulating substrate provided with the conductor is simple and highly reproducible. A material that prevents impregnation of the film material solution such as a protective tape to prevent the film material solution from adhering when there is a portion on the insulating substrate where it is not desirable to cover with the underlying polymer film such as the connection terminal part for the electric circuit. It is also possible to perform masking in advance by using.

Next, after the base polymer film is dried and formed, at least the upper part of the working electrode portion and the peripheral portion of the insulating substrate portion are included, and the selective permeation film or the like is included in the range included in the base polymer film. Form a functional film. At this time, when the solution of the film material is dropped near a portion of the dried base polymer film around the working electrode, using a device such as a pipette having a constant volume discharge capability, the dry surface of the base polymer film and the film material are dropped. It is possible to prevent the amorphous shape from spreading due to a contact angle of a certain size with the solution. If the film is gently dried and formed into a film in this state, a functional film having substantially the same thickness and size can be formed on the working electrode portions of the plurality of electrodes.
Therefore, even when a plurality of electrodes are formed, it is possible to form electrodes having uniform response values with good reproducibility.

As a hydrogen peroxide selective permeable membrane, Japanese Patent Application No. 63-1
The one disclosed in No. 82559 can be exemplified. That is, 0.0
A protein solution such as albumin containing a polyfunctional aldehyde at a concentration of 5 to 0.5% by weight, preferably 0.1 to 0.5% by weight is applied and the temperature range is 0 to 20 ° C, preferably 0 to 10 ° C. Crosslinking is performed to form a permselective membrane.

In the present invention, a permselective membrane is formed on the surface of the underlying polymer membrane, and then an immobilized enzyme membrane such as various oxidases is formed to obtain an immobilized enzyme electrode. Examples of hydrogen peroxide-forming oxidase include glucose oxidase, galactose oxidase, alcohol oxidase and the like. The immobilized enzyme layer is composed of polyfunctional aldehyde and hydrogen peroxide-forming oxidase, and if necessary, albumin, gelatin,
Or apply an aqueous solution containing proteins such as collagen
It can be formed by drying.

When an immobilized enzyme electrode is prepared, it is possible to use a pipette or the like capable of discharging a constant volume for dropping the membrane material solution. Therefore, it is possible to immobilize a high concentration only in the vicinity of the working electrode where film formation is required. It is possible to drop the membrane material solution of the enzyme membrane,
It is possible to easily prepare a highly active immobilized enzyme membrane.

[0034]

The contents of the present invention will be described in more detail with reference to the following examples, but of course the present invention is not limited thereto.

Example 1 As shown in FIG. 1 (a cross-sectional view is a cross-sectional view taken along the length of the conductor 2), the insulating substrate (1) has a length of 8 m.
Using a m × 16 mm alumina ceramic plate, platinum was used to form two film-shaped conductors (2) and (3) having a width of 1.5 mm and a length of 14 mm on the surface thereof. The center of the platinum part was sealed with an epoxy resin (5).

1) Formation of base polymer film As a protein of the film material, bovine serum albumin (Sig
ma company) was used. 10 mg of bovine serum albumin was precisely weighed and dissolved in 900 μl of distilled water. To this solution, 100 μl of a 5% glutaraldehyde solution was added to obtain a film material solution for the base polymer film. This solution was applied to the electrode surface excluding the connection terminal portion by a dip coating method and dried to obtain a base polymer film (6).

2) Formation of permselective membrane As a material for the permselective membrane, bovine serum albumin (Sig
ma company) was used. 25 mg of bovine serum albumin was precisely weighed and dissolved in 900 μl of distilled water. To this solution, 100 μl of a 5% glutaraldehyde solution was added to obtain a membrane material solution for a selective permeable membrane. A 10 μl aliquot of this solution was taken with a constant-volume pipette and added dropwise so as to cover the working electrode portion and not to deviate from the underlying polymer film. In this state, it was gently dried to obtain a selectively permeable membrane (7).

Five electrodes with a selective permeation film were prepared by the same method, and the electrode numbers A-1 to A-5 were added. 3) Measuring apparatus A flow type measuring apparatus having electrodes arranged as shown in FIG. 2 was used. In this flow type measuring device, an injector (9) for high performance liquid chromatography and a measuring cell (11) to which the measuring electrode (10) obtained in 2) above is attached have an inner diameter of 0.5 mm and a length of 1.0 m. Fluororesin piping (12)
Connected by.

These are installed inside a constant temperature bath (13) and the temperature inside the bath is kept at 37 ° C ± 0.2 ° C. A voltage of +0.6 V was applied to the platinum portion by the potentiostat (14) so that hydrogen peroxide was detected as a target for amperometric measurement. Detection signals are A / D converter (15), communication cable (16)
Data processing was performed by transferring to computer (17) via.

The buffer solution is sent from the buffer solution reservoir (18) at a flow rate of 1.0 ml / min by controlling the solution sending pump (19) by a computer. The buffer is 100 mM phosphate buffer (p
H6) was used. The buffer solution after the measurement is captured in the waste liquid reservoir (20).

4) Measuring method After the temperature of the constant temperature bath reached equilibrium, 5 μl of hydrogen peroxide standard solution was added.
1 injection. After confirming that a proportional relationship was established between the detected value and the concentration up to a concentration of 10 mM, hydrogen peroxide was injected at a concentration of 5 mM.

5) Results Table 1 shows the measurement results of Example 1.

[0043]

[Table 1]

Average fluctuation rate (CV) of measured values of five electrodes
%) Was 20%. A phosphate buffer containing glucose oxidase and bovine serum albumin was prepared, glutaraldehyde was added, the solution was dropped on the permselective membrane of each electrode, and further dried to form an immobilized enzyme membrane. The output value obtained from each electrode had little variation, and an electrochemical measurement electrode with stable quality was obtained.

COMPARATIVE EXAMPLE 1 As shown in FIG. 3, the same plate electrode as in Example 1 was used. That is, an alumina ceramic plate of 8 mm × 16 mm was used as an insulating substrate, and platinum was used to form two film-like conductive portions having a width of 1.5 mm and a length of 14 mm on the surface thereof. The center of the platinum part was sealed with an epoxy resin.

1) Formation of permselective membrane As a material for the permselective membrane, bovine serum albumin (Sig
ma company) was used. 200 mg of bovine serum albumin was precisely weighed and dissolved in 900 μl of distilled water. 10% in this solution
100 μl of the glutaraldehyde solution was added to obtain a membrane material solution for the selective permeation membrane. This solution was applied to the electrode surface excluding the connection terminal portion by a dip coating method and dried to obtain a selectively permeable membrane (7). Five electrodes with a selectively permeable film were prepared by the same method, and the electrode numbers B-1 to B-5 were added.

2) Measuring device Same as in Example 1. 3) Measurement method Same as in Example 1. 4) Results Table 2 shows the measurement results of Comparative Example 1.

[0048]

[Table 2]

Average fluctuation rate (CV) of measured values of five electrodes
%) Was 50%. An immobilized enzyme membrane was formed on the working electrode portion on the permselective membrane in the same manner as in Example 1, but the output values varied greatly.

Example 2 As shown in FIG. 4 (a cross-sectional view is a cross-sectional view taken along the length of the conductor 2), the insulating substrate (1) is 8
mm × 16 mm alumina ceramic plate, platinum on the surface of which 1.5 mm wide, 14 mm long two film-shaped conductors (2) and (3), and silver of 1.5 m wide
One film conductor (4) having a length of m and a length of 14 mm was formed.

The silver portion was oxidized in 0.1 M hydrochloric acid to generate silver chloride, which was used as a silver / silver chloride reference electrode. Next, as shown in FIG. 4, the center was sealed with an epoxy resin (5).

1) Formation of Base Polymer Film (6) Same as in Example 1. 2) Formation of permselective membrane (7) Same as in Example 1. 3) Formation of Immobilized Enzyme Membrane The enzyme to be immobilized is glucose oxidase (EC).
1.1.3.4, manufactured by Sigma) was used. Glucose oxidase (5 mg) and bovine serum albumin (5 mg) as protective proteins were precisely weighed and 100 mM phosphate buffer solution 900
It was dissolved in μl. To this solution, 100 μl of a 5% glutaraldehyde solution was added to prepare a membrane material solution for the immobilized enzyme membrane.
10 μl of this solution was collected with a constant volume pipette, and
It dripped on the permselective membrane formed by the method similar to. In this state, it was gently dried to obtain an immobilized enzyme membrane (8).

Five electrodes with a selective permeation film were prepared by the same method, and the electrode numbers C-1 to C-5 were added. 4) Measuring device Same as in Example 1. 5) Measurement method Glucose standard solution was
1 injection.

After confirming that a proportional relationship was established between the detected value and the concentration up to a concentration of 30 mM, glucose was injected at a concentration of 20 mM. 6) Results Table 3 shows the measurement results of Example 2.

[0055]

[Table 3]

Average fluctuation rate (CV) of measured values of five electrodes
%) Was 20%.

Comparative Example 2 As shown in FIG. 5, the same plate electrode as in Example 2 was used. That is, an alumina ceramic plate of 8 mm × 16 mm is used as an insulating substrate, platinum is used on the surface thereof, and a film conductor (2) having a width of 1.5 mm and a length of 14 mm,
(3) 2 pieces, using silver, width 1.5mm, length 14mm
One film-shaped conductor (4) was formed.

The silver portion was oxidized in 0.1 M hydrochloric acid to generate silver chloride, which was used as a silver / silver chloride reference electrode. Next, as shown in FIG. 5, the center was sealed with an epoxy resin (5).

1) Formation of permselective membrane Bovine serum albumin (Sig
ma company) was used. 200 mg of bovine serum albumin was precisely weighed and dissolved in 900 μl of distilled water. 10% in this solution
100 μl of the glutaraldehyde solution was added to obtain a membrane material solution for the selective permeation membrane. This solution was applied to the surface of the electrode except the connection terminal portion, and the solution was applied for 1000 r. p. m. It was treated for 3 minutes, spin-coated and dried to obtain a selectively permeable membrane (7).

2) Formation of Immobilized Enzyme Membrane The enzyme to be immobilized is glucose oxidase (EC).
1.1.3.4, manufactured by Sigma) was used. Glucose oxidase (50 mg) and bovine serum albumin (50 mg) as protective proteins were precisely weighed and 100 mM phosphate buffer 9
It was dissolved in 00 μl.

100 μl of a 10% glutaraldehyde solution was added to this solution to obtain a membrane material solution for the immobilized enzyme membrane. This solution was applied to the electrode surface excluding the connection terminal portion of the electrode on which the selectively permeable film was formed by the above-mentioned treatment, and 1000 r. p.
m. After treatment for 3 minutes, spin coating and drying, an immobilized enzyme membrane (8) was obtained.

Five electrodes with a selective permeation film were prepared by the same method, and the electrode numbers from D-1 to D-5 were added. 3) Measuring device Same as in Example 2. 4) Measurement method Same as in Example 2. 5) Results Table 4 shows the measurement results of Comparative Example 2.

[0063]

[Table 4]

Average fluctuation rate (CV) of measured values of five electrodes
%) Was 72%.

Example 3 As shown in FIG. 4, the insulating substrate (1) was 8 mm × 1.
A 6 mm alumina-based ceramic plate was used to deposit platinum on the surface of the film-shaped conductive parts (2) and (3) having a width of 1.5 mm and a length of 14 mm, and a width of 1.5 mm using silver. One film-like conductive portion (4) having a length of 14 mm was formed.

The silver portion was oxidized in 0.1 M hydrochloric acid to generate silver chloride, which was used as a reference electrode. The center of the film conductor was sealed with an epoxy resin (5).

1) Formation of base polymer film As a film material for the selective permeation film, 5% perfluoropolyethylene sulfonic acid liquid (manufactured by Aldrich: trade name Nafion, medium is lower aliphatic alcohol containing 10% water). Using. 100 μl of this solution was spread on the conductor-formed side of a flat plate electrode whose connection terminal portion was masked with tape. The solution as it was was air-dried at room temperature to form a film, which was used as a base polymer film (6). 2) Formation of permselective membrane (7) Same as in Example 1.

3) Formation of Immobilized Enzyme Membrane The enzyme to be immobilized is glucose oxidase (EC).
1.1.3.4, manufactured by Sigma) was used. Glucose oxidase (5 mg) and bovine serum albumin (5 mg) as protective proteins were precisely weighed and 100 mM phosphate buffer solution 900
It was dissolved in μl. To this solution, 100 μl of a 5% glutaraldehyde solution was added to prepare a membrane material solution for the immobilized enzyme membrane.
10 μl of this solution was collected with a constant volume pipette, and
It dripped on the permselective membrane formed by the method similar to. In this state, it was gently dried to obtain an immobilized enzyme membrane (8).

Five electrodes with a selective permeation film were prepared by the same method, and the electrode numbers from D-1 to D-5 were added. 4) Measuring device Same as in Example 1. 5) Measurement method Glucose standard solution was
1 injection.

After confirming that a proportional relationship was established between the detected value and the concentration up to a concentration of 30 mM, glucose was injected at a concentration of 20 mM. 6) Results Table 5 shows the measurement results of Example 3.

[0071]

[Table 5]

Average fluctuation rate (CV) of measured values of five electrodes
%) Was 15%.

[0073]

When a large amount of enzyme is applied in order to obtain high activity, the conventional method does not use a means such as spin coating because the viscosity of the enzyme solution increases. Further, there is a drawback that the film material solution spreads in an irregular shape and the reproducibility of the film thickness is lost. The electrode for electrochemical measurement of the present invention can solve such a problem and stably manufacture a product with stable quality with good reproducibility.

According to the present invention, it is possible to construct a measuring electrode having a particularly simple structure, high sensitivity and excellent reproducibility of response values between electrodes.

[Brief description of drawings]

FIG. 1 is a schematic view of a hydrogen peroxide electrode produced in Example 1. The cross-sectional view is a cross-sectional view taken along the length of the conductor 2.

FIG. 2 is a diagram of a measuring apparatus used in Example 1, Example 2, Comparative Example 1, and Comparative Example 2.

FIG. 3 is a schematic view of a hydrogen peroxide electrode produced in Comparative Example 1.

FIG. 4 is a schematic view of the immobilized enzyme electrodes prepared in Examples 2 and 3. The cross-sectional view is a cross-sectional view taken along the length of the conductor 2.

FIG. 5 is a schematic diagram of an immobilized enzyme electrode prepared in Comparative Example 2.

[Explanation of symbols]

 1 Insulating Substrate 2 Conductor (Platinum) 3 Conductor (Platinum) 4 Conductor (Silver) 5 Epoxy Resin Layer 6 Base Polymer Membrane 7 Selective Permeation Membrane 8 Immobilized Enzyme Membrane 9 Injector 10 Measurement Electrode 11 Measurement Cell 12 Fluorine Resin Pipe 13 Constant Temperature Bath 14 Potentiostat 15 A / D Converter 16 Communication Cable 17 Computer 18 Buffer Solution Reservoir 19 Liquid Delivery Pump 20 Waste Solution Reservoir

Claims (5)

[Claims] 1. An insulating substrate having at least a film-shaped conductor serving as a working electrode and a film-shaped conductor serving as a counter electrode, and fixing a hydrogen peroxide selective permeable film on the conductor serving as a working electrode. Electrochemical measurement electrode formed by forming a phosphatase membrane, in which a base polymer film is formed between the hydrogen peroxide selective permeable membrane and the insulating substrate adjacent to the working electrode. Electrodes. 2. The hydrogen peroxide selective permeable membrane is formed by applying a membrane material solution on the base polymer membrane, and the base polymer membrane is a hydrogen peroxide selective permeable membrane membrane material solution. The electrode for electrochemical measurement according to claim 1, which is insoluble in. 3. The electrode for electrochemical measurement according to claim 1, wherein the underlying polymer film contains a protein. 4. The electrode for electrochemical measurement according to claim 1, wherein the base polymer film contains perfluoropolyethylene sulfonic acid. 5. The electrode for electrochemical measurement according to claim 3 or 4, wherein the hydrogen peroxide selective permeable membrane contains a protein.