JPH0372254A - Enzyme electrode - Google Patents

Abstract

PURPOSE:To perform accurate measurement reduced in the irregularity of electrode output by constituting the immobilized enzyme film applied to the response part of an enzyme electrode of the first polymer layer composed of a cation exchange polymer, an enzyme layer and the second polymer layer. CONSTITUTION:A plastic film is used as an electrode support base material 2 and an acting electrode 3 and a reference electrode 4 are formed to the surface of the base material 2 and covered with an insulating protective film 5 to expose electrode response ports 3a, 4a and an electrode connection part. Lead wires are connected to the electrodes and the connection part is reinforced by an epoxy resin 8 and the response parts 3a, 4a are coated with an immobilized enzyme film 9 to constitute an enzyme electrode 1. When the immobilized enzyme film 9 is constituted of a layer 9a, an enzyme layer 9b and a polyurethane layer 9c, the sensitivity to hydrogen peroxide is enhanced by the cation exchange function of the layer 9a and the linear concn. range of a calibration curve is widened and a uniform film thickness is obtained by dip coating and the irregularity of the output between individual electrodes can be reduced.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application This invention relates to an enzyme electrode that electrically measures the concentration of a substrate in a sample using an enzyme reaction.

(b) Prior Art Conventionally, cylindrical so-called pencil-shaped enzyme electrodes have been known, but this type of enzyme electrode has had many problems in terms of performance and productivity. In order to overcome these problems, so-called planar micro-enzyme electrodes have been proposed.

A cross section of this planar microenzyme electrode is shown in FIG. 22 is a flat electrode support base material made of an insulating material such as a plastic film. On this electrode supporting base material 22, a working electrode 23 made of a metal such as platinum (pt) and a contrast type Fji 24 are formed as thin films.

Further, an insulating protective film 25 is formed on the electrode support base material 22, and the picture electrodes 23, 24 are covered and insulated except for the sensitive parts 23a, 24a and the connection parts (not shown). In addition,
Each connection has a lead for external connection (not shown).
is connected.

29 is an immobilized enzyme membrane, which includes a first polymer layer 29a,
It is composed of three layers: an enzyme layer 29b and a second polymer layer 29c. Acetyl cellulose is used for the first polymer layer 29a and the second polymer layer 29c, and is formed by dipping the electrode supporting base material 22 in an acetyl cellulose solution (dip coating). ing.

On the other hand, the enzyme layer 29b contains hydrogen peroxide (H20) during the reaction.
It is formed by dropping a solution of an enzyme that produces □), such as glucose oxidase, onto the first polymer layer 29a.

This conventional enzyme electrode 21 includes a working electrode 23 and a reference electrode 24.
It is immersed in the specimen with a predetermined voltage applied between them.

At this time, since an electrode current corresponding to the concentration of a substrate such as glucose in the sample is obtained, the glucose concentration can be determined by applying a calibration curve prepared in advance to this electrode current.

(c) Problems to be Solved by the Invention In the conventional enzyme electrode described above, the first polymer layer 29a, the second
Since both the polymer layers 29c are made of acetylcellulose, there are problems listed below.

■Low sensitivity to hydrogen peroxide produced by enzymatic reactions,
The linear concentration range in a calibration curve for substrate measurement, for example glucose measurement, is narrow.

■When the concentration of interfering substances such as ascorbic acid in the sample increases, it is affected and accurate measurements cannot be performed.■High molecular substances in the sample, such as proteins, tend to adhere to the immobilized enzyme membrane, reducing the electrode output. to be influenced.

■The mechanical strength of the immobilized enzyme membrane is weak and easily damaged.

■Although acetylcellulose can be formed into a film using a simple dip coating method, it is difficult to make the film thickness uniform, and there is large variation in electrode output between individual enzyme electrodes.

■Acetyl cellulose has different properties such as acetylation rate depending on the manufacturer or loft, which affects the electrode output.

■Preparation of acetylcellulose solution is complicated.

The present invention has been made in view of the above, and an object of the present invention is to provide an enzyme electrode that has less variation in electrode output between electrodes and can perform accurate measurements.

(d) Means and action for solving the problem In order to solve the above problem, the enzyme electrode of the first aspect comprises an electrode support base material, an electrode formed on the electrode support base film, and this electrode. Completely insulated except for sensitive parts! a membrane, and an immobilized enzyme membrane covering at least the sensitive part, the immobilized enzyme membrane comprising a first polymer layer in contact with the sensitive part, and a first polymer layer formed on the first polymer layer. In one comprising an enzyme layer and a second polymer layer covering this enzyme layer, the first
The polymer layer is a cation exchange polymer layer.

Due to the cation exchange function of this cation exchange polymer,
The sensitivity to hydrogen peroxide is increased, the linear concentration range of the calibration curve is widened, and the sensitivity to interference substances in the sample is reduced. In addition, some cation exchange polymers have excellent mechanical strength and chemical resistance, and these polymers can be used to increase the mechanical strength of immobilized enzyme membranes and create chemically stable ones. It can be done. Furthermore, solutions of cation exchange polymers are already commercially available, and even if this solution is used to form the first polymer layer by deep coating, the film thickness will be uniform, and each enzyme electrode will have a uniform thickness. It is possible to reduce the variation between the two.

The enzyme electrode according to the second aspect further includes an electrode supporting base material, an electrode formed on the electrode supporting base material, an insulating film covering and insulating the electrode except for the sensitive part, and at least the sensitive part. an immobilized enzyme membrane covering the sensitive area, the immobilized enzyme membrane comprising: a first polymer layer in contact with the sensitive area; an enzyme layer formed on the first polymer layer; It is particularly preferable that the second polymer layer is a polyurethane layer.

This polyurethane has less adhesion of protein polymers in the specimen, and has less influence on the electrode output. Furthermore, polyurethane has excellent mechanical strength and chemical resistance, and can increase the mechanical strength of the immobilized enzyme membrane and make it chemically stable. Additionally, the polyurethane solution is easy to prepare and can be used for dip coating.
Even if a polymer layer is formed, the film thickness is uniform, and variations between individual enzyme electrodes can be reduced.

(e) Embodiment An embodiment of the present invention will be explained based on FIGS. 1 to 6.

This example enzyme electrode is applied to the measurement of glucose concentration in a sample, and uses glucose oxidase (COD) as the enzyme.

Hereinafter, an example enzyme electrode will be explained while following the manufacturing process.

FIGS. 2(a) and 2(b) show a plurality of groups of working electrodes 3 and reference electrodes 4 formed on the surface of a plastic film (electrode support base material) 2o.

This plastic film is selected to be insulating and heat resistant, and has an appropriate size for the work (for example, 50m).
(m x 50 mm, thickness 0.125 mm) is used. Note that the electrode supporting base material is not limited to a plastic film, and a plate material made of other insulating materials can be used.

The working electrode 3 and the reference electrode 4 are metal thin films such as platinum arranged in parallel, and thin film forming methods such as sputtering and vacuum evaporation are applied.
゜It is formed on the surface.

FIGS. 3(a) and 3(b) show a state in which an insulating protective film 5 is formed on the surface of the plastic film 2°.

This insulating protection 1195 covers the surface of the plastic film 2° with a photosensitive resin film made of photosensitive polyimide,
After exposing this photosensitive resin film to light using a photomask (not shown), it is developed and rinsed to remove unnecessary portions (photolithography). From this removed portion, the working electrode 3 and the reference electrode 4 are exposed as sensitive parts 3a, 4a and connecting parts 3b, 4b, respectively. These areas, especially the areas of the sensitive parts 3a and 4a, can be determined with high precision since photolithography is applied. In this embodiment, the area ratio between the working electrode sensitive section 3a and the reference electrode sensitive section 4a is 1:4. Note that the shape, area ratio, etc. of the sensitive section are not limited to those described above, and the design can be changed as appropriate.

Figures 4(a) and 4(b) show the state in which the plastic film 2° is cut into individual plastic films 2 and the lead wires 7 are connected! (referred to as the base electrode).

The plastic film 2° can be cut using easy means such as scissors or a cutter knife because it is thin. Lead 4% 7.7 is supported by a strip-shaped insulator 7a, and each lead wire 7.7 is electrically connected to the working electrode connection portion 3b and the reference electrode connection portion 4b using solder or silver paste. connected to. Epoxy resin 8 is applied on the connecting portions 3b and 4b, and the connecting portions of the lead wires 7.7 are insulated and reinforced.

1(a) and 1(b) show the completed enzyme electrode 1 by forming an immobilized enzyme membrane 9 on the base electrode 6 shown in FIG. 4(aJ(b)).This immobilized enzyme The membrane 9 is
It is composed of three layers: a Nafion layer 9a, an enzyme i9b, and a polyurethane layer 9c. Hereinafter, the formation process of the immobilized enzyme membrane 9 will be explained while comparing it with the conventional enzyme electrode 21.

First, a Nafion layer 9a is formed on the base electrode 6. Nafion is one of the cation exchange polymers and has the structure shown below.

((CF2)y CFz]2 (OCF2-CF), -0CFZCF2SO111CF
! Currently, Nafion solution (5%) using ethanol as a solvent
is commercially available, and the base electrode 6 is dipped in this Nafion solution to form a Nafion layer 9a (thickness is approximately 1 μm).
) is formed.

On the other hand, in the conventional enzyme electrode 21 (see Fig. 7), 2
% acetylcellulose solution (the solvent is a mixture of acetone and cyclohexane at a ratio of 40:10), and the base electrode is dipped in this acetylcellulose solution to form the first acetylcellulose layer 29a. .

Next, an enzyme layer 9b is formed on the Nafion layer 9a. The enzyme layer 9b is formed by dropping a glucose oxidase solution onto the Nafion layer 9a and drying it. This glucose oxidase solution is prepared by mixing the following solutions A and B.

Solution A: 20 mg of nigerose oxidase dissolved in 100 uffi of 0.1 molar phosphate buffer (pH 6,0). Solution B: 100 μl of 0.5% glutaraldehyde prepared in 0.1 molar phosphate buffer (pH 6,0). The formation of the enzyme layer is exactly the same in the conventional enzyme electrode 21. In addition, in FIG. 1(b), the enzyme layer 9b
is formed only on the working electrode sensitive part 3a, but it can also be formed larger.

Finally, the polyurethane layer 9c is formed also by dip coating. What is used at this time is a 2% polyurethane solution, which is polyurethane in tetrabidrofluorocarbon? Prepared by dissolving.

In contrast, with the conventional enzyme electrode 2■ (see Figure 7),
A second acetylcellulose layer 29 is formed by dipping the base electrode in a 3% acetylcellulose solution (solvent: a mixture of acetone and ethanol in a ratio of 40:10).
c is formed.

Next, the characteristics of the example enzyme electrode 1 will be explained while comparing them with the conventional enzyme electrode 21. Before that, the measurement system 11 used for measuring the characteristics will be explained with reference to FIG. 6.

Reference numeral 12 denotes a constant temperature bath, in which a 0.1 molar phosphate buffer solution 13 adjusted to pH 7.0 is stored. The enzyme electrode 1 (21) is immersed in this phosphate buffer 13. Further, this phosphate buffer solution 13 is stirred by a stirrer I4, and 15 is a rotor of this star 14. Leads 4$7 and 7 of the enzyme electrode 1 are connected to the electron meter 16 and set to a predetermined voltage (5.5V in this measurement).
) is added and the electrode output is displayed.

A predetermined amount of glucose solution is dropped into the phosphate buffer 12 using a micropipette (not shown).

Glucose causes the following reaction within the immobilized enzyme membrane 9 of the enzyme electrode 1.

The generated H2O2 is oxidized in the working electrode sensitive section 3a, and by detecting the oxidation current as the electrode output, the concentration of glucose can be determined stoichiometrically.

Therefore, first H. It is necessary to check the electrode output for 02. FIG. 5(a) shows the electrode output with respect to the H z O z concentration, and the white circles (○) indicate the base electrode 6.
The electrode output in which only the Nafion layer 9a was formed, and the black circle (·) indicates the electrode output when only the Nafion layer 9a was formed on the base electrode.
The electrode output is shown when only 9a is formed.

Figure 5(b) shows the electrode output for several glucose concentrations. In the figure, the white circles (○) indicate the electrode output of the enzyme electrode 1 with the immobilized enzyme membrane 9 completely formed, and the black circles (·) indicate the electrode output of the conventional enzyme electrode with the immobilized enzyme membrane 29 completely formed. 21 electrode outputs are shown. The plotted points in this figure are connected to form a calibration curve, and the concentration of glucose in an unknown sample, such as blood, can be quantified. Excellent linearity is obtained compared to (・).

In this example, the first polymer layer is a Nafion layer and the second polymer layer is a polyurethane layer.
It is also possible to use the polymer layer as a Nafion layer and the second polymer layer as an acetyl cellulose layer, or the first polymer layer as an acetyl cellulose layer and the second polymer layer as a polyurethane layer. , the design can be changed as appropriate.

Furthermore, in the above examples, glucose oxidase is used as the enzyme, but the enzyme is not limited to this and can be changed as appropriate.

(f) As described in detail of the invention, since the enzyme electrode of the first claim has the first polymer layer as a cation exchange polymer layer, the enzyme electrode is listed in the following items i-v. It has the advantage of

i: A calibration curve with a wide linear concentration range can be obtained.

ii: Not easily affected by interfering substances in the sample, such as ascorbic acid.

iii: The mechanical strength of the immobilized enzyme membrane is improved, and it becomes chemically stable with excellent chemical resistance.

iv: Even with the dip coating method, a uniform layer thickness can be obtained, and variations in the electrode output of individual enzyme electrodes are reduced.

V: Cation-exchangeable polymer solutions are already commercially available and do not require preparation.

Furthermore, since the enzyme electrode according to the second aspect has the second polymer layer as a polyurethane layer, the above-mentioned paragraph 111,
It has the advantages described in section iv and the advantages described in sections vi and vii below.

vi: The polymer in the sample, such as protein, is immobilized enzyme 11
It is difficult to adhere to the surface.

■i=Preparation of mini-polyurethane is extremely easy.

[Brief explanation of drawings]

FIG. 1(a) is an external perspective view of an enzyme electrode according to an embodiment of the present invention, and FIG. 1(b) is a diagram of FIG. 1(a) of the same enzyme electrode.
The cross-sectional views along the middle Ti line and FIGS. 2 to 4 are diagrams explaining the manufacturing process of the enzyme electrode, and FIG. 2(a) is
FIG. 2(b) is a perspective view showing a state in which a working electrode and a control electrode are formed on the surface of a plastic film, and a cross-sectional view of the same plastic film taken along the line ■-■ in FIG. 2(a), and FIG. 3(a) are ) is a perspective view showing a state in which an insulating protective film is formed on the surface of the same plastic film, and FIG. 3(b) is a perspective view of the plastic film shown in III-I in FIG. 3(a).
4(a) is a perspective view of the base electrode constituting the enzyme electrode of the example, and FIG. 4(b) is a cross-sectional view taken along the line II, and FIG. 4(b) is the rV-IV line in FIG. 4(a) of the same base electrode. A cross-sectional view of
FIG. 5(a) shows a comparison of the electrode output with respect to the hydrogen peroxide concentration of the base electrode compared with the conventional one, and FIG. 5(b) shows the electrode output with respect to the glucose concentration of the enzyme electrode of the example compared with the conventional one. Figure 6 is a diagram illustrating the measurement system applied to the measurement of the characteristics of the enzyme electrode of the example, and Figure 7 is a diagram for comparison.
FIG. 2 is a cross-sectional view of a conventional enzyme electrode. 2 Niblast film, 3: Working electrode, 3a: Working electrode sensitive part, 4 = Control electrode, 4a: Control electrode sensitive part, 5: Insulating protective film, 9: Immobilized enzyme membrane, 9a: Nafion layer, 9b ;
Enzyme layer, 9C: Polyurethane layer.

Claims (2)

[Claims] (1) An electrode support base material, an electrode formed on the electrode support base material, an insulating film that covers and insulates the electrode except for the sensitive part, and an immobilized enzyme film that covers at least the sensitive part. The immobilized enzyme membrane includes a first polymer layer in contact with the sensitive part, an enzyme layer formed on the first polymer layer, and a second polymer layer covering the enzyme layer. An enzyme electrode comprising: The first polymer layer is a cation exchange polymer layer. (2) an electrode support base material, an electrode formed on the electrode support base material, an insulating film that covers and insulates the electrode except for the sensitive part, and an immobilized enzyme film that covers at least the sensitive part; The immobilized enzyme membrane includes a first polymer layer in contact with the sensitive part, an enzyme layer formed on the first polymer layer, and a second polymer layer covering the enzyme layer. An enzyme electrode comprising: The second polymer layer is a polyurethane layer.