JP2648361B2 - Permselective membrane and electrode using the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a permselective membrane suitably used for an immobilized biocatalyst electrode or the like for detecting the production or consumption of an electrochemically detectable substance, and the like. It relates to an electrode using the same.

2. Description of the Related Art In recent years, biological reactions that have not been elucidated conventionally have been elucidated due to the development of researches such as biochemistry, and have been applied to various fields of the chemical industry.

Although the application range of biochemical reactions catalyzed by cells, enzymes, and the like is wide, so-called biosensing, which applies biochemical reactions to means for detecting substances, has attracted attention. With this biosensing, it is possible to easily measure substances that could not be measured with conventional technologies or substances that have spent a great deal of time and effort in measurement, and have been able to measure environmentally, food-manufacturing, food analysis, and medical analysis. Practical application is progressing in various fields such as.

 Generally, biocatalysts such as enzymes have a high reaction specificity.

Measurement under mild conditions is possible.

It is possible to measure trace components with high sensitivity.

However, biocatalysts such as enzymes are expensive because biological materials are used as catalysts.

There are restrictions on reaction conditions such as temperature and pH.

Various factors cause loss of catalytic activity of a biocatalyst such as an enzyme, so-called inactivation.

It also has the drawback.

In order to solve such problems, it has been devised to use a biocatalyst immobilized, and many methods have been proposed. Among them, the method of immobilizing a biocatalyst by a cross-linking reaction using a covalent cross-linking agent is suitable for practical use because the bond is stronger than adsorption or ionic bond.

When such an immobilized biocatalyst is applied to the measurement of a substance to be detected, a combination of the biocatalyst and a detection mechanism capable of detecting a substance that increases or decreases due to the biochemical reaction is used. At this time, as a detection mechanism, an electrochemical detector, a fluorescence detector, a heat detector, and the like are applied. In particular, the method of electrochemically detecting the consumption of oxygen or the generation of hydrogen peroxide in a biochemical reaction by an immobilized enzyme using an oxygen electrode or a hydrogen peroxide electrode has a simple device configuration and high detection sensitivity. It has advantages such as high response speed and is most often used.

However, for example, when a hydrogen peroxide electrode is used, not only hydrogen peroxide generated by the biochemical reaction but also interfering substances such as ascorbic acid contained in the measurement sample are detected, and the measurement is not performed. It is known that reliability is reduced. Therefore, a method of interposing a permselective membrane between an immobilized enzyme membrane and an enzyme electrode or a hydrogen peroxide electrode has been put to practical use. That is, a permselective membrane that restricts the permeation of substances other than the substance to be detected via a buffer or the like via a buffer or the like near the surface of an enzyme electrode or a hydrogen peroxide electrode, and an immobilized enzyme membrane are arranged in this order or the electrodes are arranged in this order. A permselective membrane and an immobilized enzyme membrane are formed directly on the surface in this order. This prevents the detection of interfering substances and improves the measurement accuracy.

Conventionally, as such a permselective membrane, a permselective membrane using a material such as acetylcellulose has been used, as disclosed in, for example, Japanese Patent Application Laid-Open No. 60-56254. However, there is a problem that a solvent that is inconvenient to handle must be used in order to manufacture this permselective membrane. In addition, in order to obtain a desired permselectivity, it is necessary to finely adjust the drying time and the like, and to obtain a practical permselectable membrane unless a great deal of time and extremely troublesome operation are required. Can not.

In addition, the present inventors have proposed a method for easily forming a permselective membrane using a protein and a cross-linking agent (Japanese Patent Laid-Open No.
63-182559). However, it is not limited to the permselective membrane,
In general, an immobilized protein membrane formed using a protein and a cross-linking agent has a problem of low physical strength, is inconvenient to handle, and has poor durability.

The problem of the physical strength of such an immobilized protein membrane is that, for example, even if a protein is immobilized on a carrier having high physical strength, the immobilized protein membrane itself becomes brittle and may be partially peeled off. Can not be resolved.

Therefore, the problems with the permselective membrane as described above have not been proposed yet, and the present problems have not been solved.
In general, a method for improving the strength of an immobilized protein membrane in which a protein that is a biocatalyst such as an enzyme is immobilized, instead of a permselective membrane, is disclosed in, for example, Japanese Patent Publication No. 58-49821. In this method, an enzyme which is a biocatalyst is immobilized together with an amino or imino polymer, and the physical strength of the enzyme-immobilized membrane is improved. However,
When such a method is applied to a permselective membrane, there is a problem that it is difficult to obtain a desired permselectivity because the physical properties of the immobilized protein tend to change.

Further, as shown in JP-A-62-32352,
There has also been proposed a method of immobilizing a protein enzyme on a polyester cloth to reinforce the physical strength of the enzyme-immobilized membrane. However, in such a case, the thickness of the protein membrane increases, and when applied to the formation of a permselective membrane, the measurement sensitivity and measurement speed of the immobilized enzyme electrode deteriorate. is there.

Accordingly, an object of the present invention is to solve the above technical problems,
It is an object of the present invention to provide a permselective membrane having desired permselectivity, excellent physical strength, and which can be easily manufactured, and an electrode using the same.

Means for Solving the Problems The present invention develops a mixed solution containing a protein and at least one kind of cross-linking agent, to which at least one kind of polymer substance that reacts with the cross-linking agent is added in addition to the protein, into a film form. And a permselective membrane comprising the protein immobilized by a crosslinking reaction.

Further, the present invention is a component configuration comprising the protein or at least one or more types of globular proteins, wherein the at least one type of crosslinking agent includes a crosslinking agent that undergoes a crosslinking reaction with an amino group, and the polymer substance is an amino group, The above permselective membrane, characterized in that it contains a linear polysaccharide having an amino group derivative or both.

The constituent weight ratio of the protein and the linear polysaccharide is 100.
It is preferably in the range of 0: 1 to 1000: 10, and the constituent weight ratio of the protein to the crosslinking agent is preferably in the range of 100: 5 to 100: 50. The linear polysaccharide may be chitosan.

Furthermore, the present invention is an electrode characterized in that the above-mentioned permselective membrane is provided near a conductive substrate. In such an electrode, an immobilized enzyme membrane on which at least one type of oxidase is immobilized may be provided on the surface of the permselective membrane opposite to the electrode substrate.

When immobilizing a protein on the surface of a conductive substrate, using it as a permselective membrane, and then forming an immobilized enzyme membrane, the physical strength of the permselective membrane has a significant effect on the life of the immobilized enzyme electrode. give.

Generally, in order to improve the physical strength of the immobilized protein membrane, the concentration of the crosslinking agent in the immobilized protein membrane may be increased. However, when this immobilized protein membrane is formed as a permselective membrane, the physical properties of the immobilized protein membrane change depending on the concentration of the crosslinking agent.
Selectivity may deteriorate, which is not desirable.

There is also a method of improving the strength of the immobilized protein membrane by allowing a reinforcing substance that reacts with a crosslinking agent to coexist in the immobilized protein membrane. However, in such a method, the reinforcing substance may adversely affect the physical properties of the immobilized protein membrane, and may lose its original function as a permselective membrane, that is, selectivity.

The present inventors conducted an experiment in which a reinforcing substance that reacts with a cross-linking agent was present in a permselective membrane composed of a protein and a cross-linking agent in particular, and did not adversely affect the physical properties of the permselective membrane, and The present inventors have studied the reinforcing material for improving the physical strength of the permeable membrane and the manufacturing conditions thereof, and have completed the present invention.

Such a reinforcing substance must be a substance that dissolves in a solution of a protein to be immobilized or a mixed solution of the protein and a crosslinking agent.

A substance capable of improving the physical strength of the permselective membrane in a small amount so as not to adversely affect the physical properties of the permselective membrane.

A substance that has the same degree of reactivity as a protein to be immobilized to a cross-linking agent and is insolubilized by a cross-linking reaction.

And other properties are required.

Therefore, the present inventors searched for a compound that can coexist in a mixed solution of a protein and a cross-linking agent, performs a cross-linking reaction with the cross-linking agent, and does not adversely affect the permselectivity of the permselective membrane. did.

For example, using glutaraldehyde or the like as a cross-linking agent, preparing a mixed solution of a protein and a cross-linking agent at a certain concentration using distilled water or a buffer solution as a solvent, and coexisting various compounds in the mixed solution with glutaraldehyde or the like. A cross-linking reaction was performed to prepare an immobilized protein membrane. An experiment can be conducted on the physical strength and permselectivity of the immobilized protein membrane thus prepared to search for a compound that improves the physical strength of the permselective membrane and does not adversely affect the permselectivity.

As a result, the protein is made into a component composition containing at least one or more globular proteins, a compound that undergoes a cross-linking reaction with an amino group is used as a cross-linking agent, and an amino group, an amino group derivative or a derivative thereof is used as a compound to coexist in the mixed solution. It has been found that the use of a linear polysaccharide containing both of them can improve the physical strength of the immobilized protein membrane without adversely affecting the permselectivity of the immobilized protein membrane.

According to the present invention, it is possible to easily form an excellent permselective membrane. In particular, it becomes easy to provide a permselective membrane directly on the surface of the conductive substrate, and further, by forming an immobilized enzyme membrane on the permselective membrane, an immobilized enzyme electrode can be manufactured.

As the cross-linking agent used in the present invention, various cross-linking agents can be used, but a cross-linking reagent such as glutaraldehyde and hexamethylene diisocyanate is preferably used because of its high bonding strength. Particularly, glutaraldehyde is more preferably used.

The constituent weight ratio of the protein and the cross-linking agent is preferably 100: 5 to 100: 50, in terms of permselectivity of the obtained permselective membrane.
Range.

Further, if the protein used in the permselective membrane according to the present invention has a component composition containing at least one or more globular proteins such as albumin, globulin, and prolamin, a permselective membrane having excellent permselectivity can be produced. it can.

Further, the polymer substance added to the mixed solution of the protein and the crosslinking agent and reacting with the crosslinking agent is preferably an amino group, a linear polysaccharide having an amino group derivative or both, and specifically, glucosamine, It is possible to use other sugars having an amine sugar such as galactosamine or a derivative thereof as a constituent unit. Such examples include bacterial peptidoglycan, partially deacetylated hyaluronic acid, partially deacetylated chondroitin sulfate, and chitosan. Chitosan, which is easily available, is suitably used as such a polymer.

Further, in the permselective membrane according to the present invention, the constituent weight ratio of the protein and the linear polysaccharide is 1,000: 1 to 100.
It is preferably in the range of 0:10. When the constituent weight ratio of the linear polysaccharide to the protein exceeds 1000: 10, the selectivity of the permselective membrane deteriorates. The reason for this is not clear, but as the constituent weight ratio of the linear polysaccharide to the protein increases, the concentration of the crosslinking agent for the protein relatively decreases, and the crosslinking reaction for the protein becomes insufficient. Therefore, it is considered that the immobilized protein film becomes brittle, which affects the binding to the conductive substrate surface, the adhesion, and the binding between proteins, and the denseness of the film cannot be maintained. In addition, the above composition weight ratio is 100
If the ratio is less than 0: 1, the improvement of the film strength becomes insufficient.

According to the present invention, the permselective membrane is prepared by first dissolving the above-mentioned protein and cross-linking agent in distilled water or a buffer solution, adding the above-mentioned polymer substance, and applying the resultant on a surface of, for example, a conductive substrate. As a result, in the mixed solution to which the polymer substance is added, the crosslinking reaction proceeds, and an immobilized protein film is formed. The permselective membrane manufactured in this way has extremely excellent durability against physical impacts such as ultrasonic treatment. In addition, the addition of the high molecular substance does not cause a situation in which the permselectivity deteriorates.

The permselective membrane according to the present invention is arranged on a conductive substrate, whereby an electrode for measuring hydrogen peroxide can be constituted. At this time, the permselective membrane may be arranged near the conductive substrate via an impact liquid or the like, but the permselective membrane may be formed directly on the conductive substrate. Further, an immobilized enzyme electrode can be manufactured by immobilizing at least one kind of enzyme such as oxidase on the side of the permselective membrane opposite to the conductive substrate. Such an immobilized enzyme electrode has high physical strength, and is extremely excellent in durability and selectivity of a substance to be detected.

The permselective membrane according to the present invention can also be used for selectively separating only reaction products or raw materials in a bioreactor.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto. In addition,% represents weight%.

Example Measuring Device In this example, a flow type measuring device shown in FIG. 1 was used. A flow type measuring apparatus shown in FIG. 1, an injector 3 for high-performance liquid chromatography capable of injecting a sample on the order of μ, and electrodes E1 to E4 (C1 to C5) using a permselective membrane according to the present invention and references An Ag / AgCl electrode 8 as an electrode is attached, and a measuring cell 5 provided with a stainless steel conduit as a counter electrode 7 is configured. For example, a Teflon dilution line 4 with an inner diameter of 0.5 mm and a length of 1.5 m
Is connected between the injector 3 and the measuring cell 5. The inner volume of the measuring cell 5 is 40 μm, and the electrodes E1 to E4
(C1 to C5) and the Ag / AgCl electrode 8 are arranged to face each other via a buffer solution channel. The electrodes E1 to E4 (C1 to C5) are connected to the Ag / AgCl electrode 8 by the potentiostat 9 and
A voltage of 0.60V is applied.

Such a configuration is arranged in the thermostat 12 and the thermostat 12
The temperature inside is kept at 37 ° C. The pump 2 for high-performance liquid chromatography is used for sending the buffer solution 1, and a 0.1 M sodium phosphate buffer having a pH of 6.0 is sent as the buffer solution 1 at a flow rate of 1.0 ml / min. The buffer containing the sample after the measurement is captured in the waste liquid bottle 11. The measured value is recorded by the recorder 10.

Example 1 Bovine serum albumin (fraction
V, Sigma), glutaraldehyde as a cross-linking agent, and chitosan (manufactured by Tokyo Chemical Industry) as a substance that reacts with the cross-linking agent other than protein.

Chitosan was dissolved in a 100 mM aqueous hydrochloric acid solution to obtain a 0.5% chitosan solution.

 A 5% bovine serum albumin aqueous solution 400μ, a 25% glutaraldehyde aqueous solution 20μ, a 0.5% chitosan solution 20μ, and distilled water 560μ are mixed in a Watselmann test tube to a final concentration of 2% bovine serum albumin, 0.5% glutaraldehyde, 0.01% chitosan. Was prepared.

The component weight ratio of protein and chitosan in this mixed solution is
The ratio by weight is 1000: 5, and the weight ratio of protein to crosslinking agent is 100: 25.

On the other hand, the side surface of a platinum wire having a diameter of 2 mm, which is a conductive substrate, was covered with a heat-shrinkable Teflon, and the cross section was polished with 1600th emery paper. 5 μ of the above mixed solution was placed on the polished surface with a microsyringe and dried at 40 ° C. to form a protein film directly on the platinum electrode. In this way,
An electrode E1 for detecting hydrogen peroxide was created.

An electrode consisting of a platinum electrode covered with this protein membrane
E1 was incorporated into the flow-type measuring device shown in Fig. 1 and 100 mM
While flowing the phosphate buffer solution 1, the Ag / AgCl electrode 8
A potential of 0.6 V was applied. In this state from Injector 3
When 5 mM hydrogen peroxide was injected, the detected current value was 218 nA. Next, when a solution of 5 mM ascorbic acid was injected, the detected current value was 2.9 nA. Therefore, as shown in Table 1, the ratio of the detected current value of ascorbic acid to the detected current value of hydrogen peroxide of the same concentration was 1.3%.

The electrode E1 created as described above was
After processing for one minute, it was incorporated again into the flow-type measuring device shown in FIG. The detected current value when 5 mM hydrogen peroxide was injected was 218 nA, and the detected current value when 5 mM ascorbic acid solution was injected was 2.9 nA.

This result, together with the result before ultrasonic treatment, was
It is shown in the table. Table 2 shows that the sensitivity of the electrode E1 to hydrogen peroxide and ascorbic acid did not change before and after the ultrasonic treatment.

Example 2 Bovine serum albumin (fraction
V, Sigma), glutaraldehyde as a cross-linking agent, and chitosan (manufactured by Tokyo Chemical Industry) as a substance that reacts with the cross-linking agent other than protein.

Chitosan was dissolved in a 100 mM acetic acid aqueous solution to obtain a 0.5% chitosan solution.

 A 5% bovine serum albumin aqueous solution 400μ, a 25% glutaraldehyde aqueous solution 20μ, a 0.5% chitosan solution 20μ, and distilled water 560μ are mixed in a Watselmann test tube to a final concentration of 2% bovine serum albumin, 0.5% glutaraldehyde, 0.01% chitosan. Was prepared.

The component weight ratio of protein and chitosan in this mixed solution is
The ratio by weight is 1000: 5, and the weight ratio of protein to crosslinking agent is 100: 25.

On the other hand, the side surface of a platinum wire having a diameter of 2 mm, which is a conductive substrate, was covered with a heat-shrinkable Teflon, and the cross section was polished with 1600th emery paper. 5 μ of the above mixed solution was placed on the polished surface with a microsyringe and dried at 40 ° C. to form a protein film directly on the platinum electrode. In this way,
An electrode E2 for detecting hydrogen peroxide was prepared.

An electrode consisting of a platinum electrode covered with this protein membrane
E2 was incorporated in the flow-type measuring apparatus shown in the drawing, and 0.6 g was applied to the Ag / AgCl electrode 8 while flowing 100 mM phosphate buffer 1.
A potential of V was applied. In this state, Injector 3 to 5 mM
When hydrogen peroxide was injected, the detected current value was 220 nA. Next, when a solution of 5 mM ascorbic acid was injected, the detected current value was 2.9 nA. Accordingly, as shown in Table 1, the ratio of the detection current value of ascorbic acid to the detection current value of hydrogen peroxide of the same concentration was 1.3% as in Example 1. Further, even when the electrode E2 was subjected to the ultrasonic treatment, the sensitivity to hydrogen peroxide and ascorbic acid did not change. Therefore, it was found that the aqueous solution for dissolving chitosan may be hydrochloric acid or acetic acid as long as it is an acidic solution.

Example 3 Bovine serum albumin (fraction
V, Sigma), glutaraldehyde as a cross-linking agent, and chitosan (manufactured by Tokyo Chemical Industry) as a substance that reacts with the cross-linking agent other than protein.

Chitosan was dissolved in a 100 mM aqueous hydrochloric acid solution to obtain a 0.5% chitosan solution.

 In a Wassermann test tube, mix 5% bovine serum albumin aqueous solution 400μ, 25% glutaraldehyde aqueous solution 20μ, 0.5% chitosan solution 200μ, distilled water 380μ, and final concentration 2% bovine serum albumin, 0.5% glutaraldehyde, 0.05% chitosan Was prepared.

The component weight ratio of protein and chitosan in this mixed solution is
The ratio by weight of the protein and the crosslinking agent is 100: 25.

On the other hand, the side surface of a platinum wire having a diameter of 2 mm, which is a conductive substrate, was covered with a heat-shrinkable Teflon, and the cross section was polished with 1600th emery paper. 5 μ of the above mixed solution was placed on the polished surface with a microsyringe and dried at 40 ° C. to form a protein film directly on the platinum electrode. In this way,
An electrode E3 for detecting hydrogen peroxide was prepared.

An electrode consisting of a platinum electrode covered with this protein membrane
Incorporate E3 into the flow measuring device shown in Fig.
While flowing the phosphate buffer solution 1, the Ag / AgCl electrode 8
A potential of 0.6 V was applied. In this state from Injector 3
When 5 mM hydrogen peroxide was injected, the detected current value was 219 nA. Next, when a solution of 5 mM ascorbic acid was injected, the detected current value was 6.8 nA. Therefore, as shown in Table 1, the ratio of the detected current value of ascorbic acid to the detected current value of hydrogen peroxide of the same concentration was 3.1%. Although it is slightly inferior in selective permeation ability as compared with Examples 1 and 2, it is a value that can be sufficiently used. Further, even when the electrode E3 was subjected to the ultrasonic treatment, the sensitivity to hydrogen peroxide and ascorbic acid did not change.

Comparative Example 1 A side surface of a platinum wire having a diameter of 2 mm was covered with a heat-shrinkable Teflon,
The cross section is polished with emery paper of 1600th and platinum electrode C1
And

This platinum electrode C1 was incorporated into the flow-type measurement device shown in the drawing, and a potential of 0.6 V was applied to the Ag / AgCl electrode 8 while flowing 100 mM phosphate buffer 1 to inject 5 mM hydrogen peroxide. The detection current value was 530 nA, and when a 5 mM ascorbic acid solution was injected, the detection current value was 333 nA.
It was. Therefore, as shown in Table 1, the ratio of the detected current value of ascorbic acid to the detected current value of hydrogen peroxide of the same concentration was 62.9%. As a result, it was found that a sample containing ascorbic acid could not be measured with the platinum electrode C1 having no permselective membrane.

Comparative Example 2 Bovine serum albumin (fraction
V, manufactured by Sigma), glutaraldehyde as a cross-linking agent, and 1,4
-Diaminobutane was used.

 A 5% bovine serum albumin aqueous solution 400μ, a 25% glutaraldehyde aqueous solution 20μ, a 0.5% 1,4-diaminobutane solution 20μ, and distilled water 560μ are mixed in a Watselmann test tube to give a final concentration of 2% bovine serum albumin, 0.5% glutaral. A mixed solution of aldehyde and 0.01% 1,4-diaminobutane was prepared.

On the other hand, the side surface of a platinum wire having a diameter of 2 mm, which is a conductive substrate, was covered with a heat-shrinkable Teflon, and the cross section was polished with 1600th emery paper. 5 μ of the above mixed solution was placed on the polished surface with a microsyringe and dried at 40 ° C. to form a protein film directly on the platinum electrode. In this way,
An electrode C2 for detecting hydrogen peroxide was prepared.

An electrode consisting of a platinum electrode covered with this protein membrane
Incorporate C2 into the flow-type measuring device shown in Fig. 1, 100mM
While flowing the phosphate buffer solution 1, the Ag / AgCl electrode 8
A potential of 0.6 V was applied. In this state from Injector 3
When 5 mM hydrogen peroxide was injected, the detected current value was 296 nA. Next, when a solution of 5 mM ascorbic acid was injected, the detected current value was 144 nA. Therefore, as shown in Table 1, the ratio of the detected current value of ascorbic acid to the detected current value of hydrogen peroxide of the same concentration was 48.5%. The permselective membrane obtained by adding 1,4-diaminobutane was inferior in permselectivity, and it was found that the electrode C2 could not measure a sample containing ascorbic acid as an interfering substance. In addition, it was found that when the ultrasonic treatment was performed, the selectivity was further reduced, so that the physical strength was also inferior.

Comparative Example 3 Bovine serum albumin (fraction
V, Sigma), glutaraldehyde as a cross-linking agent, and triethylenetetraamine as a substance that reacts with the cross-linking agent other than protein.

 A 5% bovine serum albumin aqueous solution (400μ), a 25% glutaraldehyde aqueous solution (20μ), a 0.5% triethylenetetraamine solution (20μ), and distilled water (560μ) were mixed in a Wattsselmann test tube to a final concentration of 2% bovine serum albumin, 0.5% glutaraldehyde, A mixed solution of 0.01% triethylenetetraamine was prepared.

On the other hand, the side surface of a platinum wire having a diameter of 2 mm, which is a conductive substrate, was covered with a heat-shrinkable Teflon, and the cross section was polished with 1600th emery paper. 5 μ of the above mixed solution was placed on the polished surface with a microsyringe and dried at 40 ° C. to form a protein film directly on the platinum electrode. In this way,
An electrode C3 for detecting hydrogen peroxide was prepared.

An electrode consisting of a platinum electrode covered with this protein membrane
C3 was incorporated into the flow-type measuring device shown in the drawing, and 0.6 g was applied to the Ag / AgCl electrode 8 while flowing 100 mM phosphate buffer 1.
A potential of V was applied. In this state, Injector 3 to 5 mM
When hydrogen peroxide was injected, the detected current value was 426 nA. Next, when a solution of 5 mM ascorbic acid was injected, the detected current value was 104 nA. Therefore, as shown in Table 1, the ratio of the detected current value of ascorbic acid to the detected current value of hydrogen peroxide of the same concentration was 24.4%. The permselective membrane obtained by adding triethylenetetraamine was inferior in permselectivity, and it was found that the electrode C3 could not measure a sample containing ascorbic acid as an interfering substance. Further, it was found that when the ultrasonic treatment was performed, the selectivity was further reduced, and the physical strength was also inferior.

Comparative Example 4 Bovine serum albumin (fraction
V, Sigma) and glutaraldehyde as a crosslinking agent.

 A 5% bovine serum albumin aqueous solution (400 µm), a 25% glutaraldehyde aqueous solution (20 µm), and distilled water (580 µm) were mixed in a Watselmann test tube to prepare a mixed solution of a final concentration of 2% bovine serum albumin and 0.5% glutaraldehyde.

On the other hand, the side surface of a platinum wire having a diameter of 2 mm, which is a conductive substrate, was covered with a heat-shrinkable Teflon, and the cross section was polished with 1600th emery paper. 5 μ of the above mixed solution was placed on the polished surface with a microsyringe and dried at 40 ° C. to form a protein film directly on the platinum electrode. In this way,
An electrode C4 for detecting hydrogen peroxide was prepared.

An electrode consisting of a platinum electrode covered with this protein membrane
C4 was incorporated into the flow-type measuring apparatus shown in the drawing, and 0.6 g was applied to the Ag / AgCl electrode 8 while flowing 100 mM phosphate buffer 1.
A potential of V was applied. In this state, Injector 3 to 5 mM
When hydrogen peroxide was injected, the detected current value was 219 nA. Next, when a solution of 5 mM ascorbic acid was injected, the detected current value was 2.6 nA. Therefore, as shown in Table 1, the ratio of the detected current value of ascorbic acid to the detected current value of hydrogen peroxide of the same concentration was 1.2%. Thus, a permselective membrane consisting only of a protein and a cross-linking agent is excellent in its permselectivity.

However, after the electrode C4 was treated with an ultrasonic treatment device for 10 minutes, it was incorporated into the flow-type measurement device shown in FIG. 1 again. Detected current value is 262nA when 5mM hydrogen peroxide is injected
The detected current value when a 5 mM ascorbic acid solution was injected was 6.6 nA.

This result, together with the result before ultrasonic treatment, was
It is shown in the table. From Table 2, it can be seen that after sonication, the sensitivity of electrode C4 to hydrogen peroxide and ascorbic acid is increased, and the permselectivity is poor. Therefore, it was found that such an electrode C4 had insufficient physical strength and was inferior in durability.

Example 4 The side surface of a platinum wire having a diameter of 2 mm was covered with a heat-shrinkable Teflon,
A permselective membrane is formed on the surface polished with emery paper of No. 1600 using the same material and procedure as in Example 1.
A solution prepared by dissolving 1 mg / ml of bovine serum albumin, 1 mg / ml of glucose oxidase (Type II, manufactured by Sigma), and glutaraldehyde in a 100 mM sodium phosphate buffer (pH 6.0) to a concentration of 0.2% was added thereto. 3μ with micro syringe
Add dropwise and heat at 40 ° C for 15 minutes to form an immobilized enzyme layer,
The electrode was E4.

This electrode E4 was incorporated into the flow-type measuring device shown in the drawing, and the Ag / AgCl electrode 8 was fed while flowing 100 mM phosphate buffer 1.
, A voltage of 0.6 V was applied. Injector 3 to 30
When 5 μm of an aqueous glucose solution was injected, the detected current value was 393 nA. Next, when the same amount of 30 mM ascorbic acid aqueous solution was injected, the detected current value was 11.2 nA. That is, the detection value of ascorbic acid with respect to the same concentration of glucose is 2.8%, which is completely negligible in practical use.

Further, the electrode E4 was once removed from the flow-type measuring device, treated with an ultrasonic treatment device for 10 minutes, and then attached to the flow-type measuring device again. And as before sonication, 30
mM glucose and ascorbic acid aqueous solutions
μ was injected. The detected current value for glucose is 395 nA
The detected current value for ascorbic acid was 11.1 nA. The results are shown in Table 3. Thus, it was found that the performance of the present electrode E4 did not deteriorate due to the ultrasonic treatment.

In addition, when this ultrasonically treated electrode was stored in a buffer at room temperature, no decrease in sensitivity or selectivity was observed even after 3 months, showing excellent durability.

Comparative Example 5 An immobilized enzyme electrode C5 was prepared in the same manner as in Example 4, except that chitosan was not included when the permselective membrane was prepared.

The electrode C5 was incorporated into the flow-type measuring apparatus shown in FIG. 1, and a voltage of 0.6 V was applied to the Ag / AgCl electrode while flowing 100 mM phosphate buffer 1. 30mM glucose aqueous solution 5
When μ was injected, the detected current value was 385 nA. Next, when the same amount of 30 mM ascorbic acid aqueous solution was injected, the detected current value was 10.3 nA.

Further, the electrode C5 was once removed from the flow-type measuring device, and was treated for 10 minutes by an ultrasonic treatment device. No abnormality was visually observed in the membrane, but the electrodes were attached to the flow-type measuring device again, and 30 mM of glucose and an aqueous solution of ascorbic acid were injected at 5 μm each as before the ultrasonic treatment. The current value was 402 nA, and the detected current value for ascorbic acid was 16.2 nA. The results are shown in Table 3 together with the results of Example 4. As described above, in the electrode C5, it was found that the membrane permeability was changed by the ultrasonic treatment, and the selectivity was lowered.

When the ultrasonically treated electrode was stored at room temperature in a buffer solution, peeling of the film was visually observed after two months, and partial damage to the film caused by the ultrasonic treatment was finally completed. It is considered that the film progressed to the peeling of the film.

Effects of the Invention As described above, according to the present invention, when a functional membrane having selective permeation ability is constructed using a protein, it does not adversely affect the properties of the immobilized protein membrane, and
A permselective membrane having excellent physical strength can be easily formed.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a flow-type measuring device used in one embodiment and a comparative example of the present invention. 1 ... buffer solution, 2 ... pump, 3 ... injector, 4
... Dilution pipeline, 5 ... Measurement cell, E1-E4 ... Electrode,
7… Counter electrode, 8… Ag / AgCl electrode, 9… Potiostat, 10… Recorder, 11… Waste liquid bottle

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location G01N 27/327 G01N 27/30 341G 27/404 341J 353Q (56) References JP-A-63-182559 (JP, A) JP-A-55-164349 (JP, A) JP-A-60-56254 (JP, A) JP-A-62-32352 (JP, A)

Claims (7)

(57) [Claims] 1. A mixed solution containing a protein and at least one kind of cross-linking agent, wherein at least one kind of a polymer substance that reacts with the cross-linking agent is added in addition to the protein, and the mixture is developed into a film. A permselective membrane characterized by immobilizing a protein. 2. The composition according to claim 1, wherein the protein comprises at least one kind of globular protein, wherein the at least one kind of crosslinking agent comprises a crosslinking agent which undergoes a crosslinking reaction with an amino group, and wherein the polymer substance comprises an amino group, 2. The permselective membrane according to claim 1, comprising a linear polysaccharide having a group derivative or both. 3. The permselective membrane according to claim 2, wherein said polymer substance is chitosan. 4. The permselective membrane according to claim 2, wherein the weight ratio of the protein to the linear polysaccharide ranges from 1000: 1 to 1000: 10. 5. The composition of the present invention, wherein the weight ratio of the protein to the crosslinking agent is
The permselective membrane according to claim 2 or 4, wherein the range is from 100: 5 to 100: 50. 6. An electrode, wherein the permselective membrane according to any one of claims 1 to 5 is provided near a conductive substrate. 7. The electrode according to claim 6, wherein an immobilized enzyme membrane on which at least one type of oxidase is immobilized is provided on the surface of the permselective membrane opposite to the conductive substrate. .