JPH0122898B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a bioelectrochemical sensor having enzymes or microorganisms immobilized on the electrode surface, and a method for manufacturing the same. , a bioelectrochemical sensor characterized in that an enzyme or a living microorganism is coated and integrated with an immobilized enzyme membrane or an immobilized microorganism membrane wrapped in a water-insoluble polymer membrane via a porous membrane, and a method for producing the same. It is.

Recently, bioelectrochemical sensors have been developed that combine the reaction specificity of living organisms with electrochemical devices.Especially in the medical field, their substrate selectivity and trace reactivity make it possible to transform clinical testing methods. It is attracting attention as something that can be done.

Already, proposals have been made for a method for quantifying biological components using an enzyme electrode based on both electrode reactions of amperometry and potentiometry, and a method for quantifying trace components using a microbial electrode that applies the bioassay method.

However, conventionally proposed enzyme immobilization methods or microorganism immobilization methods (hereinafter both referred to as immobilization methods) suffer from continuous elution of the immobilized substance, inactivation, changes in steric structure, and low specific activity. They have drawbacks such as difficulty in passing through the substrate, and some immobilization methods cannot be immobilized due to molecular weight limitations, so their development has stalled. On the other hand, in conventional bioelectrochemical sensors, the electrode surface is coated with a pre-formed enzyme or microbial immobilization film, which poses a major obstacle to handling and miniaturization of the sensor. was hindering the development of In addition, bioelectrochemical sensors whose electrode surfaces are coated with membranes with immobilized enzymes or microorganisms electrochemically detect substances produced or consumed by biological reactions. In the case of enzyme electrode reactions, the presence of interfering substances that affect electrode reactions, such as Fe ⁺⁺⁺ ions, significantly reduces accuracy, and countermeasures are complicated, so it is difficult to put bioelectrochemical sensors into practical use. There were also problems such as slow progress.

The present inventors have arrived at the present invention as a result of intensive studies aimed at eliminating these inhibiting factors at once and promoting the practical use of bioelectrochemical sensors.

That is, the gist of the present invention is that a porous polymer membrane is directly coated on the electrode surface of a separate electrode made of a noble metal wire, and an immobilized enzyme membrane or an immobilized microorganism membrane is directly coated on the surface of the porous membrane. electrode,
It is a bioelectrochemical sensor in which a porous polymer membrane, an immobilized enzyme membrane, or an immobilized microbial membrane forms an integral structure, and its manufacturing method is a separate type consisting of a noble metal wire coated with a porous polymer membrane in advance. This is a method for producing a bioelectrochemical sensor in which the electrode surface of an electrode is coated with a mixture of ice cubes containing enzymes or microorganisms and an organic solvent in which a water-insoluble polymer substance is dissolved, and then the organic solvent is removed.

The present invention will be explained in detail below.

The drawing shows a specific example of the sensor of the present invention, in which a porous polymer membrane 1 and an immobilized enzyme membrane or an immobilized microbial membrane 4 (hereinafter abbreviated as immobilized enzyme membrane, etc.) are made of a noble metal coated with an insulator 2. The separable electrode 3 is coated with an integral structure.

That is, conventional electrodes using immobilized enzyme membranes are made by separately molding a porous polymer membrane or an immobilized enzyme membrane, as shown in FIG. 2 of JP-A-54-102193, for example. A method has been adopted in which this film is coated on the electrode surface.

When using such a method, if the electrode is thin such as a platinum wire, it is very difficult to coat the electrode because the film may break during coating. On the other hand, in the electrode of the present invention, since the immobilization of the enzyme and the coating of the immobilized enzyme membrane on the electrode are carried out in an integrated manner during manufacturing, it is possible to coat even extremely small electrode surfaces, and furthermore, it is possible to coat even extremely small electrode surfaces. By making appropriate selections, the thickness and porosity of the coating film can be arbitrarily selected. Furthermore, the immobilized enzyme membrane that is integrally immobilized on the electrode is unlikely to fall off during measurement, and can be used not only by immersion in an external solution, but also when inserted into living tissue for measurement.
It is safe to use.

As is clear from the figure, the second feature of the sensor of the present invention is that it has a laminated structure of a porous polymer membrane 1 that does not contain an immobilized enzyme, etc., and an immobilized enzyme membrane, etc. 4 that has enzymes and microorganisms immobilized therein. It is to have.

In an electrochemical analysis method that uses a combination of an immobilized enzyme membrane or the like and an electrode reaction, for example, in an enzyme electrode, it is generated or consumed by the reaction between a substrate and an enzyme. The electrode is used to quantitatively detect active substances such as oxygen, hydrogen peroxide, carbon dioxide, ammonia gas, carbonate ions, hydrogen ions, etc., but under measurement conditions, such as amperometric method using polarography, it is necessary to set the potential to reduce oxygen. If there are metal ions such as Fe ⁺⁺⁺ that are reduced at the same time, these become interfering substances and prevent the measurement.

Although immobilized enzyme membranes and the like allow substrates to easily pass through them, they also allow these interfering substances to pass through, or the waves of the measurement system, which is another factor that inhibits measurement accuracy, easily reach the electrode surface, making measurement difficult. Accuracy will be reduced.

In order to eliminate these interferences, there are proposals for laminating thin films and multiple electrode systems for the purpose of correction, but the former has drawbacks such as the difficulty of laminating thin films, and the latter requires complicated circuits and equipment. It had

In order to improve the above-mentioned thin film lamination, the present invention involves forming a fine porous membrane layer in advance on the electrode tip of a separate electrode made of a noble metal wire, and further forming and integrating an immobilized enzyme membrane etc. thereon. This paper proposes a sensor with high measurement accuracy that is effective and simple.

The porous polymer membrane referred to in the present invention has a pore diameter of 10 Å to
It has pores of 5 μm, preferably 20 Å to 3 μm, more preferably 50 Å to 1 μm. In addition, asymmetric membranes have a porous polymer membrane itself with a dense layer with small pores on the surface and a porous layer with large pores inside.
By forming a multilayer film, the transfer speed of the substrate and the electrode reactant can be adjusted. In this case, it is preferable that the pore diameter of the dense layer is controlled within the range of 20 Å to 1 μm, and the pore diameter of the internal porous membrane is controlled to 1 μm or more.

That is, assuming that the bioelectrochemical sensor of the present invention is most effectively used for biological information, that is, the component capacity of blood and interstitial fluid, the permeation of blood and interstitial fluid into the membrane under unloaded pressure, which is considered to be colloidal, is For this purpose, a pore size of 20 Å or more, preferably 50 Å or more is required; if the pore size is smaller than this, it will take time to obtain a stable response when a dry electrode is inserted into tissue or blood flow. On the other hand, when the pore diameter becomes larger than 3μ,
Fluid movement within the pores occurs in response to the movement of tissue or blood flow, resulting in unstable measurements and increased susceptibility to the effects of interfering substances.

It is more effective to distribute the pore diameters of the porous polymer membrane so that they become smaller in the direction of the membrane thickness from the electrode surface. On the other hand, the higher the pore volume fraction of a porous polymer membrane, the better the sensitivity of the electrode, and this is determined in correlation with the physical strength of the membrane.

Materials forming the porous membrane include hydrophilic materials such as cellulose, cellulose acetate, collodion membrane (cellulose nitrate), polyamide hydrazide, polyvinyl alcohol, polyhydroxyethyl methacrylate, or polycarbonate,
Hydrophobic polymers such as ethylene oxide block polymers, polystyrene, nylon 6, and polyester are easily dissolved and swollen by solvents and swelling agents, and are particularly suitable for the ease of controlling pore size, membrane strength, and immobilized enzyme membranes described below. It is determined by taking into consideration the relationship with the forming solvent, etc.

The thickness of the porous membrane is preferably increased or decreased depending on the pore diameter, and as the pore diameter becomes smaller, it needs to be thinner, but it is preferably approximately 10 to 100 μm.

Following this porous membrane, an immobilized enzyme membrane and the like are integrally formed. Known methods can be used to manufacture the immobilized enzyme membrane, but care must be taken to ensure that the enzymes and microorganisms to be immobilized are not deactivated.
Particularly preferred is the method described below in which enzymes and the like are included in an ice block and then further included in a polymer membrane. The immobilized enzyme membrane or the like is generally one in which the enzyme or the like is immobilized in a water-insoluble polymer matrix, and the polymer matrix is preferably a porous membrane containing a large number of micropores.

By having such a structure, the substrate in the solution that comes into contact with the sensor can freely come into contact with the enzyme, etc., and the reaction can be carried out quickly.

Next, a method for manufacturing the biochemical sensor of the present invention will be described.

Porous membranes can be created by first swelling a dense membrane created on the electrode surface with a swelling agent and then replacing it with a non-solvent to create a porous membrane, or by attaching a polymer solution to the electrode surface. After that, any method such as desolvation and coagulation in air or in a non-solvent that is compatible with the solvent may be used.Several types of solvents may be used, combinations of solvents and swelling agents, solvent-nonsolvent mixtures, etc. Any system including combinations of liquids may be used.

The pore size can be adjusted by adjusting the combination of solvent and swelling agent, temperature,
This is done depending on the dissolved concentration of the polymer, the solvent-nonsolvent ratio, the coagulation timing, etc. Further, in order to provide a distribution of pore diameters, the film formation may be performed multiple times in a sequentially stacked manner.

The obtained porous membrane may be further subjected to annealing to adjust the pore diameter or swelling strength.

Next, an immobilized enzyme membrane or the like is integrally formed on the polymer porous membrane integrally formed on the electrode surface of the separated electrode made of the noble metal wire.

Various methods have been proposed for immobilizing enzymes or microorganisms, and among them, the so-called comprehensive method is widely used as a method that does not reduce the activity of the immobilized substance.

That is, as a method for enclosing and immobilizing enzymes and microorganisms in water-insoluble polymeric substances, water-soluble monomers or water-soluble polymeric substances and water-soluble crosslinking agents are dissolved in water together with enzymes or microorganisms, and then this is passed through the water. There are methods of polymerizing with a polymerization catalyst such as potassium sulfate or radiation such as gamma rays, simultaneously giving a crosslinked structure, and enclosing enzymes and microorganisms in the resulting water-insoluble polymer gel. An aqueous solution containing enzymes or microorganisms is dispersed as minute water droplets in an organic solvent in which a molecular substance is dissolved, and then polymerization is performed, or an organic solvent in which a water-insoluble polymer substance is dissolved is removed. A method is known in which the water droplets are then surrounded by a water-insoluble polymer substance.

However, these conventional methods have the following drawbacks. That is, enzymes and microorganisms are generally relatively stable in water, but unstable in organic solvents, and in conventional methods, many of the enclosing materials are water-soluble. When using water-soluble materials, it is necessary to make the water-soluble materials insoluble by polymerization, crosslinking, etc., and these operations inevitably cause deterioration of enzymes and microorganisms. Furthermore, when attempting to use water-insoluble polymeric substances as enclosing materials, it is necessary to use organic solvents to dissolve them, and the organic solvents often deactivate enzymes and kill microorganisms. Sakimae et al., one of the inventors of the present invention, conducted research to solve the drawbacks of these conventional methods, and developed several methods for stably enclosing enzymes and microorganisms with water-insoluble polymeric substances in organic solvents. We are making proposals (JP-A-Sho
54-105289, Japanese Patent Publication No. 55-135591). Furthermore, as an improvement method, the surface of the solid structure is coated with a water-insoluble polymer substance containing enzymes or microorganisms, which has excellent mechanical strength and can be molded into various thin film shapes that match the shape of the solid structure. It has been found that immobilized enzymes or immobilized microorganisms can be easily produced.

The present inventors have found that a bioelectrochemical sensor in which enzymes or microorganisms are directly immobilized on the electrode surface by applying this method has excellent performance in terms of activity, durability, and applicability, and has developed the present invention. Reached.

That is, the electrode surface is coated with a mixture of ice cubes containing the enzyme or microorganism and an organic solvent in which a water-insoluble polymer substance is dissolved, and then the organic solvent is removed. This is a bioelectrochemical sensor and its manufacturing method.

The enzyme used in the present invention can be obtained from animal or plant tissues or produced by microorganisms, regardless of its source. Enzymes can also be purified or unpurified, such as enzyme-containing tissue homogenates or microbial cells.

The enzymes used in the present invention are not particularly limited, but for example, all of the various enzymes described in JP-A-54-105289 can be used.

Further, the microorganisms that can be used in the present invention are those classified into molds, enzymes, bacteria, actinomycetes, and Deuteromyces, and the types thereof are not particularly limited, but for example, the microorganisms described in JP-A-55-135591 All types of microorganisms can be used.

These microorganisms can be grown in a nutrient medium and then used in a live state. The living state here means that the microorganism has the ability to self-regenerate, and can be confirmed by culturing it in an environment suitable for the growth of the microorganism.

The ice block containing enzymes or microorganisms used in the present invention is obtained by freezing an aqueous solution containing the enzymes or microorganisms described above to 0° C. or lower, and incorporating them inside the ice block. This block of ice can be prepared by dispersing an aqueous solution containing enzymes and microorganisms in a deep-chilled atmosphere and simultaneously rapidly freezing the solution. The deep-cooled atmosphere may be either a cooled gas or a cooled liquid, but preferably a liquid cooling medium is used.

Examples of the liquid cooling medium include liquids having a freezing point of 0° C. or lower, such as methanol, ethanol, acetone, ethyl acetate, methylene dichloride, chloroform, carbonic acid tetrachloride, ethyl ether, tetrahydrofuran, toluene, n-hexane, petroleum ether, Liquid nitrogen, liquid oxygen, etc. can be cooled by using heat of evaporation, directly cooling by adding dry ice, or indirectly cooling by using a refrigerator.

When freezing an aqueous solution containing enzymes or microorganisms using a liquid cooling medium, the aqueous solution containing them may be placed in a container etc. and frozen indirectly, or it may be directly frozen in the liquid cooling medium. good.

When freezing directly in a cooling medium, in order to minimize the deactivation of enzymes or the death of microorganisms, the temperature of the cooling medium should be kept as low as possible, and the aqueous solution should be turned into minute water droplets using a sprayer and then frozen quickly. is desirable. Further, in order to form water droplets into minute water droplets, an aqueous solution of enzymes or microorganisms may be previously dispersed in a saturated hydrocarbon solvent such as n-hexane or petroleum ether using a surfactant.

The ice block used in the present invention may contain the following substances in addition to enzymes and microorganisms. For example, water-soluble polymer substances such as polyvinyl alcohol, polyethylene glycol, proteins, nucleic acids, and polysaccharides, polyhydric alcohols such as glycerin, polar organic solvents such as dimethyl sulfoxide, oligosaccharides such as sucrose and lactose, glutamic acid, and aspartic acid. Examples include amino acids such as α-ketocultaric acid and malic acid, metal salts such as magnesium, manganese, cobalt, and calcium, and minute solids such as powdered activated carbon, diatomaceous earth, and latex. In addition, especially in the case of an enzyme, the enzyme's substrate, reaction product, coenzyme, etc. may also be included. These substances are mainly used to protect enzymes and microorganisms during the immobilization process, or to improve the physical properties of products after immobilization.After being added to an aqueous solution containing enzymes and microorganisms, they are quickly frozen. and co-incorporated into the ice block.

The water-insoluble polymer substance used in the present invention is a high molecular weight polymer that is soluble in an organic solvent and insoluble in water, and any substance that is even slightly soluble in an organic solvent at 0°C or lower can be used in the present invention. However, preferably a water-insoluble polymeric substance that dissolves at least 0.1% by weight in an organic solvent at 0° C. or lower is suitable. Here, the fact that the water-insoluble polymeric substance is dissolved in the organic solvent means that the water-insoluble polymeric substance is uniformly mixed with the organic solvent at a concentration that does not cause phase separation from the organic solvent.

Typical water-insoluble polymeric substances used in the present invention include homopolymers such as polyacrylonitrile, polyacrylic esters, polymethacrylic esters, polystyrene, polyvinyl acetate, polyvinyl chloride, and polycarbonate, or homopolymers composed of these homopolymers. Examples include copolymers containing monomers that contain monomers such as those described above, and cellulose derivatives such as cellulose acetate and ethyl cellulose, but of course they are not limited to these. These water-insoluble polymer substances at temperatures below 0°C
Organic solvents that dissolve 0.1% by weight or more are selected from those that exist in liquid form below 0°C, such as methanol, ethanol, propanol, acetone,
Methyl ethyl ketone, ethyl acetate, methylene dichloride, chloroform, carbon tetrachloride, ethyl ether, toluene, xylene, n-hexane, petroleum ether, tetrahydrofuran, cyclohexane,
N,N'-dimetalformamide, γ-butyrotictone, acetonitrile and the like are preferably used, but the present invention is not limited thereto.

The water-insoluble polymeric substance is dissolved in these organic solvents and then cooled to 0° C. or lower before use.

A mixture of ice cubes containing enzymes or microorganisms and an organic solvent in which a water-insoluble polymeric substance is dissolved is a mixture of ice cubes dispersed in a suspended state in an organic solvent at 0°C or lower in which a water-insoluble polymeric substance is dissolved. It is. For dispersion, separately prepared ice cubes may be added to an organic solvent in which a water-insoluble polymer substance has been dissolved, and the mixture may be dispersed into a suspension by rapid stirring.
Alternatively, an aqueous solution containing enzymes or microorganisms may be directly dispersed in the form of minute water droplets in an organic solvent in which a water-insoluble polymeric substance is dissolved under cooling, and then rapidly frozen to generate ice cubes containing the enzymes or microorganisms.

In order to uniformly disperse ice blocks in an organic solvent, the smaller the particle size of the ice blocks, the more effective it is, and it is preferable to use ice blocks with a diameter of 1 mm or less. In order to maintain the dispersed ice blocks stably in the organic solvent, an appropriate amount of a non-solvent of the water-insoluble polymer substance may be added together with the ice blocks during the ice block dispersion. In particular, if the specific gravity of the ice block and the specific gravity of the organic solvent in which the water-insoluble polymer substance is dissolved are different, the dispersed ice block will separate from the organic solvent when stirring is stopped.

In such cases, the ice cubes can be stably dispersed in the organic solvent by adding a nonsolvent. When adding a non-solvent for the water-insoluble polymeric substance together with ice blocks, the ice blocks are once slurried in the non-solvent for the water-insoluble polymeric substance, and then this slurry is rapidly added to an organic solvent in which the water-insoluble polymeric substance has been dissolved. Just add it while stirring. In this case, the ice block is dispersed together with a non-solvent in an organic solvent in which a water-insoluble polymer substance is dissolved, so the water-insoluble polymer substance solidifies around the ice block, and furthermore, this solidified water-insoluble polymer substance becomes an excess of the water-insoluble polymer substance. A partially dissolved state is formed in the organic solvent, and as a result, the ice block is stably dispersed in the organic solvent in which the water-insoluble polymer substance is dissolved.
Furthermore, in order to make this more effective, water-soluble polymeric substances such as polyvinyl alcohol and polyethylene glycol, or polyhydric alcohols such as glycerin and ethylene glycol may be included in the ice block. Nonsolvents used for the purpose of improving the dispersibility of ice cubes are solvents that do not dissolve water-insoluble polymeric substances and are liquid at temperatures below 0°C, and are miscible with organic solvents in which water-insoluble polymeric substances are dissolved. To be elected.

In the present invention, a dope, which is a mixture of the ice block containing the enzyme or microorganism obtained in this way and an organic solvent in which a water-insoluble polymer substance is dissolved, is attached to the tip of a separable electrode covered with a porous membrane, and then The organic solvent is removed from the dope, and a film in which enzymes or microorganisms are immobilized in a water-insoluble polymer matrix is formed directly on the electrode surface. When the electrode tip coated with this porous membrane is coated with the mixture containing ice blocks, for example, the electrode tip coated with the porous membrane is immersed in a mixture of ice blocks and an organic solvent containing a water-insoluble polymer substance. The mixture can be coated on the surface of the electrode tip by applying the mixture to the surface. These operations can be carried out continuously or batchwise. Next, to remove the organic solvent from the mixture coating the surface of the electrode tip, the organic solvent may be evaporated under reduced pressure, or it may be coagulated in a non-solvent of a water-insoluble polymeric substance. .

While enzymes and microorganisms are contained inside the ice, they are stable even when organic solvents coexist, but when the ice melts, there is a risk that the enzymes will be inactivated or the microorganisms will die due to the organic solvent. Preferably, the organic solvent is removed from the inclusions before melting.

The ice inclusions from which the organic solvent has been removed in this way can be stored frozen and thawed before use to be used as immobilized enzymes or immobilized microorganisms. By freeze-drying, it can be made into a form that is convenient for storage and transportation.

The present invention provides a completely new bioelectrochemical sensor and its manufacturing method in which a water-insoluble polymer substance containing enzymes and microorganisms is directly formed on the surface of the electrode tip of a separate electrode made of a noble metal wire. . Conventional methods do not encapsulate enzymes or microorganisms in a stable state in an organic solvent, resulting in various drawbacks as mentioned above, whereas the present invention overcomes these conventional drawbacks. This solution has made it possible to use various water-insoluble polymer substances that are currently widely used industrially, and has an immobilized enzyme membrane or immobilized microbial membrane with excellent mechanical strength supported by a solid structure. Bioelectrochemical sensors have the advantage of being easy to manufacture. Furthermore, the present invention forms a thin film of water-insoluble polymer material containing enzymes and microorganisms on the surface of the electrode tip, thereby providing an efficient bioelectrochemical sensor with significantly improved permeability to substances involved in the reaction. It also has the advantage that it can be obtained freely and reproducibly regardless of the type of enzyme or microorganism. In addition, since the comprehensive method is used, the obtained sensor has high activity expression and durability.

In addition, according to the present invention, the sensor can be miniaturized, so it is particularly useful for quantitative determination in the medical field of blood, urine, tissue fluid, etc., which requires measurement of small amounts of samples.
Furthermore, it becomes possible to measure by directly inserting it into blood vessels, tissues, etc.

The present invention will be explained in more detail with reference to Examples below.

Example 1 Glucose oxidase (manufactured by Boehringer)
After dissolving 100 units/mg (hereinafter abbreviated as GOD) in ion-exchanged water, spraying it into minute water droplets into -70°C n-hexane cooled with dry ice and quickly freezing the product containing GOD. It produced tiny ice blocks. This ice block was quickly separated and collected using a Buchner funnel. About 50g of this ice block and about 65g of n-hexane are cooled with methylene dichloride.
An ice block slurry was prepared by adding it to 200gr. This slurry was gradually added to 400 gr of methylene dichloride at -20°C in which 3.0% by weight of cellulose triacetate (hereinafter abbreviated as CTA), which had been prepared separately, had been dissolved, under rapid stirring to prepare a CTA dope with ice blocks dispersed therein. This dope was cooled to around -40°C to adjust its viscosity.

On the other hand, the tip of the polyurethane-coated platinum wire with a diameter of 300 μm was cut with a sharp knife, perpendicular to the length direction of the platinum wire, to expose a new cross section of the platinum. The tip of this platinum wire was brought into contact with the surface of a CTA formic acid solution dissolved in 98% formic acid with a CTA concentration of 5.0% by weight, and after the CTA formic acid solution was attached to the cross section of the tip of the platinum wire, it was immersed in ion-exchanged water at room temperature. Then, the solvent was removed. This operation was repeated three times to form a porous CTA membrane at the tip of the platinum wire. This membrane has a thickness of approximately 15 μm and an average pore diameter of 0.5 μm.
It was hot.

The platinum wire coated with this CTA porous membrane was immersed in the CTA dope in which the GOD ice blocks were dispersed, and the dope was adhered onto the CTA porous membrane of the platinum wire, and then CTA was applied in a cooled toluene bath.
was solidified.

Repeat this adhesion operation 5 times and attach the platinum tip.
A CTA coagulation film containing ice blocks was formed on a CTA porous film. The toluene impregnated in this coagulated film is extracted and removed with cooled n-hexane, then the n-hexane is extracted and removed with cooled ethanol, and the platinum wire is then removed with M/10 phosphate buffer ( A platinum enzyme electrode coated with a CTA film having an average thickness of 150 μm was obtained by immersing the electrode in PH7.0) to melt the ice blocks contained within the coagulated film.

The immobilized GOD platinum electrode and silver/silver chloride electrode were dissolved in 100% M/10 phosphate buffer (PH7.2) at a temperature of 37.0°C.
ml, and while stirring with a stirrer, the electrolytic current value of oxygen was determined at an applied voltage of -0.6V, and then 1 ml of M/10 phosphate buffer (PH7.2) in which 1.0% glucose was dissolved was added. Then, the oxygen electrolytic current value at each glucose concentration was measured. As a result, glucose concentration (0mg/100ml~500mg/
100ml) and the electrolytic current value,
It was found that it can be used as a glucose measurement electrode. In addition, the relationship between the glucose concentration and the oxygen electrolytic current value was determined using the same method as above using M/10 phosphate buffer (PH7.2) containing 1% by weight of lactic acid and 4% by weight of albumin. Obtained the same results as above.

Furthermore, the glucose concentration was measured once a day for 6 months using this electrode, and as a result, the activity reduction rate of this enzyme electrode was 3%. The noise caused by the stirring of this electrode was about 2%, and the time until stabilization was about 20 seconds.

Example 2 The CTA 5% formic acid solution of Example 1 was brought into contact with the tip of a polyurethane-coated platinum wire with a diameter of 300μ with a new platinum cross section exposed using the same method as in Example 1, and the CTA formic acid solution was attached to the tip of the cross section. after,
After regenerating the CTA membrane in ion-exchanged water at about 60°C, it is brought into contact with the above CTA formic acid solution, slightly air-dried at room temperature, and then immersed in ion-exchanged water at about 50°C to apply CTA to the tip of the platinum wire. A porous membrane was formed. As a result of scanning electron microscopy observation, this porous membrane has a two-layer structure, with the inner layer having a thickness of approximately
The outermost layer had a thickness of about 8μ and an average pore diameter of about 0.3μ.

The platinum wire coated with the CTA porous membrane prepared by the above method was coated on the surface of the CTA porous membrane by the same method as in Example 1 using the CTA dope in which ice blocks containing GOD used in Example 1 were dispersed. ni GOD
A platinum enzyme electrode was obtained which had a comprehensively immobilized platinum enzyme electrode. The film thickness of this platinum wire electrode was approximately 180μ. This immobilized GOD
Using a platinum electrode, the relationship between glucose concentration (0 mg/100 ml to 500 mg/100 ml) and oxygen electrolytic current value was determined using the same method as in Example 1. As a result, a linear relationship existed, and the electrode was used as a glucose measurement electrode. It turns out it can be done.

In addition, compared to the electrode of Example 1, this electrode has a lower noise effect of about 1.5% due to stirrer agitation.
The time required for stabilization was reduced to approximately 15 seconds.

[Brief explanation of drawings]

The figure is a sectional view of a specific example of the biochemical sensor of the present invention. 1... Polymer porous membrane, 2... Insulator, 3...
Noble metal electrode, 4...immobilized enzyme membrane, etc.

Claims (1)

[Scope of Claims] 1. A porous polymer membrane is directly coated on the electrode surface of a separate electrode made of a noble metal wire, and an immobilized enzyme membrane or an immobilized microorganism membrane is directly coated on the surface of the porous membrane, A bioelectrochemical sensor characterized in that the electrode, a porous polymer membrane, and an immobilized enzyme membrane or an immobilized microorganism membrane form an integral structure. 2. The bioelectrochemical sensor according to claim 1, wherein the porous polymer membrane has pores with a pore diameter of 20 Å or more and 3 μm or less. 3 The polymeric porous membrane has an outer layer (the side that contacts the immobilized enzyme membrane or immobilized microorganism membrane) consisting of a thin dense layer having pores with a pore size of 20 Å or more and 1 μm or less, and a pore size of 1 μm that is continuous and integrated with this. The bioelectrochemical sensor according to claim 1, comprising an inner layer (the side in contact with the electrode) having the above-mentioned pores. 4. After coating the electrode surface of a separate electrode made of a noble metal wire, which has been previously coated with a porous polymer membrane, with a mixture of ice cubes containing enzymes or microorganisms and an organic solvent in which a water-insoluble polymer substance is dissolved, organic A method for producing a bioelectrochemical sensor, characterized by removing a solvent. 5. The method for producing a bioelectrochemical sensor according to claim 4, wherein the porous polymer membrane has pores with a pore diameter of 20 Å or more and 3 μm or less. 6. Claim 4, in which the porous polymer membrane comprises an outer layer consisting of a thin dense layer having pores with a pore diameter of 20 Å or more and 1 μm or less, and an inner layer continuously integrated with this thin dense layer and having pores with a pore diameter of 1 μm or more. Method for manufacturing the bioelectrochemical sensor described in Section 1.