JPS645251B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a novel bioelectrochemical sensor having enzymes or microorganisms immobilized on an electrode surface, and more specifically, the present invention relates to a method for producing a novel bioelectrochemical sensor having enzymes or microorganisms immobilized on an electrode surface. The present invention relates to a method for producing a bioelectrochemical sensor in which an electrode surface is directly coated with an immobilized enzyme membrane or an immobilized microbial membrane surrounded by a substance.

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 methods for quantifying biological components using enzyme electrodes based on both electrode reactions of amperometry and potentiometry, and methods for quantifying trace components using microbial electrodes that apply bioassay methods.

However, conventionally proposed enzyme immobilization methods or microbial immobilization methods (hereinafter referred to as "immobilization methods") suffer from continuous elution of immobilized substances, inactivation, changes in steric structure, low specific activity, and There are disadvantages such as difficulty in passing, and some immobilization methods cannot be immobilized due to molecular weight restrictions, and their development has stalled. On the other hand, in conventional bioelectrochemical sensors, the electrode surface is coated with a separately prepared enzyme or microorganism immobilized membrane, which is complicated to handle, and furthermore, there are other problems such as difficulty in miniaturizing the electrode. Ta.

In view of these points, the present inventors have studied a method in which enzymes and microorganisms can be freely selected and in which the electrode surface is directly coated with an immobilized membrane, and as a result, the present invention has been achieved.

Among the immobilization methods, a method called the inclusion method is often used as a method that does not reduce the activity of the immobilized substance. 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 filtrated. A method 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, or using water-insoluble monomers or water-insoluble polymers An aqueous solution containing enzymes or microorganisms is dispersed as minute water droplets in an organic solvent in which a polymer substance is dissolved, and then polymerization is performed, or an organic solvent in which a water-insoluble polymer substance is dissolved is removed. A known method is to enclose these water droplets with 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 encapsulating enzymes and microorganisms with water-insoluble polymeric substances in organic solvents. (Japanese Unexamined Patent Application Publication No. 1989-1999)
105289, Japanese Patent Application Publication No. 55-135591). Furthermore, as a shaping method, the surface of the solid structure is coated with a water-insoluble polymer substance containing enzymes or microorganisms, which provides excellent mechanical strength and various molded thin films 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 gist of the present invention is to coat the surface of an electrode made of platinum wire 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 remove the organic solvent. A method for producing a bioelectrochemical sensor, characterized in that an enzyme or microorganism encapsulated in a water-insoluble polymer matrix is directly coated on an electrode surface via the polymer matrix.

The feature of the electrochemical sensor of the present invention is that, as shown in Figures a and b, enzymes or microorganisms 3 enclosed in a water-insoluble polymer matrix 2 are directly attached to the electrode surface via the polymer matrix 2 on the surface of the electrode 1. It must be covered. 4 is an insulator covering the electrode surface.

That is, in conventional electrodes using immobilized enzymes, for example, as shown in Figure 2 of JP-A No. 54-102193, an immobilized enzyme membrane is separately formed, and this membrane is coated on the electrode surface. A method is being taken to do so.

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. In particular, when a platinum wire is used as an electrode, the detected current value is stable and provides favorable results. When the enzyme membrane is integrally immobilized on the electrode as described above, it is difficult for the immobilized enzyme membrane to fall off during measurement, and it 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.

The polymer matrix enclosing the enzyme or microorganism is preferably porous and must allow the substrate in the solution to be measured to permeate through it at a sufficient rate. The thickness of the polymer matrix is preferably as thin as possible, but from the viewpoint of strength, it is preferably in the range of 20 to 300 μm.

The enzyme used in the present invention can be obtained from animal or plant tissues or produced by microorganisms, regardless of its source.

Further, the enzyme may be purified or unpurified, such as a homogenate of an enzyme-containing tissue or a microbial cell.

Enzymes used in the present invention are not particularly limited, but include, for example, oxidoreductases such as glucose oxidase described in JP-A-54-105289, transferases such as aspartate transcarbamylase, hydrolyzing enzymes such as amylase, urease, etc. All kinds of enzymes such as enzymes can be used.

Further, the microorganisms that can be used in the present invention are those classified into molds, yeasts, bacteria, actinomycetes, and Deuteromyces, and the types thereof are not particularly limited, but for example, the microorganisms described in JP-A-55-135591 are 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 it is preferable to use a liquid cooling medium.

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, carbon 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 oxygen or microorganisms using a liquid cooling medium, the aqueous solution containing these 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 rapidly frozen by using a sprayer or the like to drop small drops of water. It is desirable to do so. Furthermore, in order to form fine 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 if necessary.

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'-dimethylformamide, N,N'-dimethylacetamide, 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 the organic solvent in which the water-insoluble polymer substance has been dissolved, and the water-insoluble polymer substance may be dispersed into a suspension by rapid stirring, or the water-insoluble polymer substance may be dissolved under cooling. An aqueous solution containing enzymes or microorganisms may be directly dispersed in the form of minute water droplets in an organic solvent, and then quickly frozen to produce 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. The nonsolvent used for the purpose of improving the dispersibility of ice blocks is a solvent that does not dissolve the water-insoluble polymeric substance and is liquid at 0°C or lower, and is selected from those that are miscible with the organic solvent in which the water-insoluble polymeric substance is dissolved. It will be done.

The present invention involves depositing a dope, which is a mixture of ice cubes containing enzymes or microorganisms thus obtained, and an organic solvent in which a water-insoluble polymer substance is dissolved, on the tip of an electrode, and then removing the organic solvent from the dope.
A membrane in which enzymes or microorganisms are immobilized in a water-insoluble polymer matrix is formed directly on the electrode surface. When the electrode tip is coated with the mixture containing ice blocks, for example, the electrode tip is immersed in a mixture of ice blocks and an organic solvent containing a water-insoluble polymer, or the mixture is applied to the surface of the electrode. By doing this, the surface of the electrode tip can be coated with the mixture. These operations can be carried out continuously or batchwise. To remove the organic solvent from the mixture coating the electrode tip surface, the organic solvent may then be evaporated under reduced pressure.
Alternatively, a method such as coagulation in a non-solvent of a water-insoluble polymer substance may be used. Noble metals such as platinum are generally used as electrodes.

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 novel method for producing a bioelectrochemical sensor, in which a water-insoluble polymer substance containing enzymes and microorganisms is directly formed on the surface of an electrode tip. 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. enzyme,
It also has the advantage that it can be obtained freely and reproducibly regardless of the type of microorganism. In addition, since the comprehensive method is used, the obtained sensor has high activity expression and durability.

Furthermore, by applying the method of the present invention, it has become possible to develop bioelectrochemical sensors such as substrate electrodes, immunosensors, and enzyme thermistors.

This will be explained in detail below using examples.

Example 1 Glucose oxidase (manufactured by Boehringer)
After dissolving 100 units/mg) in ion-exchanged water, the microscopic water droplets were sprayed into n-hexane at -70°C cooled with dry ice and rapidly frozen to form microscopic ice blocks containing glucose oxidase. It was generated. This ice block was quickly separated and collected using a Buchner funnel. Approximately 50g of this ice block and approximately 65g of n-hexane were added to 200g of cooled methylene dichloride to prepare an ice block slurry.
This slurry was gradually added under rapid stirring to 400 gr of methylene dichloride at -20°C in which 3.0% by weight of cellulose triacetate, which had been prepared separately, had been dissolved, to prepare a cellulose triacetate dope with ice blocks dispersed therein. This dope was cooled to around -40°C to adjust its viscosity.

Cut the tip of a polyurethane-coated platinum wire with a diameter of 300 μm at right angles to the length of the platinum wire to form a new platinum cross section. A dope was attached to the tip, and then cellulose triacetate was coagulated in a cooled toluene bath.

This adhesion operation was repeated three times to coat the platinum tip with a coagulated cellulose triacetate film containing ice blocks. The toluene impregnated in this coagulated film was extracted and removed with cooled n-hexane, and then the n-hexane was removed by evaporation under reduced pressure. Thereafter, the platinum wire was immersed in M/10 phosphate buffer (PH7.0) to melt the ice blocks contained in the coagulated film, and the wire was coated with a cellulose triacetate film having an average thickness of 100 μm. A platinum enzyme electrode was obtained.

The immobilized glucose oxidase platinum electrode and silver/silver chloride electrode were immersed in 100 ml of M/10 phosphate buffer (PH7.0) at a temperature of 37°C, and an applied electrode pressure of -0.6 V was applied while stirring with a stirrer. The electrolytic current value of oxygen was determined, and then 1 ml of M/10 phosphate buffer (PH7.0) in which 1.0% by weight of glucose had been dissolved was added, and the electrolytic current value of oxygen at each glucose concentration was measured.

As a result, the glucose concentration (0mg/100ml~500
It was found that there was a linear relationship between the electrolytic current (mg/100 ml) and the electrolytic current value, and that it could be used as an electrode for measuring glucose. As a result of measuring the glucose concentration using this enzyme electrode once a day for 6 months, the activity reduction rate of this enzyme electrode was 3%.

[Brief explanation of drawings]

The figure shows a specific example of the bioelectrochemical sensor of the present invention. 1... Electrode, 2... Polymer matrix, 3...
...Enzyme or microorganism, 4...Insulator.

Claims (1)

[Claims] 1. The electrode surface 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. A method for producing a bioelectrochemical sensor in which an enzyme or microorganism encapsulated in a water-insoluble polymer matrix is directly coated via the polymer matrix.