JPS61274253A - Oxygen film and manufacture thereof - Google Patents

Abstract

PURPOSE:To enable precision analysis of biochemical substance stably for a long time, by a method wherein the surface of a high polymer film undergoes a specified oxidation processing to activate the surface thereof and a porous high polymer reinforcing material is placed thereon to form an enzyme gel layer with a specified thickness. CONSTITUTION:A high polymer film 2 made of acetylcellulose or the like which allows only a specified substance such as water and salts to pass is provided on a smooth substrate 6 and the surface thereof 2 undergoes a specified oxidation processing by a physical oxidation processing such as oxygen plasma and a chemical oxidation processing such as ozone to form and activate a high polymer oxidation layer 3 on the surface of the high polymer film 2. Then, a high polymer reinforcing material 4 having a hole with a specified mesh range and a void is laminated on the surface of the layer 3 and is coated with an enzyme solution of a specified component composition to form an enzyme gel layer 5. Before the formation of the enzyme gel layer 5, the high polymer film 2 is oxidized to form an oxidation layer 3 which is activated. This can intensify the adhesivety between the enzyme gel layer 5 and the high polymer film 2 and the porous high polymer reinforcing material 4 and the high polymer film 2.

Description

[Detailed Description of the Invention] [Field of Application of the Invention] This invention relates to an enzyme membrane of an enzyme electrode used for measuring the components of biochemical substances such as biological fluids by combining an enzyme membrane and an electrode, and is particularly suitable for being attached to a Clark type electrode. The present invention relates to a suitable enzyme membrane and a method for producing the same.

[Background of the invention]

In recent years, enzyme electrodes have been developed that combine enzyme membranes and electrodes and can measure the components of various biochemical substances, and some of these electrodes have been put into practical use as sensors for measuring components in blood. The principle of this enzyme electrode is to convert the substance to be measured into a substance that can be detected by the electrode through the action of an enzyme membrane, and then to measure this substance with the electrode.

Enzyme electrodes can be roughly divided into the potentiometric method and the amperometric method, depending on the measurement method.The former mainly uses an ion electrode as the electrode.
Clark electrodes are often used for the latter. When using the above-mentioned ion electrodes, the selectivity of the electrode itself is generally good, so the enzyme membrane itself is not required to have the ability to eliminate substances that interfere with measurement; rather, the enzyme membrane is required to be permeable to the substance to be measured. . On the other hand, Clark (C1ark)
When using a type electrode, the enzyme membrane is required to have the ability to eliminate substances that interfere with measurement. The reason for this is that the measurement principle using the Clark type electrode is based on the principle of polarography. In other words, the Clark type electrode detects the substance to be detected (H2
02, Oz) By applying a specific redox potential between the anode and cathode of the electrode and measuring the current that flows when the substance to be detected contacts the electrode and is oxidized or reduced, the amount of the substance to be detected can be determined. are being measured. Therefore, if there is a substance in the measurement system whose redox potential is close to that of the substance to be detected, it will be detected by the electrode, resulting in an error in measurement.
will no longer be able to fulfill its role. For this purpose, Clark type electrode,
In particular, when constructing an enzyme electrode using a H2O2 electrode, the enzyme membrane used is free from measurement-interfering substances (e.g., when measuring components in blood, reducing substances such as endogenous ascorbic acid, uric acid, and glutathione). It is necessary to have the property of eliminating substances (such as substances). That is, H2
Use an enzyme membrane that has the ability to permeate compounds with small molecular weights such as O2, H2O, and salts, and exclude high molecular weight compounds with relatively large molecular weights such as endogenous ascorbic acid, uric acid, and glutathione. is necessary.

In addition to the above-mentioned enzyme membrane properties, enzyme membranes used in enzyme electrodes are generally required to have the following properties.

(1) Enzyme membrane has high permeability and quick response; (2)
) Has sufficient mechanical strength and will not be damaged during enzyme membrane replacement or use; (3) Enzyme membrane has high activity; (4)
(5) It is easy to produce and the reproducibility of the enzyme membrane properties is high; (6) The production cost is low.

If we review the enzyme membranes according to the prior art based on the above-mentioned required characteristics of enzyme membranes, there are very few enzyme membranes that can be used in H2O2 electrodes, which are Clark type electrodes, and have characteristics that eliminate substances that interfere with measurement. For example, as the prior art (a) JP-A-52-
There are enzyme membranes disclosed in JP-A No. 55691, (b) JP-A-54-102193, and (C) JP-A-55-98347. The enzyme membrane has a laminate structure in which an enzyme gel is sandwiched between a homogeneous thin film with a thickness of about 1 to 2 μm and a porous polycarbonate thin film with a thickness of several μm. This enzyme membrane uses a homogeneous acetyl cellulose thin membrane to eliminate substances that interfere with measurement, and has the advantage that the enzyme membrane is strong despite its thin thickness due to its laminate structure. Although the required properties (1) to (4) are satisfied, there are problems in that the reproducibility (5) of the enzyme membrane properties is low and the production cost (6) is high. In addition, the enzyme membranes disclosed in the above-mentioned known examples (b) and (C) use an asymmetric membrane consisting of a dense and thin skin layer and a porous sponge layer, so they have a tendency to interfere with measurement. The substance exclusion property is sufficient, which is a necessary property of the enzyme membrane (11. +31 to (5
) is satisfied, but the mechanical strength (2) is low,
There was a problem that the production cost (6) was high.

In order to eliminate the drawbacks of the enzyme membranes according to the above-mentioned conventional technology,
The present inventors previously proposed an enzyme membrane that is easy to manufacture, inexpensive, and has excellent performance (Japanese Patent Application No. 59-268467
). However, this enzyme membrane has a problem in that defects tend to occur inside the membrane depending on the manufacturing conditions, resulting in a slightly inferior product yield.

[Purpose of the invention]

The purpose of the present invention is to solve the problems of the prior art described above,
An enzyme membrane with excellent performance when used in combination with a Clark-type electrode, especially an H202 electrode, to form an enzyme electrode, and a method for producing it easily, inexpensively, and with good reproducibility and high yield. It is about providing.

[Summary of the invention]

The enzyme membrane for enzyme electrodes according to the present invention is a thin, dense polymer membrane that allows a specific substance to pass through, and the surface of the polymer membrane is activated by performing a specific oxidation treatment, and then porous The main idea is to place a polymer reinforcing material on the substrate and form an enzyme gel layer of a predetermined thickness.

That is, the enzyme membrane of the present invention, as shown in FIG. 1, has a structure made of acetylcellulose, etc., which allows only specific substances such as water and salts to pass, on a smooth substrate 6 such as glass or silicon wafer. A polymer membrane 2 is provided,
The surface of the polymer film 2 is subjected to a specific oxidation treatment using a physical oxidation treatment method such as oxygen plasma or a chemical oxidation treatment method such as ozone to form a polymer oxide layer 3 on the surface of the polymer film 2. Then, a porous polymer reinforcing material 4 having pores and voids in a predetermined mesh range is layered on top of the reinforcing material 4.
An enzyme solution having a predetermined component composition is applied thereon to form an enzyme gel layer 5. The enzyme gel layer 5 is formed by adding a crosslinking agent having two or more functional groups capable of reacting with the enzyme to the enzyme solution to be immobilized, and then adding the crosslinking agent to the polymer reinforcing material 4. It is obtained by coating from the top using a known coating method. Before forming this enzyme gel layer 5, as described above, the polymer membrane 2 is oxidized in advance to form and activate the oxidized layer 3 (and the enzyme gel layer 5, polymer membrane 2, and porous The adhesion between the polymer reinforcing material 4 and the polymer membrane 2 becomes stronger, and the enzyme membrane 1 with excellent mechanical strength and performance can be manufactured easily, inexpensively, and with a high yield.

The enzyme membrane of the present invention obtained by the above production method has the following characteristics. In other words, (a) the polymer membrane is reinforced by a porous polymer reinforcing material even though it is thin, so it has sufficient mechanical strength, which is a necessary condition for enzyme membranes mentioned above, and does not break during use. Satisfy condition (2). (b) Also, since the polymer membrane is dense,
It allows limited low-molecular substances such as water, H2O2, and salts to pass through, but it can sufficiently exclude measurement-interfering substances such as endogenous ascorbic acid, uric acid, and glutathione. (The C1 enzyme gel layer is also reinforced with a porous polymer reinforcing material like the polymer membrane, so it has sufficient strength even if the enzyme gel layer is thin. Since it is embedded in the pores and voids of the porous polymer reinforcing material, it is possible to make the enzyme load sufficiently large and the activity high.

(e) Furthermore, in the conventional technology, it is very difficult to control the thickness of the enzyme gel layer, which causes large variations in the properties of the produced enzyme membrane, making it difficult to ensure reproducibility of the performance of the enzyme membrane. Although it was extremely difficult, according to the enzyme membrane manufacturing method of the present invention, it is possible to control the thickness of the enzyme gel layer with sufficient accuracy by adjusting the thickness of the porous polymer reinforcing material or the concentration of the enzyme solution. . (f) In addition, a porous polymer reinforcing material is placed on a polymer membrane, and an enzyme solution containing a crosslinking agent is applied thereon to form an enzyme gel layer. It is possible to produce enzyme membranes with excellent reproducibility and excellent performance at low cost.

Materials for the polymer membrane used in the enzyme membrane of the present invention include:
Examples include cellulose and its derivatives, polyamide, polyethyleneimine, polybutyral, polyvinyl alcohol and its derivatives, polycarbonate, polyacrylate derivatives, polyacrylonitrile and their copolymers, but generally hydrophilic cellulose derivatives, poly Butyral, polyvinyl alcohol derivatives, and the like are preferred. In addition, these materials may be used alone or in combination.
It is also possible to use a mixture of more than one species.

The polymer membrane used in the enzyme membrane of the present invention can be obtained by a generally known method. That is, it is possible to dissolve the above-mentioned material for the polymer film in an appropriate solvent to form a polymer solution, and form a polymer film using a coating method such as casting, brushing, spinner method, or spray method. Although it is possible, it is preferable to use the spinner method. In addition, the thickness of the polymer membrane is preferably in the range of 0.5 to 5 μm, more preferably 1 to 5 μm in terms of its performance as an enzyme membrane.
A range of 3 μm is more preferable.

The polymer oxidation layer constituting the enzyme membrane of the present invention can be obtained by oxidizing and activating the polymer membrane, and methods for oxidation and activation include ozone, which is a common chemical oxidant, In addition to chemical oxidation treatment methods such as hydrogen peroxide, potassium permanganate, potassium dichromate, and nitric acid, physical oxidation treatment methods include irradiation with ultraviolet rays, electron beams, gamma rays, etc.
Examples include corona discharge, low frequency or high frequency plasma discharge treatment. Among these, low-temperature plasma discharge treatment using low or high frequency waves is more desirable because the oxidation treatment is simple and the conditions of the oxidation treatment are easy to control. The formation of a polymer oxide layer by this low-temperature plasma discharge treatment involves applying low-frequency or high-frequency power of 50 W or more and 5 kW or less under a pressure of I Torr or less without introducing oxygen or carbon dioxide gas into the plasma generator. , by performing an oxidation treatment for at least 10 seconds, more preferably for at least 1 minute.

Examples of the porous polymer reinforcing material used in the enzyme membrane of the present invention include nonwoven fabrics, woven fabrics, meshes having a network structure, and porous membranes. Any material may be used as long as it is water-insoluble, insoluble in the solution of the polymer membrane dissolved in the above-mentioned solvent, and has sufficient strength. Specific examples of suitable porous polymer reinforcing materials include polyester nonwoven fabrics, polyamide or polyester meshes, and the like. The thickness of this polymer reinforcing material is preferably 1 to 100 μm, more preferably 1 to 30 μm, considering the performance of the enzyme membrane. Also,
Network mesh (pores) of polymer reinforcement material with porous network structure
The size of the membrane is preferably in the range of 100 to 500 mesh in view of the performance of the enzyme membrane.

Enzymes used in the enzyme gel layer of the present invention include enzymes that generate or consume H202 through enzymatic reactions, such as glucose oxidase, uricase, alcohol oxidase, pyruvate oxidase, bovine santhin oxidase, amino acid oxidase, lactate oxidase, and cholesterol oxidase. Oxidases such as , or enzymes and coenzymes that generate substrates for enzymatic reactions that involve production and consumption of H2O2 can also be used in combination. Additionally, as an incidental matter to the present invention, when an electrode other than the H2O2 electrode is used as an enzyme electrode, the enzyme membrane of the present invention may be manufactured using another enzyme unrelated to the production or consumption of H2O2. Needless to say, it can be done.

The enzyme solution to be applied to the porous polymer reinforcing material is prepared by dissolving the enzyme that forms the enzyme gel layer of the desired enzyme membrane in a buffer solution suitable for the enzyme, and adding a crosslinking agent to it. Obtained by mixing well. The enzyme concentration of this enzyme solution is preferably in the range of 5 to 100 mg/ml in terms of controlling the thickness of the enzyme gel layer, and more preferably in the range of 20 to 50 mg/ml. Examples of crosslinking agents that carry out the crosslinking reaction include polyfunctional aldehydes such as glutaraldehyde, polyfunctional incyanates such as hexamethylene diisocyanate, and N.

N'-ethylene bismaleimide, N,N'-polymethylene iodoamide, etc. can be used. The amount of these crosslinking agents added to the enzyme solution is preferably in the range of 0.1 to 1% by weight. Furthermore, it is also possible to increase the strength of the enzyme membrane by adding a protein such as albumin to the enzyme solution, if necessary. Furthermore, additives such as other coenzymes may be added.

The enzyme solution can be applied onto the porous polymeric reinforcing material using known methods such as brushing, spraying, and spinner methods. It is also possible to apply multiple coats after the crosslinking reaction of the solution is completed. In this case, a plurality of enzyme gel layers may be formed by overcoating other types of enzyme solutions.

[Embodiments of the invention]

An example of the present invention will be described below in more detail.

(Example 1) Acetyl cellulose (average acetyl value 39.8%.

Eastman Kodak Company, USA) was dissolved in cyclohexanone to obtain a cyclohexanone solution of acetylcellulose with a concentration of 10% by weight. A 2.9 μm thick acetyl cellulose film was formed on the wafer by dropping 1 ml of the above cyclohexanone solution of cellulose acetate onto a silicon wafer with a diameter of 5 crn, and coating it with a spinner at a rotational speed of LOOORPM. formed. The Unihara with this acetyl cellulose film formed was set in a normal plasma etching device, and under an oxygen partial pressure of 0.5 Torr, an output of 55 W and 13.56 MHz (D high frequency was applied, and plasma irradiation was performed for 1.5 minutes to remove acetyl cellulose). The surface of the cellulose membrane was activated by oxidation treatment. Then, a polyester mesh having a diameter of 47 mm, a thickness of 60 μm, and a mesh structure of 200 mesh was placed on top of the activated acetyl cellulose membrane. 20 mg glucose oxidase (manufactured by Toyobo Co., Ltd., 100 U/mg),
15 mg bovine serum fulbumin (Sigma, USA)
and 300 μl of phosphate buffer (pH 6, 8, concentration 0)
．． 100 μl of 2.5% by weight glutaraldehyde M was added to an enzyme solution consisting of 1 mol/l) under ice cooling, and 120 μl of the mixed solution was added dropwise and the thickness of the mixed solution was averaged with a brush. Thereafter, the membrane was air-dried at room temperature for 3 hours to prepare an enzyme membrane. This enzyme membrane was glued to a type 0 ring and a commercially available glucose analyzer (manufactured by Yellow Spring, type 23A,
(USA) H202 electrode, g/l/-t
-su (200 mg/d/) was analyzed. At this time, 95
It took about 13 seconds to reach the equilibrium value of the ratio, indicating that the membrane had high permeability and good responsiveness.

(Example 2) An enzyme membrane prepared in the same manner as in Example 1 above was attached to the electrode of the glucose analyzer shown in Example 1 and heated at 37°C.
The device was operated for one month at a temperature of 4,000 samples including blood. In this case, the activity of the enzyme membrane decreased by about 10%, and the enzyme membrane could be used for a long time with high activity.

(Example 3) An enzyme membrane prepared in the same manner as in Example 1 above was attached to and detached from the electrode of the glucose analyzer used in Example 1 10 times at intervals of 1 hour for analysis. However, no damage to the enzyme membrane or change in activity was observed.

(Comparative Example) For the purpose of comparison with Example 3 of the present invention, a method disclosed in Japanese Patent Application Laid-open No. 55-98347, which is a prior art, was used.
Glucose okintase was immobilized on an asymmetric membrane of acetylcellulose, and the membrane was attached to and detached from the electrode of a glucose analyzer in the same manner as in Example 3 above. As a result, the asymmetric membrane of acetylcellulose described above was damaged after the fifth use and could no longer be used.

(Example 4) Ten enzyme membranes were produced in the same manner as in Example 1 above, and these were attached to the electrodes of the glucose analyzer used in Example 1 to examine variations in the output.

As a result, the coefficient of variation of the output of these 10 enzyme membranes was as small as a factor of 5, indicating good reproducibility of the enzyme membrane properties.

(Example 5) The enzyme membrane prepared by the method of Example 1 above was attached to the electrode of the glucose analyzer used in Example 1, and the
Although a 0 mg/di ascorbic acid aqueous solution was injected, the glucose analyzer showed no output at all, indicating that ascorbic acid was completely blocked, indicating good properties for eliminating substances that interfere with measurement.

(Example 6) The enzyme membrane prepared by the method of Example 1 above was immersed in distilled water and left at room temperature for 3 months, but no peeling between the acetylcellulose membrane and the enzyme gel layer was observed. It showed excellent mechanical strength.

As explained above in the examples of the present invention, the enzyme membrane for enzyme electrodes according to the present invention has a quick response, completely blocks the permeation of measurement interfering substances, has high activity, and has high mechanical strength. This enzyme membrane has excellent properties, showing good reproducibility.

〔Effect of the invention〕

As explained in detail above, the enzyme membrane for enzyme electrodes according to the present invention has good permeability and excellent responsiveness, has good ability to exclude substances that interfere with measurement, and has small output fluctuations. The enzyme membrane exhibits high reproducibility of enzyme membrane properties, has especially high mechanical strength, and has almost no damage to the enzyme membrane during enzyme membrane replacement or component measurement, so it can be used stably for long periods of time to produce biochemical substances. A precise analysis can be performed. The method for manufacturing an enzyme membrane according to the present invention involves oxidizing and activating the surface of a polymer membrane prepared to a predetermined size, placing a porous polymer reinforcing material on top of the surface, and By simply applying an enzyme solution to the top, a thin, strong, and highly accurate enzyme film can be created.
Since it can be easily manufactured with good reproducibility and a high yield, the cost of the product is low and its industrial value is extremely high.

[Brief explanation of drawings]

FIG. 1 is a sectional view showing the schematic structure of an enzyme membrane in an example of the present invention. 1... Enzyme membrane 2... Polymer membrane 3...
Polymer oxide layer 4: Porous polymer reinforcement material

Claims (1)

[Scope of Claims] 1. An enzyme membrane for an enzyme electrode for electrochemically analyzing substances in a sample solution, wherein the enzyme membrane is homogeneous and has a predetermined thickness that allows only a specific low-molecular substance to pass through. The polymer membrane has a polymer oxide layer formed by performing oxidation treatment to activate its surface, and the above-mentioned enzyme is provided on the polymer oxide layer. A porous polymer reinforcing material of a predetermined thickness having pores and voids of a predetermined size for reinforcing a membrane, and an enzyme gel configured to hold a predetermined enzyme cross-linked and immobilized on the polymer reinforcing material. An enzyme membrane for an enzyme electrode, characterized by comprising a layer. 2. The polymer membrane is selected from the group of cellulose and derivatives thereof, polyamide, polyethyleneimine, polybutyral, polyvinyl alcohol and derivatives thereof, polycarbonate, polyacrylate and derivatives thereof, polyacrylonitrile and copolymers thereof. The enzyme membrane according to claim 1, characterized in that it comprises at least one type of enzyme. 3. The enzyme membrane according to claim 2, wherein the polymer membrane has a thickness of 0.5 to 5 μm. 4. The porous polymer reinforcing material is made of a water-insoluble polymer compound and has pores and voids with a size of 100 to 500 mesh, and the polymer reinforcing material has a thickness of 1 to 100 μm.
The enzyme membrane according to claim 1, which is a nonwoven fabric, a woven fabric, a mesh having a network structure, or a porous membrane. 5. The enzyme membrane according to claim 4, wherein the porous polymer reinforcing material is made of polyester, polyamide, or a mixture thereof. 6. The enzyme constituting the enzyme gel layer is an enzyme that generates or consumes H_2O_2 through an enzymatic reaction,
The enzyme membrane according to claim 1, comprising at least one member selected from the group of glucose oxidase, uricase, alcohol oxidase, pyruvate oxidase, xanthine oxidase, amino acid oxidase, lactate oxidase, and cholesterol oxidase. . 7. In a method of manufacturing an enzyme membrane for an enzyme electrode that electrochemically analyzes substances in a sample solution, a property that allows only a homogeneous specific low-molecular substance to pass through a predetermined substrate, which is a smooth support base. forming a polymer film with a predetermined thickness, and forming a polymer oxide layer by a physical oxidation treatment method or a chemical oxidation treatment method to activate the surface of the polymer film,
A porous polymer reinforcing material of a predetermined thickness having pores and voids of a predetermined size for reinforcing the enzyme membrane is placed on the polymer oxide layer, and the upper part of the polymer reinforcing material is A method for manufacturing an enzyme membrane for an enzyme electrode, comprising applying an enzyme solution containing a predetermined enzyme, a buffer solution, and a crosslinking agent to the membrane to form an enzyme gel layer by crosslinking and immobilizing the enzyme. 8. The method for producing an enzyme membrane according to claim 7, wherein the method for producing the polymer membrane is spinner coating. 9. Claims characterized in that the physical oxidation treatment method for oxidizing the surface of the polymer film is by low-frequency or high-frequency low-temperature plasma discharge, corona discharge, or irradiation with ultraviolet rays, electron beams, or gamma rays. The method for producing an enzyme membrane according to item 7. 10. The method for producing an enzyme membrane according to claim 9, wherein the physical oxidation treatment method is a low-frequency or high-frequency low-temperature plasma discharge treatment. 11. The method according to claim 7, wherein the chemical oxidation treatment method for oxidizing the surface of the polymer film is performed using ozone, hydrogen peroxide, potassium permanganate, potassium dichromate, or nitric acid. Method for producing enzyme membrane. 12. The concentration of the enzyme contained in the enzyme solution is 5 to 10.
8. The method for producing an enzyme membrane according to claim 7, wherein the concentration is 0 mg/ml. 13. The method of applying the enzyme solution is a brushing method, a spray method, or a spinner method, and the number of times of application of the enzyme solution is one or more times, according to claim 7. A method for producing an enzyme membrane.