JPH029794B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an immobilized enzyme membrane.

In recent years, enzymes have been used in the field of clinical analysis to analyze serum,
BACKGROUND ART Analysis of components, particularly trace components, contained in body fluids such as urine has begun to be actively conducted, and enzyme electrodes that combine immobilized enzymes with polarographic electrodes, ion-selective electrodes, etc. are attracting particular attention. As carriers for immobilized enzymes used in these enzyme electrodes, glass beads and the like are available for columns, but membrane carriers are widely used because of their high measurement accuracy.

Conventionally, membrane carriers for immobilized enzymes include cellulose esters such as cellulose acetate, porous ultrafiltration membranes such as nylon, polyvinyl chloride, and Teflon, and homogeneous membranes of cellulose acetate casted on a stationary water surface. Collagen membranes formed by electrophoresis or electrophoresis have been used. However, conventional membranes are difficult to handle, have low mechanical strength, break easily, and swell in water.

Furthermore, the membrane manufacturing method has drawbacks such as poor working efficiency due to the limited membrane size and difficulty in processing accompanying reactions such as enzyme immobilization.

An object of the present invention is to provide a method for producing an immobilized enzyme membrane that is simple and easy to handle, allows continuous and efficient membrane production, and facilitates processing associated with reactions such as enzyme immobilization. .

That is, the present invention is a membrane having a thickness of 2.0 to 100 μm in which a dense layer and a porous layer are integrally formed by casting a membrane raw material dope onto a carrier, drying it, and immersing it in a poor solvent. layer is 5 to 50% of the total film thickness,
Producing a membrane in which the porous layer is 95-50% of the total membrane thickness,
The membrane is rolled up from a carrier onto a cylinder so that the porous layer of the membrane is in contact with the cylinder surface, the membrane is spread on the support so that the dense layer of the membrane is in contact with the support surface, and then This is a method for producing an immobilized enzyme membrane, characterized in that the enzyme is immobilized by bringing an enzyme solution into contact with the porous layer of the membrane.

The membrane raw materials of the present invention are polymeric substances such as polyamide, polyurethane, cellulose, cellulose acetate, polyvinyl chloride, polyvinyl alcohol, polypropylene, polymethyl methacrylate, and polyvinyl acetate, which may be used alone or in a mixture of two or more. It may be.

These membrane raw material dopes are these polymeric substances dissolved in a suitable solvent. Suitable solvents include acetone, methylene chloride, acetone/dimethylformamide, acetone/dimethylformamide/methanol, and acetone/cyclohexanone. etc.

Any carrier may be used as a carrier for casting the membrane raw material dope as long as it is smooth and clean.
For example, there are glass plates, plastic plates, stainless steel plates, chrome plated steel plates, etc.
A glass plate is particularly preferred.

As the poor solvent used in the present invention, water, n-hexane, etc. are used.

The cylindrical body used in the present invention may be any cylindrical body with a smooth surface, and may be made of glass or plastic. Particularly preferred are those made of Teflon and glass. The diameter of the cylinder is
100-200mm is preferred.

Supports used in the present invention include polyethylene films, polypropylene films, polystyrene films, polyvinylidene chloride films, polytetrafluoroethylene films with a thickness of 10 μm or more,
Polyester film, polyamide film, etc. can be used. Particularly preferred is a polyester film having a thickness of 20 to 100 μm.

The enzyme solution used in the present invention is a solution in which enzymes such as glucose oxidase, cholesterol oxidase, uricase, sarcosine oxidase, pyruvate oxidase, galactose oxidase, choline oxidase, l-amino acid oxidase, alcohol oxidase, peroxidase, and urease are dissolved in a buffer solution. It is. This enzyme solution may contain stabilizers such as EDTA and BSA. Further, the enzyme solution may contain a crosslinking agent. Furthermore, the enzyme solution contains polyacrylamide, poly-N-methylol acrylamide, chitosan, polyhydroxyethyl methacrylate,
It may also contain collagen or the like.

The present invention consists of the following steps.

(1) A membrane with a thickness of 2.0 to 100 μm in which a dense layer and a porous layer are integrally formed by casting a membrane raw material dope onto a carrier, drying it, and immersing it in a poor solvent.
A membrane is produced in which the dense layer is 5-50% of the total membrane thickness and the porous layer is 95-50% of the total membrane thickness.

(2) The membrane is wound up from the carrier onto the cylinder so that the porous layer of the membrane is in contact with the surface of the cylinder.

(3) Spread the wound membrane on the support so that the dense layer of the membrane is in contact with the surface of the support.

(4) Next, the enzyme solution is brought into contact with the porous layer of the membrane spread on the support to immobilize the enzyme.

In the above step (1), the membrane raw material dope is cast onto a carrier, dried for a certain period of time, and then immersed in a poor solvent. surface coating method, graft polymerization method,
A similar film can be manufactured by forming a dense layer using plasma polymerization.

The thickness of the film obtained in step (1) above is usually
It is 2.0 μm to 100 μm. If the thickness is less than 2.0 μm, it is difficult to manufacture the membrane itself, and breakage or pinholes may occur. Moreover, if it exceeds 100 μm, the permselectivity becomes low and it is difficult to use it as an enzyme electrode.

The dense layer is a uniform layer formed from membrane raw materials, and does not allow the substrate of the immobilized enzyme to pass through, but allows electrode sensing of oxygen consumed or hydrogen peroxide produced by the enzyme reaction. Allows substances to permeate selectively. This dense layer is usually used facing the electrode.

On the other hand, the porous layer is formed from a membrane raw material, has a large number of pores with a pore diameter of about 1 to 50 μm, and allows the substrate of the enzyme to be immobilized to pass therethrough. Usually, this porous layer is brought into contact with the substance to be measured.

The composition ratio of the dense layer and the porous layer is such that the dense layer accounts for 5 to 50% of the total film thickness, and the porous layer accounts for 95% of the total film thickness.
~50% is necessary to maintain mechanical strength.

In the above step (4), the method for immobilizing the enzyme is to cast an enzyme solution onto the porous layer of the membrane, and then cast a bifunctional compound such as glutaraldehyde or hexamethylene diisocyanate to cause a crosslinking reaction. A method in which an enzyme solution containing the above-mentioned crosslinking agent is cast onto the porous layer of the membrane to cause a crosslinking reaction, or a method in which a functional group such as an amino group or an aldehyde group is introduced onto the surface of the membrane, and then the above-mentioned There are methods such as casting an enzyme solution.

The steps of the present invention will be explained using figures. These figures are one specific example of the present invention.

In the above step (1), the obtained film has a cross-sectional structure as shown in FIG.

In step (2), the membrane is wound onto a cylindrical body as shown in FIG. 2 so that the porous layer of the membrane on the carrier is in contact with the upper surface of the cylindrical body. In FIG. 2, arrows indicate the direction of rotation of the cylinder and the direction of movement of the cylinder. FIG. 3 shows a cross-sectional view of the membrane wound onto a cylinder.

In step (3), as shown in FIG. 4, the wound membrane is spread on the support so that the dense layer of the membrane is in contact with the surface of the support. In FIG. 4, arrows indicate the direction of rotation of the cylindrical body and the direction of movement of the cylindrical body.

In step (4), an enzyme solution is cast onto the porous layer of the membrane spread on the support, or the membrane is immersed together with the support in the enzyme solution.

In Fig. 1, 1 is the membrane, 2 is the dense layer side of the membrane,
3 indicates the porous layer side of the membrane, and 4 indicates the carrier.

In FIG. 2, 5 indicates a cylindrical body. In FIG. 4, 6 indicates a support.

In the present invention, a membrane manufactured on a carrier, in which a thin dense layer and a thicker porous layer are integrally formed, is wound onto a cylindrical body, and then the membrane is ruptured by spreading it out again on the support. Immobilized enzyme membranes can be efficiently produced without any problems.

The immobilized enzyme membrane obtained by the production method of the present invention selectively permeates electrode-sensing substances such as oxygen, hydrogen peroxide, carbon dioxide, and ammonia.
It can be effectively used in enzyme electrodes, densitometry, etc.

Next, the present invention will be explained by examples.

Example 2 g of cellulose acetate was dissolved in 50 ml of a mixed solvent of 30 ml of acetone and 20 ml of cyclohexane to prepare a dope for a membrane raw material. Apply this dope onto a clean and smooth glass plate to a thickness of 100 μm using a knife coater.
It was cast and applied in the form of a film. After standing at room temperature for 1 minute, the cast film together with the glass plate was immersed in n-hexane, and the solvent (mixed solvent of acetone and cyclohexane) was extracted to form a thin film. The dense layer is about 10% of the total film thickness, and the porous layer is about 10% of the total film thickness.
It was 90%. After air-drying, the thin film was placed in water together with the glass plate, one end of the thin film was peeled off, and the thin film was wound up so that the porous layer side was in contact with the bobbin. At this time, care was taken to prevent air bubbles from entering the wound membrane. The dense layer of thin film wound on the bobbin was then spread out against the polyester support film.

On the other hand, glucose oxidase (50IU/mg)
25mg, pore former 50mg, albumin 75mg 0.05M
Dissolved in 8 ml of sodium acetate buffer (PH5.1)
An enzyme solution was prepared by adding 0.3 ml of 25% glutaraldehyde aqueous solution.

Apply this enzyme solution to the porous layer side of the thin film,
After the immobilization reaction was completed by leaving it at 5°C for 8 hours, it was washed with a sodium acetate buffer and air-dried.

[Brief explanation of drawings]

FIG. 1 shows a cross-sectional view of a membrane produced on a carrier in which a thin dense layer and a thicker porous layer are integrally formed. FIG. 2 shows a cross-sectional view of the membrane wound up so that the porous layer is in contact with the surface of the cylinder. FIG. 3 shows a cross-sectional view of the membrane rolled up onto a cylinder. Fourth
The figure shows a cross-sectional view in which the dense layer of the membrane is spread out in contact with the surface of the support. In the figure, 1 is the immobilized enzyme membrane, 2 is the compact layer side of the membrane,
3 indicates the porous layer side of the membrane.

Claims (1)

[Claims] 1. A membrane with a thickness of 2.0 to 100 μm, in which a dense layer and a porous layer are integrally formed, is produced by casting the membrane raw material dope onto a carrier, drying it, and immersing it in a poor solvent. A membrane having a porous layer of 5 to 50% of the total membrane thickness and a porous layer of 95 to 50% of the total membrane thickness is produced, and the membrane is transferred from a carrier to a cylinder so that the porous layer of the membrane is in contact with the surface of the cylinder. The membrane is rolled up onto a support, the membrane is spread on a support so that the dense layer of the membrane is in contact with the surface of the support, and the porous layer of the membrane is then brought into contact with an enzyme solution to immobilize the enzyme. Characteristic method for producing an immobilized enzyme membrane.