JPS5948651A - Immobilized enzyme membrane - Google Patents

Abstract

PURPOSE:To obtain a single layer immobilized enzyme membrane high in strength, by containing enzyme in a polymer matrix having one porous surface permeable to an enzyme substrate and an opposite surface permeable to only low molecular substance being substance to be detected in a granular form. CONSTITUTION:For example, a water contained org. solution containing cellulose triacetate and cellulose diacetate is applied to a glass plate and, after allowed to stand for a while, the coating layer is immersed in n-hexane along with the glass plate to be gelled and the gelled membrane is peeled off while the whole is immersed in an aqueous solution to be dried. The dried gel is immersed in an enzyme E-containing aqueous solution to absorb the same. On the other hand, a cellulose diacetate aqueous solution is applied to a glass plate and the aforementioned enzyme contained membrane P is put on the coated glass plate before the solvent therein is volatilized and the whole is allowed to stand in dry air to partially transfer the solvent of the membrane P to the dense layer A on the glass plate to integrate both of them. The enzyme E is subjected to phase separation during this process and dispersed in a granular form to be held in a cellulose acetate matrix. By this method, one layer membrane having high strength and good properties is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention This invention relates to an immobilized enzyme membrane, and more particularly to a membrane attached to an electrode surface and separating the electrode from a test liquid, in which an enzyme is immobilized and the membrane is When it comes into contact with the test liquid, the analyte contained in the test liquid diffuses into the membrane, and is electrochemically detected as an increase or decrease in the electrode active substance corresponding to the concentration of the analyte due to the reaction of the immobilized enzyme in the membrane. This invention relates to an immobilized enzyme membrane used in an enzyme electrode.

Description of the prior art In recent years, enzyme electrodes have become one of the analytical tools, especially in the field of clinical chemistry analysis.
It has come to be widely applied as one.

With conventional colorimetric methods, measurements may be difficult if the body fluid sample to be measured is colored, but the enzyme electrode method can be applied to specimens such as whole surfaces, and requires almost no special reagents. It has many advantages, such as being able to perform simple, relatively accurate analysis quickly. The enzyme electrode method, which has these advantages, saves expensive enzyme reagents (and it is common sense to use the enzyme immobilized). Active research and development is being carried out on a method in which an immobilized enzyme film formed in the above-mentioned manner is placed on the working surface of a base electrode as a practical method.

FIG. 1 is an illustrative cross-sectional view showing an example of a conventional immobilized enzyme membrane. This immobilized enzyme membrane contains an electrode active substance (e.g. H2).
A thin film A that selectively permeates O2) and a porous support film P that excludes polymers, and these thin films A and support N
This is an immobilized enzyme membrane with IP laminated and bonded.

FIG. 2 is an illustrative cross-sectional view showing another example of a conventional immobilized enzyme membrane, which is the background of the present invention. This enzyme membrane is made by laminating an asymmetric membrane A that contains an immobilized enzyme E in a sponge layer of an asymmetric membrane A that includes a layer that selectively permeates an electrode active substance (H2O2), and a porous support membrane P that excludes polymers.

In this way, the immobilized enzyme membranes known to date are polymer membranes that have a dense thin layer that selectively permeates only the immobilized enzyme and the analyte at the underlying electrode, such as the H2O2 electrode. It is tightly integrated with a porous support membrane using an adhesive or a porous layer formed on one side of the polymer membrane.

On the other hand, an immobilized enzyme membrane for an enzyme electrode must have at least the following functional elements.

(1) The membrane surface facing the test solution does not allow macromolecules such as enzymes to pass through, preventing leakage of enzyme molecules contained inside and preventing infiltration of proteolytic enzymes, etc. contained in the test solution. And enzyme substrate molecules should be freely permeable.

(2) The membrane surface facing the surface of the base electrode must selectively allow the detection substance of the base electrode to pass therethrough, while effectively eliminating substances that would interfere with the electrode.

(3) Inside the membrane that satisfies the functional elements of (1) and (2) above, there is a catalyst, i.e., an immobilized enzyme, that chemically converts the electrode inert substance into the electrode's detectable substance in an efficient manner. I & ill be fully satisfied.

(4) In order to be practical, it must have excellent mechanical stability.

(5) In order to obtain a rapid response as an enzyme electrode, the film thickness should be as thin as possible.

An immobilized enzyme membrane that completely satisfies all of the above (1) to 5) has not yet been obtained. This is because (4) above
and (5), while satisfying the requirements of (1) and (2) above.
) and (3) at the same time would involve contradictory conditions in production. According to 1c,
5- In order to solve the trade-off that exists in the manufacturing method, the above functional elements (1) or 3) are divided into two or three parts, as shown in Figure 1 or Figure 2, and each of the divided parts is divided into two or three parts. It was constructed by laminating layers with adhesive or mechanical means.

However, as shown in Figure 1, conventional immobilized enzyme membranes are laminated with strong adhesives added at the stage of enzyme immobilization, resulting in deactivation of the enzyme and porous membranes that exclude polymers. There is a problem of blocking the pores of the membrane, and in the case of mechanically laminated membranes as shown in FIG. 2, there is a problem that non-negligible voids are created between the membrane layers due to differences in physical properties between the membrane layers.

Moreover, since the two membrane layers that have the functions (1) and (2) above are made of completely different substances, they may peel off during storage or use and become unusable. There was also.

Purpose of the Invention Therefore, the present invention provides a method for an enzyme electrode that can completely eliminate the conventional problems by forming the functional elements required for an immobilized enzyme membrane as one inseparable membrane. The purpose is to provide an immobilized enzyme membrane.

Structure of the Invention Simply put, this invention consists of one membrane, one side of which has a large number of pores that exclude high molecular weight substances and through which substances with a molecular weight similar to that of the substrate for immobilized enzymes can freely pass through. The other side is formed as a dense surface that allows only the low molecular weight substance that is the analyte of the base electrode to pass through and excludes molecules that interfere with the enzyme substrate and electrode response. An immobilized enzyme in which the functional structure of the membrane is physically and chemically inseparably integrated, by forming a synthetic polymer matrix between the surfaces of the membrane and dispersing the immobilized enzyme in granules. It is a membrane.

The invention will be explained in more detail below along with embodiments shown in the drawings.

Description of examples FIG. 3 is an illustrative cross-sectional view showing one embodiment of the present invention.

In this example, the immobilized enzyme membrane is constructed as a single membrane. That is, the polymer-excluding IMF and the membrane layer A that selectively allows the electrode active substance to permeate are integrally continuous. The fused portion of this membrane layer P and A changes continuously into the polymer component that forms this membrane layer A as the composition of the polymer composite forming the membrane layer P shifts to the membrane layer A side. It's fused. A synthetic polymer matrix made of a polymer hybrid is formed between the porous membrane layer P that excludes polymers and the dense membrane layer A that is permeable to the electrode active substance. This synthetic polymer matrix has anisotropy in its thickness direction. That is, both surfaces of this single sheet and the synthetic polymer matrix formed between the two surfaces are formed as a continuous layer of materials selected from a combination of two or more compatible synthetic polymer substances. . Furthermore, the enzyme is dispersed in the form of granules in the synthetic polymer matrix. The granule portion containing the enzyme is contained in a state of phase separation from the synthetic polymer matrix. That is, the enzyme is retained and immobilized in the membrane by physical entrapment.

In this way, each function of the immobilized enzyme membrane of this example exists in a single membrane that is physically and chemically inseparable, so there are no gaps or peeling between the membrane layers during storage or use. will not occur.

In contrast, with conventional products, the side in contact with the base electrode and the side in contact with the test liquid are not physically and/or chemically continuous, so the respective membrane layers may peel off during operation. In particular, porous support membranes often peel off even by immersion in water. However, in the case of an immobilized enzyme membrane constructed as one indivisible membrane as described above, such possibility of peeling does not occur at all.

Such an immobilized enzyme membrane can be produced as follows.

K1N1 (a) 3 to 6 parts of triacetylcellulose (hereinafter referred to as rTACl) and diacetylcellulose (hereinafter referred to as rDAcJ)
A casting liquid was prepared by dissolving a mixed cellulose ester prepared by mixing 9 to 12 parts of the following in a solvent consisting of 50 parts of acetone, 30 parts of cyclohexanone, and 5 parts of water. This casting solution was uniformly cast onto a 9-glass plate with a knife coater to a thickness of 30 μm, left in the air for 10 to 30 seconds, then poured into n-hexane to gel, and further distilled. An asymmetric membrane was obtained by immersing it in water and peeling it off from the vitreous plate. After drying, this asymmetric membrane was cut into circles with a diameter of 2 to 3 clll, and the circular asymmetric membrane was placed in an aqueous solution containing glucose oxidase (rGODJ) and bovine serum albumin (rBsAJ) kept under reduced pressure overnight. I left it alone.

(b) Separately from the membrane described in (a) above, a second casting solution consisting of 5 parts of DAC, 60 parts of acetone, and 35 parts of cyclohexanone was prepared. This second casting solution was poured onto the glass plate. After spreading, the glass plate was immediately coated in a high-speed rotary machine in a uniform and thin layer. Before the solvent of this second casting liquid evaporated, the hot water side containing GOD prepared in (a) above was coated. The asymmetric membrane was placed with the sponge layer on the inside on a glass plate coated with the second casting solution and left in dry air for several hours.Then, the thus formed immobilized enzyme membrane In this way, by placing the asymmetric membrane prepared in (a) on top of the second casting liquid and drying it before the solvent evaporates, the composition of the polymer composite shown in Figure 3 is obtained. As it moves from the P side to the A side, it continuously changes and fuses with the polymer component that forms A, and is physically and chemically integrated.

The surface of the thus formed immobilized enzyme membrane formed with the second casting solution, that is, the surface coated on the glass plate, was mounted facing the base electrode to prepare an enzyme electrode. The performance obtained as a result is shown in the table together with comparative examples (described later).

Example 2 (a) 10 parts of DAC and nitrocellulose (r
NcJ) was dissolved in dimethylformamide saturated with lithium nitrate to obtain a casting liquid. This casting solution was cast onto a glass plate, left to stand in dry air for 30 to 50 seconds, and then poured into distilled water to obtain an asymmetric membrane. This No.
- The asymmetric membrane consisting of DAC was air-dried and then cut into circular shapes, which were then treated with GOD and BSA under reduced pressure in the same manner as in Example 1 above.
It was immersed in a solution containing it and left overnight.

(b) The asymmetric membrane prepared in (a) above was treated in the same manner as in (b) of Example 1 to obtain an immobilized enzyme membrane. Performance etc. are shown in the table along with comparative examples.

Comparative Example: 10 parts of DAC, 55 parts of acetone, 3 parts of cyclohexanone
A casting solution consisting of 5 parts was cast onto a glass plate, left in the air for about 10 minutes, and then poured into n-hexane to form an asymmetric membrane. In the sponge layer of this asymmetric membrane, GOD
A solution containing glutaraldehyde and glutaraldehyde was applied, and a porous polycarbonate membrane was laminated thereon to obtain an immobilized enzyme membrane as shown in FIG. This was attached to the base electrode surface to create an enzyme electrode. The resulting performance is shown in the table together with the results of Examples 1 and 2 above.

In addition, compatible materials that can be used in this invention include:
In addition to a combination of cellulose acetates such as cellulose diacetate and cellulose triacetate, or a system of nitrocellulose and cellulose acetate, a system of nitrocellulose and polyvinyl acetate, and a system of nitrocellulose and polymethyl acrylate.

Examples include a nitrocellulose and polymethyl methacrylate system or a polymethacrylic acid and polyvinyl acetic acid system.

In addition, in the previous example, the enzyme was not chemically immobilized using a polyfunctional reagent, but instead was physically encased, but an enzyme membrane in which the enzyme is dispersed in the membrane is prepared During film formation, the enzymes may be roughly immobilized by covalent bonding with each other or with a protective substance such as BSA using chemicals.

However, it goes without saying that this invention should not be limited to immobilization methods that can be carried out by physically entrapping the above-mentioned materials and enzymes into a composite polymer matrix.

Effects of the Invention As described above, according to the present invention, different required functional elements can be constructed by a single membrane that is physically and chemically inseparable. will not occur.

[Brief explanation of drawings]

FIG. 1 and FIG. 2 are sectional views showing different conventional examples which form the background of the present invention. FIG. 3 is an illustrative cross-sectional view showing one embodiment of the present invention. In the figure, P indicates a porous membrane layer and A indicates a dense membrane layer. 14- Figure 1 Spear 2 Figure 3 289-

Claims (4)

[Claims] (1) An immobilized enzyme membrane used in an enzyme electrode having an electrode for electrochemically detecting a test substance, consisting of one membrane, one side of which excludes and immobilizes high molecular weight substances. A substance having a molecular weight comparable to the substrate of the enzyme to be detected is formed as a surface with pores that freely permeate therein, and the other surface is formed as a surface having pores through which only the low molecular weight substance, which is the analyte of the electrode, passes through and is sensitive to the enzyme substrate and the electrode response. A synthetic polymer matrix is formed between the two surfaces, and a synthetic polymer matrix is formed between the two surfaces.
This is an immobilized enzyme membrane in which enzymes immobilized in this synthetic polymer matrix are dispersed in granular form. (2) A patent claim in which the synthetic polymer matrix on both surfaces of the one membrane and between them is formed as a continuous phase of a material selected from a combination of two or more compatible synthetic polymer substances. The immobilized enzyme membrane according to item 1. (3) The granule portion containing the immobilized enzyme is contained in a state of phase separation from the synthetic product molecular matrix.
An immobilized enzyme membrane according to claim 2. (4) The immobilization method according to claim 2 or 3, wherein the matrix is composed of a polymer hybrid of the two or more compatible synthetic polymers and has anisotropy in the thickness direction. Enzyme membrane.