JPS58206960A - Enzyme membrane used for enzyme electrode and its preparation - Google Patents

Abstract

PURPOSE:To enable to control optionally the thickness of a membrane produced by plasma polymerization and to obtain easily the membrane with the prescribed diameter of holes which transmit selectively the electrode sensitive materials, by binding chemically to a base plate made of a porous material by using a prescribed cross-linking or condensing agent. CONSTITUTION:A central section A in a vacuum vessel 8 are made to a plasma condition, and a polymer membrane of allylamine is grown up on the supporting plates 10a-10c in this condition. For example, a crude plasma polymerization membrane of allylamine (the 1st plasmapolymerization membrane 2) having a thickness of 900-1,000Angstrom , and the prescribed diameter of holes which transmit a substrate (e.g. glucose) in the material to be measured, is formed. The base plate 1 of membrane having the 1st polymerization membrane 2 on the one side is dipped, for example, in a 10% glutarakdehyde solution and allowed to react sufficiently wth glutaraldehyde which acts as a cross-linking agent, or with dicyclohexylcarbodiimide acting as a condensing agent to immobilize the desired enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an enzyme membrane for an enzyme electrode and a method for producing the same. For more details, see anode and cathode.

The present invention also relates to an enzyme membrane for an enzyme electrode, in which a predetermined enzyme is immobilized, and which is configured to distribute to the anode surface of a hydrogen peroxide detection type polarographic cell made of an electrolytic solution, and a method for producing the same.

The following are known as conventional examples of such enzyme membranes for enzyme electrodes and methods of manufacturing the same. That is, the specification of JP-A-52-55691 (hereinafter referred to as "No. 1")
According to the conventional example), a predetermined enzyme is immobilized on a selectively permeable dense membrane, and then a porous support sheet is bonded with an adhesive. A technique for producing an enzyme membrane for elementary electrodes has been disclosed. Also, JP-A-54-1021
According to specification No. 93 (hereinafter referred to as "second conventional example"),
After bathing the membrane base material in a solvent, the bath agent is evaporated to form a skin layer, and then the solvent is placed in the bathing liquid to form a sponge layer within the membrane, and the sponge layer is formed in the membrane. A technique for producing an enzyme membrane for an enzyme electrode is disclosed by a method of impregnating a layer with a predetermined # element and chemically bonding it.

However, in the above-mentioned first conventional example V, since the enzyme is not chemically bonded to the porous support sheet (for example, polycarbonate membrane with the trade name "Nuclear Bore"), the enzyme There were drawbacks such as easy peeling. In addition, in the conventional method of @2, since it is considered safe to have a functional group in the membrane itself, there is a drawback that it is greatly limited by the characteristics of the single membrane and the solvent. On the other hand, in the plasma 1 method, which is famous in the technical field of thin film semiconductors, the first
It is known that it has the following advantages: secondly, it is possible to make thin films of organic polymers, and thirdly, it is possible to polymerize at low temperatures while reacting under high energy. There is. However, the use of such a plasma polymerization method in enzyme sensors has not yet been known.

The present invention has been made in view of the above circumstances, and its purpose is to eliminate the drawbacks of the conventional examples by utilizing the plasma polymerization method described above, and to provide an enzyme membrane suitable for use in enzyme electrodes. and its manufacturing method.

A feature of the present invention is that in an enzyme membrane for an enzyme electrode and a method for producing the same, a predetermined crosslinking agent (
A predetermined enzyme is immobilized by chemical bonding using a condensing agent (e.g., glutaraldehyde) or a condensing agent (e.g., dicyclohexylcarbodiimide), and allylamine is plasma-polymerized on the part on which the cough enzyme is immobilized. A dense plasma polymerized membrane is formed on top of the membrane to have a predetermined pore size that allows the electrode sensing substance to pass through but not the interfering substance.

Hereinafter, the present invention will be explained in detail using figures. 1st
The figure is an enlarged cross-sectional view of the main part of the embodiment of the present invention, and in the figure, 1 is made of polypropylene/(trade name "Duraguard") or polycarbonate (trade name "Nuclear Bore").
Thickness 5-25 μm and pore diameter 0.05-0.5
Isn't it porous with μm'Jk? A membrane substrate 2 having no active groups is formed roughly on one side of the membrane substrate 1 to have a predetermined pore size that allows a substrate (eg, glucose) in a substance to be measured (eg, blood) to pass through. 3 is an enzyme immobilization layer attached to the plasma polymerization membrane 2 and has a predetermined enzyme (for example, glucose oxidase) immobilized thereon; 4 is an enzyme immobilization layer 3VC attached; It is a 21g2 plasma polymerized membrane densely formed to have a predetermined pore size that selectively transmits an electrode sensing substance (for example, H20□) but prevents an interfering substance (for example, ascorbic acid) from passing through. Usually, the membrane substrate 1 is attached to the enzyme electrode so that it is in contact with the substance to be measured (for example, blood), and the second plasma polymerized membrane 4 faces the electrode.

Further, FIG. 2 is an enlarged cross-sectional view of a main part of another embodiment of the present invention, and in the figure, 5 is, for example, CM-1 having a carboxyl group.
A porous membrane substrate 6 having a predetermined active group (e.g. carboxyl group or amino group) made of cellulose or AE-cellulose having an amine group is attached to the membrane substrate 5 and a predetermined enzyme (e.g. glucose oxidase) becomes fixed. An enzyme immobilization layer is attached to the 7Vi enzyme immobilization layer 6 and an electrode sensing substance (for example, H, 02
) is a plasma polymerized membrane that is densely formed to have a predetermined pore size that selectively creates a vortex but does not allow interfering substances (for example, ascorbic acid) to pass through.

Note that the membrane substrate 5 is usually attached to the enzyme electrode so that it is in contact with the substance to be measured (for example, blood), and the plasma polymerized membrane 7 is opposed to the electrode.

Hereinafter, a manufacturing method for manufacturing an embodiment of the present invention and other embodiments will be described in detail with reference to the drawings.

FIG. 3 is a manufacturing method explanatory diagram showing a method for manufacturing the embodiment of the present invention or other embodiments, and in the figure, an 8 ha empty container,
9a and 9b are discharge electrodes, 10a to 10C are support substrates, 1
1 is a power source that applies a predetermined voltage to the discharge electrodes 9a and 9b;
12Fi, a monomer inlet device in which, for example, allylamine heptamine is stored, 13F'i, a vacuum pump for reducing the pressure in the vacuum vessel 8, 14a.

14bll'' are the first and second opening/closing pulps. In FIG. 3, first, only the membrane substrate 1 shown in FIG. With the opening/closing pulp 14a closed and the second opening/closing pulp 14b open, the vacuum pump 13 is driven to pump the inside of the vacuum container, for example, I
to reduce the pressure. Thereafter, by closing the second opening/closing pulp 1.4b and gradually opening the first opening/closing pulp 14a, 7lylamine 1 is introduced from the monomer introducer 12 into the vacuum container 8, and the vacuum The pressure inside the container 8 is adjusted to, for example, 0-2 Torr. Next, the power supply 11 is turned on and, for example, 70
When an alternating current electric field of 0 V and 5 KHz is adjusted and applied, the central portion A in the vacuum container 8 becomes a plasma state. In this state, a polymer film of allylamine grows on each negative side surface of the supporting substrates 10a to 10c, and has a thickness of, for example, 900 to 1.5 mm.
000X and a predetermined pore size that allows the substrate (for example, glucose) in the substance to be measured to pass through, a rough allylamide/plasma polymerized membrane (112 ZMA 1 composite membrane 2 in FIG. 1) is formed.

Next, the support substrate 10 taken out from inside the vacuum container 8
A desired r# element is immobilized on the first plasma polymerization 1112 (or the membrane substrate 5 in FIG. 2) attached to the axl Oc in the following manner.

That is, the membrane substrate 1 (or the membrane substrate 5 having a predetermined active group), on which the island 1 plasma polymerized membrane 2 is formed on one side, is immersed in, for example, a 10% glutaraldehyde solution. The desired enzyme is immobilized by sufficiently reacting with glutaraldehyde, which acts as a crosslinking agent, or with dicyclohexylcarbodiimide, which acts as a condensing agent. To explain the case of glutaraldehyde in detail, first, the first plasma polymerized film 2 (or the film substrate 5) is washed with a predetermined pif buffer solution to remove excess glutaraldehyde. After that, @1 above
The plasma polymerized membrane 2 (or the membrane substrate 5) is immersed in a predetermined enzyme solution such as glucose oxidase (or uricase, cholesterol oxidase, etc.) to combine the enzyme and the first plasma polymerized membrane (or the membrane substrate 5). (the above-mentioned active group) are chemically bonded to each other. The enzyme is
Thereafter, it is sufficiently washed with water and then dried to form the enzyme fixed layer 3 shown in FIG. 1 (or the enzyme fixed layer 6 shown in FIG. 2).

Incidentally, when dicyclohexylcar-posiimide, which acts as a condensing agent, is used instead of the above-mentioned glutaraldehyde, which acts as a cross-linking agent, the above-mentioned enzyme and the above-mentioned first plasma polymerized membrane (or the above-mentioned active group of the membrane substrate 5) can be used. ), or by interposing a main chain substance (for example, 1,8-diaminooctane) between the enzyme and the first plasma polymerized membrane (or the active group of the membrane substrate 5). You may also do so.

Next, referring again to FIG. 3, a method for forming the second plasma polymerized film 4 in FIG. 1 (or the plasma polymerized film 7 in FIG. 2) will be described. In FIG. 3, the enzyme immobilization layer 3
and the film substrate 1 on which the first plasma 1 composite film 2 is attached.
(or the film substrate 5 on which the #element fixed layer 6 is attached)
) are installed in the vacuum container 8 instead of the support substrates 10a to 10c. Next, the first opening/closing pulp 14m is closed and the second opening/closing pulp 14m is closed.
The opening/closing pulp 14b is opened, and in the next state, the vacuum pump 13 is driven to reduce the pressure in the vacuum container to, for example, 1×10 TOrr. Thereafter, by closing the second opening/closing pulp 14b and gradually opening the first opening/closing pulp 14a, the amount of hair in the vacuum container 8 is removed from the monomer introducer, and the pressure in the vacuum container 8 is reduced to, for example, 0.1.
5 TOrrVc regulation. Next, turn on the power supply 11,
For example, 900V between the radiation poles 9m and 9b.

When an alternating current electric field of 5KZ is adjusted and applied, the vacuum vessel 8
The central part A inside becomes a plasma state. In this state, the enzyme fixed layer 3 or the enzyme fixed layer 6 is provided with, for example, a thickness xsoo
A dense allylamine plasma-polymerized membrane (second plasma-polymerized membrane 4 in FIG. 1 or plasma-polymerized membrane 7 in FIG. 2 ) will be formed.

By the way, FIG. 4 is an explanatory diagram showing an example of use of the embodiment of the present invention, and in the figure, 15 is an anode (usually made by Nichikin).
, 16 is a cathode (usually made of steel), 17 is a mold body supporting the Fit cypress, and 18.18' is an anode 15 and a cathode 16., respectively. 19 is a first lead wire connected to the first wire and leads the detection signals from these electrodes to a signal processing section (not shown).
The enzyme membrane 20 according to the present invention, which has been explained in detail with reference to FIG. In FIG. 4, when a substance to be measured (for example, blood) comes into contact with the enzyme membrane 19, a substrate (for example, glucose) in the substance to be measured (for example, glucose) is transferred to the membrane substrate (
First, it passes through 5) and reaches the enzyme immobilization layer 3 (or 6), where the enzyme (e.g., glucose oxidase) and the substrate come into contact and react, releasing a predetermined electrode sensing substance (e.g., H2O2). It becomes like this. The electrode sensing substance and interfering substance (e.g. ascorbic acid) are
Upon reaching the plasma polymerized membrane 4 (or the plasma polymerized membrane 7), only the electrode sensing substance is transmitted, without interfering substances, acting on the anode 15 and cathode 16 to generate a detection signal. The detection signal is connected to lead wire 18.1.
The signal is transmitted to the signal processing unit via 8' and subjected to predetermined signal processing, and the substrate concentration etc. are calculated and displayed.

According to the embodiments of the present invention or the embodiments of the pond of the present invention as described in detail above, even a chemically inert membrane substrate having no active groups can be surface-modified to become active. , has the advantage that a desired enzyme can be chemically bonded (t!iJ determination) to the membrane substrate. Furthermore, there is also the advantage that there is no possibility of peeling of the constituent members as in the first prior art example or that there is no major limitation due to the type of solvent, etc. as in the second prior art example. Further, there is an advantage that the film thickness of the plasma polymerized film can be changed arbitrarily, and a film having a predetermined pore size that selectively transmits only a highly sensitive substance such as H2O2 can be easily obtained.

[Brief explanation of drawings]

FIG. 1 is an enlarged sectional view of the main part of an embodiment of the present invention, FIG. 2 is an enlarged sectional view of the main part of another embodiment of the invention, and FIG. 3 shows a method for manufacturing the embodiment of the invention or another embodiment. An explanatory diagram of the manufacturing method shown,
FIG. 4 is a detailed explanatory diagram of the present invention. 1.5... Membrane substrate, 2.4.7... Plasma polymerized membrane, 3.6... Enzyme immobilization layer, 8... Vacuum container, 9a,
9b...discharge electrode, 10a-10b...support substrate,
11... Power supply, 12... Monomer introducer, 13...
・Vacuum bo/p, 14th section・, 14b...Opening/closing pulp, 1
5... Anode, 16, --... Cathode, 17...
1 pole body, 18.18'...lead wire, 19...
#Membrane, 20...Fixing O-ring

Claims (1)

[Scope of Claims] +11 A membrane substrate made of a porous material, an enzyme immobilization layer attached to the membrane substrate and having a predetermined enzyme immobilized thereon, and an enzyme immobilization layer attached to the enzyme immobilization layer. a plasma polymerized membrane formed densely so as to have a predetermined pore size that allows an electrode sensing substance to pass through but not an interfering substance; the membrane surface of the plasma polymerized membrane faces the electrode; An enzyme membrane for an enzyme electrode, characterized in that it is configured so as to be in contact with a substance. (2) The membrane substrate is made of a porous material having no active groups, and a plasma polymerized membrane roughly formed to have a predetermined pore diameter that allows the substrate in the substance to be measured to pass through is connected to the membrane substrate. The enzyme membrane for an enzyme electrode according to claim 111, which is sandwiched between an enzyme immobilization layer. +3) The enzyme membrane for an enzyme electrode according to claim 1, wherein the membrane substrate is made of a porous material having active groups. (4) A predetermined # element is immobilized by chemically bonding it to a membrane substrate made of a porous material using a predetermined crosslinking agent or condensing agent, and then the enzyme is immobilized and 'fc
By plasma polymerizing allylua ξ/ on the part,
A method for producing an r# deposited film for an enzyme electrode, which comprises forming a dense plasma polymerized film having a predetermined pore size that allows an electrode-sensing substance to pass through but not an interfering substance. (5) Plasma of allylamide is roughly combined with a porous membrane substrate having no active groups so as to have a predetermined pore diameter that allows the substrate in the substance to be measured to pass through, and then - the enzyme immobilization and the dense The manufacturing method according to claim 4, characterized in that a plasma polymerized film is formed. (6) Produce the membrane substrate by adding amine groups to the porous membrane substrate using a predetermined activating agent, and then,
The patented chemotactic film substrate characterized by the enzyme immobilization and the formation of the dense plasma polymerized film is installed, and after reducing the pressure in the vacuum container, the allylamine monomer is introduced, and the #vacuum container is The plasma polymerized film is formed on the membrane substrate or the membrane substrate on which the enzyme is immobilized by applying a predetermined alternating current electric field to discharge electrodes provided at positions sandwiching the membrane substrate in the membrane substrate. Claim No. 4 (4) characterized in that
) to (6) above.