JPS6285853A - Enzyme electrode - Google Patents

Abstract

PURPOSE:To extend the life of enzyme activity by depositing a pulverized powder carrier immobilized with enzyme by a covalent bond method on a holding film having a fractionation mol.wt. at which protein molecules of the enzyme can pass the film as an immobilized enzyme film. CONSTITUTION:A glucose enzyme electrode 1 is constituted of a detecting electrode 3 of an enzyme electrode 2, a gas permeable membrane 4 disposed to the top end of the electrode 2, an internal liquid 5 and the immobilized enzyme film 6 formed by depositing fine powder-like glass 8 immobilized with glucose oxidase by the covalent bond method on the surface of a hydrophilic filter membrane 7 having about 1-10mum micropore size. The enzyme electrode 6 is formed to approximately the same shape as the shape of the top end face of the detecting electrode 3 and is disposed to the top end of the electrode 2 in the state in which the surface on the carrying side of the glass 8 thereof is in contact with the membrane 4 while the membrane faces the top end face of the detecting electrode 3. A PP net 9 is attached to the electrode 2 in covering the enzyme electrode 6. Since the functional group is introduced into the surface of the glass 8 and the enzyme is immobilized to the surface of the glass 8 of the electrode 1 in the above-mentioned manner, the life of the enzyme activity is extended.

Description

[Detailed Description of the Invention] The present invention is characterized by attaching an immobilized enzyme membrane to the detection end of a base electrode,
The present invention relates to an enzyme electrode that detects substance changes due to the action of an enzyme using a base electrode, and more specifically, to an enzyme electrode that has a long enzyme activity life and can be easily regenerated when the enzyme activity decreases.

U's technology and call or calculation (Yee, - Hair) Problem 2: The enzyme electrode consists of an immobilized enzyme membrane that immobilizes enzymes on a water-insoluble membrane carrier, which enables the enzyme to be used repeatedly, and a substrate. It is a combination of electrodes and has been put into practical use in fields such as clinical chemical analysis and food analysis.

In this case, the method of immobilizing the enzyme on the carrier is to introduce a cross-linking functional group onto the carrier surface, and to connect the A covalent bonding method in which the enzyme is immobilized by reacting with the terminal group of the enzyme protein is preferable because it prevents the enzyme from falling off and increases the long-term stability of the enzyme. Therefore, as an immobilized enzyme membrane for an enzyme electrode, a membrane-like carrier in which an enzyme is immobilized by a covalent bonding method has been widely used.

As mentioned above, enzyme electrodes allow the repeated use of enzymes, but there are limits to this, and in reality, enzyme activity decreases with long-term use of the electrodes. in this case,
Causes of decreased enzyme activity include inactivation of the enzyme due to gradual loss of stability of the higher-order structure of the enzyme, detachment of the enzyme from the carrier, denaturation of the enzyme due to heat changes and PL+ changes, and activity inhibitors. There are many possible causes, such as contact with other people, adhesion of proteins, and decomposition of enzymes by microorganisms.

In addition, when enzyme activity decreases in this way, conventional practice has been to discard the enzyme membrane and replace it with a new one, but if such a method is adopted, the running cost of the enzyme electrode is high and However, it is troublesome to store replacement enzyme membranes, and when the enzyme membrane is replaced, the state of attachment of the enzyme membrane to the underlying electrode changes slightly, causing a problem in that the snow removal characteristics change.

For this reason, it is desired to have an enzyme electrode that has as long a lifespan as possible for enzyme activity and that can regenerate and reuse the enzyme membrane without having to replace it when enzyme activity decreases. The reality is that no effective proposals have been made for regenerating electrodes.

The present invention was made in view of the above circumstances, and an object of the present invention is to provide an enzyme electrode in which enzyme activity is stable for a long period of time, and in which a degraded enzyme membrane can be easily regenerated when enzyme activity decreases. shall be.

Namely, in order to achieve the above object, the present inventors have conducted various studies on regenerating electrodes using enzyme membranes using covalent bonding methods. If the enzyme membrane produced by the covalent bonding method satisfies the following conditions (a) to (C), if the enzyme membrane deteriorates, simply immersing the sensing end of the electrode in the enzyme solution will remove the crosslinking functional groups on the carrier surface. They came up with the idea that the enzyme can be immobilized on a carrier by reacting with the terminal group of the enzyme, and that the enzyme membrane can be regenerated.

(a) The crosslinking functional group itself on the surface of the carrier is detached,
If a negative image occurs, reproduction will be impossible.

Therefore, the functional group introduced into the carrier must be sufficiently stable under the conditions of normal use of enzyme electrodes.

(b) The reaction between the crosslinking functional group on the carrier and the end group of the enzyme does not require special conditions and proceeds under mild conditions near room temperature in water or a buffer solution.

(c) When the electrode is immersed in the enzyme solution, the enzyme protein molecules easily reach the crosslinking functional group site of the carrier.

However, since ordinary enzyme membranes in which enzymes are immobilized on membrane-like carriers by covalent bonding do not fully satisfy the above conditions, further studies have revealed that micropowder carriers such as glass, in which enzymes are immobilized by covalent bonding, have been developed. By supporting the enzyme on a retention membrane having a molecular weight cut-off through which the protein molecules of the enzyme can pass, it is possible to obtain an enzyme membrane that satisfies each of the above conditions and in which the enzyme is stable for a long time. The inventors discovered that it is possible to obtain an electrode that has a long enzyme life and can be easily regenerated by simply immersing the detection end in an enzyme solution, leading to the present invention.

Therefore, the present invention provides an enzyme electrode in which an immobilized enzyme membrane is attached to the detection end of a base electrode, as the immobilized enzyme membrane.
It is an object of the present invention to provide an enzyme electrode characterized in that it uses a fine powder carrier on which an enzyme is immobilized by a covalent bonding method and supported on a holding membrane having a molecular weight cut-off through which protein molecules of the enzyme can pass. purpose.

In the enzyme electrode of the present invention, a functional group is introduced onto the surface of the fine powder carrier and the enzyme is immobilized on the surface of the carrier, so the effective surface area is large and the life of the enzyme activity is very long. In addition, the functional groups are stable under normal electrode usage conditions, and the enzyme protein molecules can pass through the retention membrane, so if the enzyme activity decreases, the enzyme protein molecules can be removed by immersing the detection end of the electrode in the enzyme solution. passes through the retention membrane and reaches the position of the carrier, where it is immobilized on the carrier and the enzyme membrane is easily regenerated.

The present invention will be explained in more detail below.

As described above, the enzyme electrode of the present invention uses an enzyme membrane in which a fine powder carrier on which an enzyme is immobilized by a covalent bonding method is supported on a retention membrane having a molecular weight cut-off through which protein molecules of the enzyme can pass. In this case, the material and properties of the fine powder carrier are not particularly limited, but it is particularly preferable to use fine powder glass. For example, porous glass with a micropore size of about 450 micrometers is made into fine powder with a particle size of 3 to 4 μm. Those prepared can be suitably used. In the present invention, the covalent bonding method refers to the introduction of a crosslinking functional group that reacts with the end group of the enzyme protein on the surface of the carrier under mild conditions, and the reaction between the functional group on the carrier surface and the end group of the enzyme protein. Refers to a method of immobilizing an enzyme on a carrier, such as a method of introducing an alkyl aldehyde group to the carrier surface and immobilizing it by Schiff base reaction, a method of introducing a carboxyl group, etc. and immobilizing it by peptide bonding, etc. .

Furthermore, the material and properties of the holding film are not particularly limited. In the present invention, the retention membrane has the purpose of spreading the fine powder carrier into a membrane shape, and also functions as an outer membrane that comes into direct contact with the sample solution. Any material through which enzyme protein molecules can pass during regeneration can be used. For example, when glucose oxidase is used as the enzyme, the retention membrane is made of acetyl cellulose, regenerated cellulose, mixed cellulose, etc. and has a hydrophilic membrane with a micropore diameter of about 1 to 10 μm. P iU II can be suitably used.

Note that there is no restriction on the method for holding the fine powder carrier on the holding film. Further, it is preferable that the fine powder assembly is uniformly dispersed in the form of a film on the surface of the holding film. Furthermore, electrode sensitivity can be adjusted by appropriately selecting the micropore diameter of the holding membrane.

Next, the present invention will be specifically illustrated by examples.

"M FIG. 1 shows a glucose enzyme electrode 1 according to an embodiment of the present invention.
In the figure, 2 is an oxygen electrode (base electrode), 3 is a detection electrode of the oxygen electrode 2, 4 is a gas permeable membrane disposed at the tip of the oxygen electrode 2, and 5 is an internal liquid. In addition, as shown in Fig. 2, 6 is a micropowder porous glass (fine powder) with glucose oxidase immobilized on the surface of the hydrophilic membrane (retention membrane) 7 with a micropore diameter of about 1 to 10 μm by a covalent bonding method. This is an immobilized enzyme membrane on which carrier) 8 is supported. This enzyme membrane 6 is formed in almost the same shape as the tip surface of the sensing electrode 3, and is in a state where it is opposed to the tip surface of the sensing electrode 3, and with its support 8 supporting side in contact with the gas permeable membrane 4. Oxygen electrode 2
A polypropylene net 9 is attached to the oxygen electrode 2 to cover the enzyme membrane 6, and the enzyme membrane 6 is inserted and fixed to the oxygen electrode 2 tip by the net 9. .

Note that the enzyme membrane 6 was specifically manufactured by the following method.

That is, first, a porous glass having a micropore size of about 450 is ground in a ball mill, and then particles having a particle size on the order of 3 to 4 μm are classified and collected, and this is used as the carrier 8. Next, the fine powder glass 8 is suspended in pure water, and this suspension is sucked into a hydrophilic membrane 7 made of acetyl cellulose, regenerated cellulose, mixed cellulose, etc. with a micropore size of about 1 to 10 μm. By filtering and filling the fine pores on the surface of the membrane 7 with powdered glass 8, the powdered glass 7 is uniformly dispersed and supported on the surface of the membrane 7 in the form of a film. Next, the holding film 7 supporting the glass 8 is treated with γ-aminopropyltriethoxysilane, which is a silane coupling agent, to introduce an alkylamino group therein, and then further treated with glutaraldehyde to form an alkyl aldehyde group. will be introduced. Furthermore, by immersing the retaining membrane 7 in a phosphate buffer solution containing glucose oxidase at a pH of 7.0, the crosslinking functional group and the amino group at the end of the enzyme undergo a Schiff base generation reaction. This immobilizes the enzyme,
An enzyme membrane 6 is obtained. In this case, the enzyme may be immobilized on the finely powdered glass 8 in advance in the same manner as described above, and then this glass 8 may be supported on the filtration membrane 7 in the same manner.

In the enzyme electrode 1 of the above embodiment, a functional group is introduced onto the surface of the glass powder 8 and the enzyme is immobilized on the surface of the glass powder 8, so that the life of the enzyme activity is extremely long. In addition, the functional group is stable against acids, alkalis, and high temperatures of about 100° C., and is therefore extremely stable under normal usage conditions. In addition, since glucose oxidase enzyme protein molecules can pass through the retention membrane 7, the enzyme membrane 6 If the enzyme activity of the enzyme electrode 1 decreases, by immersing the tip of the enzyme electrode 1 in a glucose oxidase enzyme solution at room temperature, the enzyme protein molecules can be transferred to the retention membrane 7.
The enzyme passes through and reaches the position of the fine glass powder 8 on the upper surface of the holding membrane 7, where it is immobilized on the glass powder 8 and the enzyme membrane is easily regenerated. Moreover, since the enzyme membrane 6 is formed in substantially the same shape as the tip surface of the sensing electrode 3 and is disposed opposite to the tip surface of the sensing electrode 3, regeneration can be carried out extremely well.

In the above example, a filtration membrane with a micropore diameter of about 1 to 10 μm was used as the retention membrane 7, but this was to enable the glucose oxidase enzyme protein molecules to reach the fine powder carrier 8 during regeneration. be. However, during normal measurements, the function of this retention membrane 7 is to allow glucose, which is a substrate, to pass through it, so it is preferable in order to protect the enzyme membrane 6 that other macromolecules such as proteins do not pass therethrough.

Therefore, the outside of this enzyme membrane 6 has micropore diameters of 0 and 01μ.
It is also possible to normally attach a guard film of about 1.0 m and to remove this guard film only when regenerating the electrode 1.

Next, the linear response, reproducibility, and active life of the enzyme electrode 1 were examined.

(1) Linear response Electrodes 1 were prepared using two types of membranes 7 with micropore diameters of 0.015 μm and 12.0 μm, and the linear response was examined. The results are shown in Figure 3 (micropore diameter 0.015 μm).
) and FIG. 4 (in the case of micropore diameter ``i'' of 2.0 μm).

(2) Renewable electrode 1 is 0. IN) II aqueous solution, 0. I N NaO
The enzyme membrane 6 is degraded by immersing it in an H aqueous solution and water at about 90° C. for 1 hour, respectively. Next, it deteriorates to 100p
The tip of electrode 1, which showed almost no output in response to the 200 ppm glucose standard solution, was soaked in 0.01 molar phosphate buffer (pH 7.0) 50 m7! Contains 10mg of glucose oxygen (140 units/■)
After immersing the electrode in the dissolved solution at room temperature and stirring the solution for 1 hour to regenerate it, the electrode was thoroughly washed with pure water, and then 100 pp
m, the output was examined by immersing it in a 200 ppm glucose standard solution.

The results are shown in Figure 5 (when immersed in II solution), Figure 6 (when immersed in NaOII solution), and Figure 7 (when immersed in hot water). b represents the output after deterioration, and C represents the output after regeneration. The output after regeneration is approximately the same level as the output before deterioration, and it is recognized that the electrode 1 has been successfully regenerated.

In addition, when the stability over time of the output of the regenerated electrode 1 was examined, it was confirmed that it was stable for more than one month, and it was confirmed that the above regeneration operation is practically effective.

(3) Active life The above electrode 1 is constantly immersed and maintained in a glucose solution with a concentration of about 300 ppm at room temperature.
When the output was examined using a 200 ppm glucose standard solution, the enzyme activity hardly decreased over a year as shown in Figure 8, indicating that the enzyme membrane of the present invention had an extremely long active life. Ta.

DETAILED DESCRIPTION OF THE INVENTION As described in detail, the enzyme electrode of the present invention has a long lifetime of enzyme activity and can be easily regenerated when the enzyme activity decreases.

[Brief explanation of drawings]

FIG. 1 is a partial cross-sectional view showing an enzyme electrode according to an embodiment of the present invention, FIG. 2 is a cross-sectional view showing the enzyme membrane on the same side, and FIGS. 3 and 4 respectively show the linear response of the electrode of the present invention. FIG. 5, FIG. 6, and FIG. 7 are graphs showing the results of regenerating the electrode of the present invention, and FIG. 8 is a graph showing the change over time in the output of the electrode of the present invention. ■...Enzyme electrode, 2...Oxygen bridge (base electrode), 6
...immobilized enzyme membrane, 7.retention membrane, 8. fine powder body.

Claims (1)

[Claims] 1. In an enzyme electrode in which an immobilized enzyme membrane is attached to the detection end of a base electrode, a fine powder carrier on which an enzyme is immobilized by a covalent bonding method is used as the immobilized enzyme membrane to carry protein molecules of the enzyme. An enzyme electrode characterized by using an enzyme electrode supported on a retention membrane having a molecular weight cut-off through which the enzyme can pass. 2. The enzyme electrode according to claim 1, wherein the fine powder carrier is fine powder porous glass.