JPH0518378B2 - - Google Patents

Description

[Detailed Description of the Invention] Industrial Application Field The present invention is characterized in that an immobilized enzyme membrane on which an enzyme is immobilized is attached to the detection end of a base electrode, so that changes in substance due to the action of the enzyme are detected by the base electrode. This invention relates to a method for regenerating an enzyme electrode.

Prior Art and Problems to be Solved by the Invention An enzyme electrode is a combination of an immobilized enzyme membrane that immobilizes an enzyme on a water-insoluble membrane carrier and enables repeated use of the enzyme, and a base electrode. It has been put into practical use in fields such as clinical chemistry analysis and food analysis.

In this case, the method for immobilizing the enzyme on the carrier is to introduce a cross-linking functional group onto the carrier surface, and to connect this functional group to the enzyme, rather than the entrapment method or physical adsorption method, in which the enzyme is physically sealed on the carrier. A covalent bonding method in which the enzyme is immobilized by reacting with the terminal group of the protein is preferable because it prevents the enzyme from falling off and increases the long-term stability of the enzyme. Therefore, as the immobilized enzyme membrane for enzyme electrodes, membrane-like carriers in which enzymes are immobilized by covalent bonding have been frequently 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, the causes of the decrease in 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 changes in heat and pH, etc. Many possible causes include contact with activity-inhibiting substances, adhesion of proteins, and decomposition of enzymes by microorganisms.

Conventionally, when the enzyme activity decreases in this way, the enzyme membrane is discarded and replaced with a new one. However, when such a method is adopted, the running cost of the oxygen electrode is high, it is troublesome to store the enzyme membrane for replacement, and when replacing the enzyme membrane, it is difficult to attach the enzyme membrane to the underlying electrode. This causes a problem in that the electrode characteristics change slightly.

For this reason, it is desired to regenerate and reuse the enzyme membrane without replacing it when the enzyme activity decreases, but there has been no research on regenerating enzyme electrodes.

The present invention was made in view of the above circumstances, and an object of the present invention is to provide a method by which an enzyme electrode can be easily regenerated when the enzyme activity of the enzyme electrode decreases.

Means and Effects for Solving the Problems That is, in order to achieve the above object, the present invention provides a fine powder porous glass carrier on which an enzyme is immobilized by a covalent bonding method, through which the protein molecules of the enzyme can pass. When regenerating an enzyme electrode in which an immobilized enzyme membrane supported on a retention membrane having a specific molecular weight is attached to the detection end of a base electrode, the immobilized enzyme membrane attached part is immersed in an enzyme solution to prevent deterioration due to use. The enzyme in the enzyme solution is immobilized on the immobilized enzyme membrane.

In this case, the covalent bonding method involves introducing a cross-linking 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 in which an alkyl aldehyde group is introduced onto the carrier surface and immobilized by Schiff base reaction, a method in which a carboxyl group, etc. is introduced and immobilized by peptide bonding, etc. However, according to the present invention, when the enzyme membrane is degraded due to deactivation of the enzyme or falling off from the carrier surface, the enzyme membrane can be removed from the carrier by immersing the enzyme membrane-attached detection end of the enzyme electrode in the enzyme solution. The crosslinking functional group on the surface reacts with the enzyme protein terminal group in the enzyme solution, and the enzyme is newly immobilized on the carrier, thereby regenerating the electrode.

Here, there are no particular limitations on the method or conditions for immersing the enzyme membrane attachment part of the enzyme electrode in the enzyme solution;
It is preferable to immerse at room temperature and further stir the enzyme solution. Note that the immersion time is not limited and can be varied depending on the type of enzyme membrane, the type of enzyme, etc.

The method of the present invention also provides an immobilized enzyme membrane in which a micropowdered porous glass 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. is used in an enzyme electrode attached to the detection end of a base electrode, and this enzyme electrode can be effectively regenerated. Here, the term "molecular weight cutoff" refers to the molecular weight of particles that indicates how large particles a separation membrane can separate. Therefore, a retention membrane with a molecular weight cut-off through which enzyme protein molecules can pass is a retention membrane with a molecular weight cut-off larger than the molecular weight of the enzyme protein molecules, that is, a retention membrane with a molecular weight cut-off that is larger than the molecular weight of the enzyme protein molecules, i.e., a retention membrane with a molecular weight cut-off that is larger than the molecular weight of the enzyme protein molecules, i.e., a retention membrane that has a molecular weight cut-off that is larger than the molecular weight of the enzyme protein molecules. A retention membrane through which protein molecules can pass. In such enzyme electrodes, functional groups are introduced onto the surface of a micropowdered porous glass carrier, and the enzyme is immobilized on the surface of this carrier. Therefore, the functional groups are extremely stable under normal electrode usage conditions, and are retained easily. Enzyme protein molecules can pass through the membrane, so if the enzyme activity decreases, by immersing the detection end of the electrode in the enzyme solution, the enzyme protein molecules will pass through the retention membrane and reach the carrier position. It is immobilized on a carrier and the enzyme membrane is easily regenerated.

The enzyme electrode described above uses an enzyme membrane in which a micropowdered porous glass 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. It was
In this case, the properties of the finely powdered porous glass carrier are not particularly limited, but for example, porous glass with a micropore size of about 450 Å adjusted to fine powder with a particle size of 3 to 4 μm can be suitably used. Furthermore, the material and properties of the holding film are not particularly limited. In this enzyme electrode, the retention membrane has the purpose of spreading the fine powder porous glass carrier into a membrane shape, and also functions as an outer membrane that comes into direct contact with the sample solution. can pass, and
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 may be acetylcellulose, regenerated cellulose,
Made of mixed cellulose, etc., with a micropore size of 1 to 10μ
Hydrophilic membranes having a certain degree of hydrophilicity can be suitably used. Note that there is no restriction on the method for holding the fine powder porous glass carrier on the holding film. Further, it is preferable that the fine powder porous glass carrier is uniformly dispersed in the form of a film on the surface of the holding membrane. 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.

EXAMPLE FIG. 1 shows an example of an enzyme electrode used to carry out the method of the present invention. In this glucose enzyme electrode 1, 2 is an oxygen electrode (base electrode), 3 is an oxygen electrode 2
4 is a gas permeable membrane disposed at the tip of the oxygen electrode 2, and 5 is an internal liquid. Also, 6 is the second
As shown in the figure, immobilization is made by supporting a micropowder porous glass carrier 8 on which glucose oxidase is immobilized by a covalent bonding method on the surface of a hydrophilic membrane (retention membrane) 7 with a micropore diameter of about 1 to 10 μm. It is a enzyme membrane. This enzyme membrane 6 is formed in almost the same shape as the tip surface of the sensing electrode 3, and is exposed to oxygen while facing the tip surface of the sensing electrode 3 and with its enzyme-carrying side surface in contact with the gas permeable membrane 4. A polypropylene net 9 is attached to the oxygen electrode 2 to cover the enzyme membrane 6, and the enzyme membrane 6 is attached and fixed to the oxygen electrode 2 tip by the net 9. has been done.

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

That is, first, porous glass having a micropore diameter of about 450 Å is ground in a ball mill, and then particles having a particle diameter on the order of 3 to 4 μm are classified and collected, and this is used as the carrier 8. Next, the fine powder porous glass carrier 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 diameter of about 1 to 10 μm. By filling the fine pores on the surface of the membrane 7 with the finely powdered porous glass carrier 8,
A finely powdered porous glass carrier 8 is uniformly dispersed and supported on the surface of the membrane 7 in the form of a membrane. Next, the holding film 7 supporting the fine powder porous glass carrier 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 introduce an alkyl aldehyde group. Furthermore, glucose oxidase was dissolved in the retention membrane 7 at room temperature.
By immersing it in a phosphate buffer solution of pH 7.0, the crosslinking functional group and the amino group at the end of the enzyme perform a Schiff base generation reaction, thereby immobilizing the enzyme and obtaining the enzyme membrane 6. It is. In this case, the enzyme may be immobilized on the fine powder porous glass carrier 8 in advance in the same manner as above, and then the fine powder porous glass carrier 8 may be supported on the membrane 7 in the same manner. do not have.

Next, a regeneration experiment was conducted using the enzyme electrode 1 according to the method described below. That is, electrode 1 is 0.1NHCl
The enzyme membrane 6 is degraded by immersing it in an aqueous solution, a 0.1N NaOH aqueous solution, and water at about 90° C. for 1 hour, respectively. Next, the tip of electrode 1, which has deteriorated and no longer shows almost any output for glucose standard solutions of 100 ppm and 200 ppm, was soaked in 0.01 molar phosphate buffer (PH).
7.0) Immerse the electrode in a solution of 10 mg of glycose oxidase (140 units/mg) dissolved in 50 ml at room temperature, stir the solution for 1 hour to regenerate it, wash the electrode thoroughly with pure water, and reconstitute it with 100 ppm and 200 ppm. The output was examined by immersing it in a glucose standard solution.

The results are shown in FIG. 3 (when immersed in HCl solution), FIG. 4 (when immersed in NaOH solution), and FIG. 5 (when immersed in hot water). In the figure, a is before deterioration;
b represents the output after deterioration, and c represents the output after regeneration, and the arrows in each figure indicate that the output decreased from state a before deterioration to state b after deterioration, and further increased to state c after regeneration. The output after regeneration was approximately the same level as the output before deterioration, and it was confirmed that the electrode 1 was successfully regenerated according to the method of the present invention.

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

Effects of the Invention As explained above, according to the method of the present invention, when the enzyme activity of an enzyme electrode using an enzyme membrane formed by a covalent bonding method decreases, it can be easily regenerated.

[Brief explanation of drawings]

FIG. 1 is a partial sectional view showing an example of an enzyme electrode used in carrying out the present invention, FIG. 2 is a sectional view showing an enzyme membrane of the same electrode, and FIGS. It is a graph showing the results of processing the electrodes. 1...Enzyme electrode, 2...Oxygen electrode (base electrode),
6... Immobilized enzyme membrane, 7... Retention membrane, 8... Finely powdered porous glass carrier.

Claims (1)

[Claims] 1. An immobilized enzyme membrane formed by supporting a micropowder porous glass carrier on which an enzyme has been immobilized by a covalent bonding method on a retention membrane having a molecular weight cut-off through which protein molecules of the enzyme can pass is detected by a base electrode. A method for regenerating an enzyme electrode attached to the end of the enzyme electrode, in which the immobilized enzyme membrane attached part of the enzyme electrode is immersed in an enzyme solution, and the enzyme in the enzyme solution is immobilized on the immobilized enzyme membrane that has deteriorated due to use. A method for regenerating an enzyme electrode, characterized in that: