JPS61187792A - Production of immobilized living body catalyst - Google Patents

Abstract

PURPOSE:A specific platinum base is treated with an aldehyde, then bonded to a biocatalyst whereby the titled biocatalyst with high activity is obtained regardless of kinds of biocatalysts to be immobilized. CONSTITUTION:A platinum base such as a platinum plate is treated with aminosilane to provide amino groups on the base 1 and coated with a film- forming solution containing albumin and glutar aldehyde as a crosslinking agent to form a crosslinked albumin film 3. Then, the film is coated with a solution containing a polyfunctional aldehyde such as glutar aldehyde to provide aldehyde groups on the crosslinked film 3, washed with a buffer solution of 7.5pH, then the solution is removed and the coated film is dried by standing. The coated film 3 is coated with a solution containing enzymes or microorganisms such as uricase to allow the aldehyde group to bond to the enzymes or microorganism by standing to give the objective biocatalyst A immobilized on the crosslinked films 3.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a method for producing an immobilized biocatalyst used in biosensors and the like.

[Background technology]

In biosensors and the like, platinum substrates on which biocatalysts such as enzymes are immobilized are often used as electrodes. As a method for immobilizing a biocatalyst on the surface of a platinum substrate, a mixed solution containing a biocatalyst, albumin, and glutaraldehyde as a crosslinking agent is applied to the surface of the platinum substrate, which has been previously treated with aminosilane, and the biocatalyst is immobilized by a crosslinking reaction of glutaraldehyde. There is a method of forming a film. When immobilizing highly stable substances such as glucose oxidase, this method is very simple and suitable. However, when immobilizing something that is easily deactivated, such as uricase, it is not preferable to use this method because the activity of the biocatalyst is lost due to the glutaraldehyde used as a crosslinking agent.

[Purpose of the invention]

The present invention has been made in view of the above circumstances, and aims to provide a production method that can always obtain a highly active immobilized biocatalyst regardless of the type of biocatalyst to be immobilized.

[Disclosure of the invention]

In order to achieve the above objectives, the inventors have conducted repeated research. As a result, if an aldehyde group is provided on a platinum-based membrane on which a cross-linked albumin membrane is formed, and a biocatalyst is bonded to this aldehyde group, the biocatalyst can be immobilized without loss of activity. They discovered that it is possible to do this, and have now completed this invention.

Therefore, in order to obtain an immobilized biocatalyst in which a biocatalyst is immobilized on a platinum substrate, the platinum substrate on which a crosslinked film of albumin is formed is treated with aldehyde to provide an aldehyde group on the film, and then The gist of this paper is a method for producing an immobilized biocatalyst, which is characterized by bonding a catalyst with an aldehyde group and immobilizing it on a membrane. The present invention will be explained in detail below.

A platinum substrate having a crosslinked film of albumin formed on its surface can be produced, for example, as follows. first,
A platinum substrate such as a platinum plate is treated with aminosilane to give the shape shown in Figure 1 (
As shown in a), amino groups 2 are provided on the surface of a platinum substrate 1. As the aminosilane, γ-aminopropyltriethoxysilane or the like is used. Next, a film-forming treatment solution containing albumin and a cross-linking agent is applied to the surface of the platinum substrate 1 by coating, dipping, etc., and the cross-linking agent cross-links the albumin, the amino group 2, and the albumin together, and as shown in FIG. As shown in (middle), a crosslinked film 3 of albumin is formed on the surface of the platinum substrate 1. As the crosslinking agent, glutaraldehyde or the like is used.

A biocatalyst is immobilized on the membrane-attached platinum base membrane thus obtained in the following manner. First, (C) in Figure 1
As shown in C, aldehyde treatment is performed by applying a solution 4 containing a polyfunctional aldehyde such as glutaraldehyde to the membrane 3 of the platinum substrate 1 by coating, dipping, etc., and then leaving it to stand. As shown in FIG. 1(d), aldehyde groups 5 are provided on the film 3 of the platinum substrate 1. After removing the solution by washing with a buffer solution (pH 7.5), air drying is performed as necessary. After this, as shown in FIG. 1(e), enzymes such as uricase are added.

The biocatalyst is covalently bonded to the aldehyde group 5 by applying a solution 6 containing a microorganism or other biocatalyst to the membrane 3 of the platinum substrate 1 by coating, dipping, etc. and then leaving it to stand. Then, as shown in FIG. 1(f), an immobilized biocatalyst in which the biocatalyst A is immobilized on the membrane 3 is obtained.

If you create an immobilized biocatalyst in this way,
Since the biocatalyst does not come into direct contact with the crosslinking agent, its activity is not impaired and it is fixed while maintaining its original activity. Moreover,
As shown in FIG. 1(f), the immobilized biocatalyst A is always highly active because it is immobilized so that it is lined up on the surface of the membrane 3 and can easily come into contact with the substrate. It is possible to obtain a catalyst. Therefore, when the immobilized biocatalyst obtained by the production method according to the present invention is used as a working electrode of a biosensor, the response sensitivity will be extremely high. When a uricase electrode is made by immobilizing uricase using the manufacturing method according to the present invention, it can be used for more than -weeks, has an excellent response to uric acid solution, and has a
A response linearly related to concentration over a wide range of 100 mg/df can be obtained.

In addition, when creating a platinum substrate with a cross-linked film of albumin on the surface, if necessary, a biocatalyst is included in the treatment solution for film formation, and at the same time a cross-linked film of albumin is formed on the surface of the platinum substrate. The biocatalyst may also be immobilized. By immobilizing the biocatalyst inside the membrane in this way, the effect of obtaining an immobilized biocatalyst with even higher activity can be obtained.

Next, Example 1 to explain Examples and Comparative Examples
Then, immobilized biocatalysts were prepared as shown in (al to (f)) in Figure 1. That is, 4X15X0.
05 (Thickness) A Tsuru platinum plate was treated with aminosilane to produce an aminosilane-treated platinum plate having amino groups on the surface. Then, 1 μl of a film forming treatment solution containing 0.5% by weight (hereinafter referred to as "%") of glutaraldehyde and 10% of albumin was applied to the platinum plate for over 2 hours. An albumin-glutaraldehyde film (base film) was formed on the surface of the platinum plate by air drying.

Next, the platinum plate was soaked in a 2% glutaraldehyde solution (pH 8).
, 1) for 2 hours at room temperature for aldehyde treatment.

After the treatment, the platinum plate was washed with a phosphate buffer solution of pH 7.5,
Air-dried immediately. After this, 20% uricase solution (p
4 μl of H9,2) was applied to a platinum plate membrane and left at 5° C. for 2 hours to obtain an immobilized biocatalyst.

The composition of the film-forming treatment solution used in Example 1, the concentration of the glutaraldehyde solution and the concentration of the uricase solution used in the aldehyde treatment are summarized in Table 1.

In Examples 2 and 3, as shown in Table 1, the immobilized living organisms were prepared in the same manner as in Example 1, except that the composition of the treatment solution for film formation and the concentration of the glutaraldehyde solution were changed. Created a catalyst.

In a comparative example, 1 μl of a film-forming treatment solution having the composition shown in Table 1 was applied to the surface of an aminosilane-treated platinum plate prepared by the same method as in Example 1, and then air-dried for more than 2 hours to immobilize uricase. An immobilized biocatalyst was created using the membrane.

The immobilized biocatalysts obtained in Examples 1 to 3 and Comparative Example were used as working electrodes, and the response sensitivity to a uric acid solution with a concentration of 5 mg/dl was measured. However, the applied voltage is 0.
7V (vs. Ag/AgC1). The results are shown in Table 1.

From Table 1, it can be seen that when the immobilized biocatalysts obtained in Examples 1 to 3 are used, the response sensitivity is better than when the immobilized biocatalysts obtained in Comparative Examples are used. From this, it can be seen that those obtained in Examples 1 to 3 have higher activity than those obtained in Comparative Examples.

〔Effect of the invention〕

The method for producing an immobilized biocatalyst according to the present invention includes the steps of obtaining an immobilized biocatalyst in which a biocatalyst is immobilized on a platinum substrate.
The platinum substrate on which a cross-linked film of albumin is formed is treated with aldehyde to provide aldehyde groups on the film, and then the biocatalyst is bonded to the aldehyde group and fixed to the film. Immobilized biocatalysts with high activity can always be obtained regardless of the type.

[Brief explanation of drawings]

FIGS. 1(a) to 1(f) are explanatory diagrams of one embodiment of the method for producing an immobilized biocatalyst according to the present invention. 1...Platinum substrate 3...Crosslinked membrane of albumin 5.
...Aldehyde group A...Biocatalyst agent Patent attorney Takehiko Matsumoto (a) 2 t] (C) Figure 1 (b) (d) (f)

Claims (5)

[Claims] (1) To obtain an immobilized biocatalyst in which a biocatalyst is immobilized on a platinum substrate, the platinum substrate on which a crosslinked membrane of albumin is formed is treated with aldehyde to provide an aldehyde group on the membrane, and then the biocatalyst is A method for producing an immobilized biocatalyst, characterized by binding it to a membrane and immobilizing it on a membrane. (2) The method for producing an immobilized biocatalyst according to claim 1, wherein the biocatalyst is an enzyme. (3) The method for producing an immobilized biocatalyst according to claim 2, wherein the enzyme is uricase. (4) The method for producing an immobilized biocatalyst according to any one of claims 1 to 3, wherein the crosslinking agent for crosslinking albumin is glutaraldehyde. (5) The method for producing an immobilized biocatalyst according to any one of claims 1 to 4, wherein the aldehyde treatment is a glutaraldehyde treatment.