JPS6156659A - Immobilization of biologically active substance - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a method for immobilizing biologically active substances.

More specifically, the present invention relates to a method for immobilizing biologically active substances such as enzymes and microorganisms on the surface of fluororesin molded articles, glass β, and other biologically active substance immobilizing materials.

[Conventional technology]

BACKGROUND ART Biologically active substances such as enzymes and microorganisms have been immobilized on various materials and used for applications such as biosensor membranes, affinity chromatography, and bioreactors.

On the other hand, for example, if urokinase enzyme is immobilized on a manufactured product of fluororesin such as polytetrafluoroethylene, which has traditionally been used as a material for artificial blood vessels, catheters, and artificial organs, problems with biomaterials may arise. Is there an antithrombotic drug? It is expected that the effects such as the following can be achieved.

In other words, if it is possible to add biological properties to fluorine 1fJJ resin products, which have excellent properties such as heat resistance, chemical resistance, and oxygen permeability, its application would be qualitative. It is expected that the company will be able to expand dramatically both in Japan and in Japan.

In addition, glass molded products have traditionally been used as a typical immobilization material for biologically active substances, and spherical ones in particular are effectively used in bioreactors, etc. If substances can be combined more effectively, their usefulness will be further enhanced.

In order to provide these various uses, biologically active substances are immobilized on fluororesin molded products as biologically active substance immobilizing materials [7] One way to immobilize biologically active substances is to etch the surface of the molded product with potassium metal to make it unsaturated. A method of forming a bond and attaching a single phosphor having a binding property to a biologically active substance to the bond, or a physical adsorption method, etc. are used. In addition, as a method of immobilizing a biologically active substance on a glass plate, a method using a 'C--' run coupling agent or a physical adsorption method is used. however,
The method using metallic potassium and a silane coupling agent is
Not only is the process complicated, but the former is particularly dangerous, and physical adsorption methods have concerns about their binding strength.

[Problem that the invention seeks to solve]

In view of the current situation, the present inventor has conducted various studies on methods for easily and firmly bonding biologically active substances to immobilizing materials, which are difficult to immobilize biologically active substances on. It has been found that this problem can be effectively solved by plasma polymerizing an aldehyde compound or an isocyanate compound, which is a compound capable of bonding with the amide 7 group of a biologically active substance, on the surface.

[Means for Solving the Problem] and [Operation] Accordingly, the present invention relates to a method for immobilizing a biologically active substance, and the immobilization of a biologically active substance is performed by applying an aldehyde compound or an isocyanate to the surface of a biologically active substance immobilizing material. This is done by plasma polymerizing the compound and then attaching the biologically active substance thereto.

The plasma polymerization treatment is carried out using, for example, an apparatus as schematically shown in FIG. That is, the vacuum pump 1,
For example, a polytetrafluoroethylene resin membrane or a glass plate 6 is housed in a plasma reaction vessel 5 connected to the leak pulp 2 and the main valve 3 and equipped with a vacuum gauge 4t, and the pressure inside the reaction vessel is maintained at about 0.001 to After setting the IQ Torr, the pulp 7 is opened and the aldehyde compound or isocyanate compound from the tank 8 is introduced into the reaction vessel. With these compounds being equal in size, using a high frequency power source consisting of a high frequency generator R (13,56 MHz) 9 and a matching unit 10, an effective power of about 50 ~
Plasma is irradiated from the oscillation coil 11 under conditions of 200 W and a time of about 5 to 600 seconds. As a reaction vessel, either a tube-shaped or a Pelger type can be used, and as a discharge electrode, in addition to a coil-shaped one,
External or internal parallel electrode plates can also be used.

As mentioned above, polytetrafluoroethylene resin molded products, glass molded products, etc. are preferably used as biologically active substance immobilization materials, and the shapes can be arbitrary such as plate-like, membrane-like, spherical, and fibrous. It can take the form of

Examples of the aldehyde compounds to be plasma-polymerized on the surface of these immobilizing materials include glutaraldehyde, formaldehyde, acetaldehyde, etc., and examples of the isocyanate compounds include toluene diisocyanate, isophorone diisocyanate, hexamethylene diisocyanate, and the like. These polymerizable single-plate compounds are used as they are or in the form of an aqueous solution, and some of them have high boiling points, but depending on the degree of vacuum in the plasma reaction vessel as described above, By appropriately heating it to ~100° C., it becomes gaseous and is subjected to a plasma reaction.

Such biologically active substances bound to plasma heavy metals include enzymes, microorganisms, etc. For example, in the case of enzymes, they are generally used as an aqueous solution of about 0.1 to 10 m<9>/-.

Examples of enzymes include oxidases such as glutenose oxidase, amino acid oxidase, cholesterol oxidase 7, and uricase, uriase, flakininase, glutaminase, benicillinase, catalase,
Examples include proteases such as peroxidase, invertase, mutarotase, amylase, papain, and trypsin, glucose isomerase, and urokinase. In addition, examples of microorganisms include Pseudomonas
Bacteria such as Bacillus fluorescens, Bacillus subtilis, and Pseudomonas aeruginosa, filamentous fungi such as Aspergillus niger, Rhizopus hormo, and Sensis, actinomycetes such as Streptomyces griseus, yeast,
Examples include mold. In addition, the method of the present invention can also be applied to other biologically active substances such as proteins having amine groups in their structures.

〔Effect of the invention〕

1. The method of the present invention in which biologically active substances are immobilized on the surface of fluororesin molded products, glass molded products, etc., especially on the surface of glass molded products, not only can the immobilization be easily and firmly carried out, but also Since the properties of the immobilization material are further taken into account, it can be effectively used for applications such as artificial blood vessels, medical materials such as sutures, bioreactors, and biosensors.

〔Example〕

Next, the present invention will be explained with reference to examples.

Examples 1-4 Plasma interaction was carried out according to the illustrated embodiment. As the immobilizing material, polytetrafluoroethylene resin (p
ryx+)tfC are each molded from glass.
A reactor with a size of X 10 X 0.5 m was used, and after setting the pressure inside the reaction vessel to Q
．． Introducing the plasma combinable single n substance vaporized from the aqueous solution of 5-, using a 13.56 MHz high frequency generator, plasma irradiation was performed under conditions of an effective power of 150 W and a time of 10 minutes to cause a polymerization reaction. Ta.

After the irradiation, the immobilization material with a plasma polymerized layer formed on its surface was taken out from the reaction vessel and was heated to an enzyme concentration of 1 my
/ml of Bacillus subtilis-derived alkaline protease (product sold by Nagase Sangyo, enzyme number: 0.3.4.4.16) at 4'°C.
It was immersed in an aqueous solution for 24 hours. Thereafter, it was washed with 100 g of distilled water.

The activity of the obtained immobilized #element was measured by a modified Anson-Ogiwara method using a casein substrate.

The measurement conditions were: in acetate buffer, pH 7.5, temperature 35°C;
The ultraviolet absorption wavelength is 660 nm. In addition, the presence or absence of degeneration of the immobilized enzyme was determined by immersing the immobilized enzyme in acetate buffer solution 5 at 4° C. for 24 hours and then measuring the enzyme activity in the same manner. The results obtained are shown in the following table.

Table same concentration C%) 50 25
50 25 Enzyme Naoko mTJ/2j) 3
゜4 3.0 9.5 5
．． 0-eliminating enzyme None None None None

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram showing one embodiment of a plasma polymerization apparatus used in the present invention. (Explanation of symbols) l...Vacuum pump 5...Plasma reaction vessel 6...Bioactive substance immobilization material 9...High frequency generator 11... ...oscillation coil

Claims (1)

[Scope of Claims] 1. A method for immobilizing a biologically active substance, which comprises plasma polymerizing an aldehyde compound or an isocyanate compound on the surface of a biologically active substance immobilizing material, and then bonding the biologically active substance thereto. 2. The method for immobilizing a biologically active substance according to claim 1, wherein the biologically active substance immobilizing material is a fluororesin molded article. 3. The method for immobilizing a biologically active substance according to claim 2, wherein the fluororesin molded product is a polytetrafluoroethylene resin molded product. 4. The method for immobilizing a biologically active substance according to claim 1, wherein the biologically active substance immobilizing material is a glass molded article. 5. The method for immobilizing a biologically active substance according to claim 1, wherein the biologically active substance is an enzyme. 6. The method for defining a biologically active substance according to claim 1, wherein the biologically active substance is a microorganism.