JPH0575430B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] 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 biologically active substance immobilizing materials such as fluororesin molded articles and glass molded articles. [Prior 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, if, for example, urokinase enzyme is immobilized on a molded product of fluororesin such as polytetrafluoroethylene, which has traditionally been used as a material for artificial blood vessels, catheters, and artificial organs,
It is expected to have effects such as being able to impart antithrombotic properties, which have been a problem with biomaterials. In other words, if biological properties can be added to fluororesin molded products that exhibit excellent properties such as heat resistance, chemical resistance, and oxygen permeability, the range of applications will be qualitatively wide. It is expected that it will be able to expand dramatically in terms of quantity. 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, the method of immobilizing biologically active substances on fluororesin molded products as biologically active substance immobilization materials involves etching the surface of the molded product with metallic potassium to form unsaturated bonds. A method in which a monomer having a binding property with a biologically active substance is bonded to the biologically active substance, or a physical adsorption method is used. Furthermore, as a method for immobilizing biologically active substances on a glass plate, a method using a silane coupling agent or a physical adsorption method is used. however,
The method using metallic potassium and a silane coupling agent is not only complicated in terms of process, but the former is particularly dangerous, and the physical adsorption method has concerns about its binding strength. [Problems to be Solved by the Invention] In view of the current situation, the present inventor has conducted various studies on methods for easily and firmly binding biologically active substances onto immobilization materials that are originally difficult to immobilize biologically active substances. As a result, we found that this problem can be effectively solved by plasma polymerizing an aldehyde compound or isocyanate compound, which is a compound that can bond with the amino group of a biologically active substance, on the surface of a biologically active substance immobilization material. . [Means for Solving the Problem] and [Operation] Therefore, 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 It is carried out by plasma polymerizing the isocyanate 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,
For example, a polytetrafluoroethylene resin membrane or a glass plate 6 is housed in a plasma reaction vessel 5 connected to a vacuum pump 1, a leak valve 2, and a main valve 3 and equipped with a vacuum gauge 4, and the pressure inside the reaction vessel is maintained at approximately After setting the pressure to 0.001 to 10 Torr, the valve 7 is opened and the aldehyde compound or isocyanate compound from the tank 8 is introduced into the reaction vessel. With these compounds introduced, the high frequency generator (13.56MHz) 9 and matching unit 1
Using a high frequency power source consisting of 0, the effective power is approximately 50 ~
Plasma is irradiated from the oscillation coil 11 under conditions of 200 W and a time of about 5 to 600 seconds. As the reaction vessel, either a tube type or a bell jar type can be used, and as the discharge electrode, in addition to a coil type, external or internal parallel electrode plates can also be used. As mentioned above, as biologically active substance immobilization materials,
Polytetrafluoroethylene resin molded articles, glass molded articles, and the like are preferably used, and the shape thereof can be arbitrary, such as a plate-like body, a membrane-like body, a spherical body, and a fibrous body. 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 monomer 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 about 40 to 100°C, it becomes gaseous and is subjected to a plasma reaction. Biologically active substances bound to such a plasma polymerized layer 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 mg/ml. Examples of enzymes include oxidases such as glucose oxidase, amino acid oxidase, cholesterol oxidase, and uricase, uriase, creakininase, glutaminase,
Examples include proteases such as penicillinase, catalase, peroxidase, invertase, mutarotase, amylase, papain, and trypsin, glucose isomerase, and urokinase. Examples of microorganisms include bacteria such as Pseudomonas fluorescens, Bacillus subtilis, and Pseudomonas aeruginosa, filamentous fungi such as Aspergillus niger and Rhizopus hormocensis, actinomycetes such as Streptomyces griseus, yeasts, and molds. Examples include. In addition, the method of the present invention can also be applied to other biologically active substances such as proteins having amino groups in their structures. [Effects of the Invention] The method of the present invention in which biologically active substances are immobilized on the surface of immobilization materials such as fluororesin molded products and glass molded products, especially on the surface of glass molded products, can be immobilized easily and firmly. Not only is this possible, but the characteristics of the immobilization material are also taken into account, so artificial blood vessels,
It can be effectively used for medical materials such as sutures, bioreactors, biosensors, etc. [Example] Next, the present invention will be explained with reference to an example. Examples 1-4 Plasma polymerizations were carried out according to the illustrated embodiment. As the immobilization material, one molded from polytetrafluoroethylene resin (PTFE) or glass with a size of 10 x 10 x 0.5 mm was used, and after setting the pressure inside the reaction vessel to 0.01 Torr,
A plasma polymerizable monomer vaporized from 0.5 ml of an aqueous solution was introduced, and plasma irradiation was performed using a 13.56 MHz high frequency generator under conditions of an effective power of 150 W and a time of 10 minutes to cause a polymerization reaction. After the irradiation, the immobilization material with a plasma polymerized layer formed on its surface was taken out from the reaction vessel and treated with Bacillus subtilis-derived alkaline protease (product sold by Nagase Sangyo, enzyme number EC0.3.4.16) with an enzyme concentration of 1 mg/ml. It was immersed in a 4°C aqueous solution for 24 hours. then 100
ml of distilled water. The activity of the obtained immobilized enzyme was measured by a modified Anson-Ogiwara method using a casein substrate. Measurement conditions are in acetate buffer, pH 7.5, temperature 35
℃, and the ultraviolet absorption wavelength is 660 nm. In addition, by immersing the immobilized enzyme in 5 ml of acetate buffer at 4°C for 24 hours, and measuring the enzyme activity in the same manner,
The presence or absence of detachment of the immobilized enzyme was determined. The results obtained are shown in the following table. 【table】

[Brief explanation of drawings]

FIG. 1 is a schematic diagram showing one embodiment of a plasma polymerization apparatus used in the present invention. (Explanation of symbols), 1... Vacuum pump, 5... Plasma reaction vessel, 6... Biologically active 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 immobilizing a biologically active substance according to claim 1, wherein the biologically active substance is a microorganism.