JPH0372274B2 - - Google Patents

Description

[Detailed description of the invention]

TECHNICAL FIELD This invention relates to glass coated tubes. More specifically, the present invention relates to an immobilized glass coating tube on which a peptide-containing functional compound such as an enzyme, an antigen, or an antibody is immobilized, and a method for producing the same. Recently, immobilized glass in which peptide-containing compounds such as enzymes are immobilized on glass carriers has been used as bioreactors for diagnosis and synthesis.
In particular, fixed glass coated tubes in which the inner surface of the tube is coated with such fixed glass have come to be used in columns for liquid chromatographs and gas chromatographs. The manufacturing method for these coating tubes is the melting method.
A glass coating tube is obtained by creating SiO ₂ -based glass, drawing it into a suitable thin tube or coating the surrounding area with polyimide resin, etc., and treating the glass surface with an alkali to generate hydroxyl groups. A method is known and has been put into practical use, in which a silane coupling agent is introduced into the silane via an ester bond, and then a peptide-containing compound such as an enzyme is added to the coupling agent, either directly or after denaturation, for immobilization. However, the conventional method described above is disadvantageous in that the production of the glass coating tube itself requires special equipment and high-temperature heating, and also requires a step to generate hydroxyl groups on the glass surface. There is a limit to the amount of hydroxyl groups that can be generated per unit surface area, and there is also a limit to the amount of enzymes etc. immobilized, making it difficult to increase the amount immobilized per glass coated surface and obtain highly active immobilized glass-coated tubes. It was hot. Therefore, special surface roughening treatments such as honeycomb treatment (whisker generation treatment) are performed on coated glass to increase the glass surface area as much as possible. This invention was made in view of such problems. The inventors of this invention came up with the idea of using a metal alkoxide as a raw material and hydrolyzing it under mild conditions to produce a gel-like substance as a carrier for immobilizing enzymes, etc., thereby eliminating the need for heat treatment etc. The present invention was achieved by discovering the fact that an immobilized glass coating tube with an increased amount of immobilization per surface area of the carrier can be obtained without performing a step of generating hydroxyl groups. Thus, according to the present invention, a peptide-containing compound can be immobilized using a glass-like gel body made of a hydroxide metal compound and/or its condensate produced by hydrolysis of a metal alkoxide as an immobilization carrier coated on the inner surface of a tube. A chemically modified glass coating tube is provided. The metal alkoxide in this invention includes:
Various metal alkoxides known in the glass manufacturing field and ceramic manufacturing field can be applied, and specifically, Si
(OCH ₃ ) ₄ , Si (OC ₂ H ₅ ) ₄ , Ti (OC ₃ H ₇ ) ₄ , V
Examples include lower alkoxy metals such as (OC ₂ H ₅ ) ₃ , Al(OC ₃ H ₇ ) ₃ , and NaOCH ₃ , and among these, it is usually preferable to use lower alkoxysilanes. Note that a mixture of two or more of these may also be used. In manufacturing the tube of the present invention, a solution of the metal alkoxide described above is first coated inside the desired tube. Usually, coating can be easily carried out by passing the above solution into the tube. This coats the inner surface of the tube with a thin layer of metal alkoxide-containing solution. At this time, it is necessary to use a metal alkoxide-containing solution in which the metal alkoxide is not substantially hydrolyzed. As this solvent, it is appropriate to use an easily volatile hydrophilic solvent or a water-containing substance thereof, and for example, water-containing methanol or water-containing ethanol is suitably used. It is also possible to use only water as a solvent, but in this case, depending on the metal alkoxide selected, there is a risk that hydrolysis may proceed too rapidly, resulting in gelation, making the coating operation difficult, and it is also disadvantageous in terms of drying, which is undesirable. . The metal alkoxide within the coated thin layer forms a glass-like gel body upon hydrolysis. Such hydrolysis proceeds under mild conditions and with evaporation of the solvent. For example, when lower alkoxysilane is used, the tube is coated with an aqueous (water-containing) solvent adjusted to a pH of 1 to 3, and then left at room temperature for rapid hydrolysis (usually within a few minutes). ) can be done. In particular, according to this method, since a thin layer of solution is coated inside the tube, evaporation of the solvent is accelerated, and it is thought that hydrolysis, which hardly progresses before coating, proceeds. However, it is necessary to perform the above hydrolysis under mild conditions such as at room temperature and avoid heating to form a gel body that prevents high condensation of the hydrolyzate (for example, Si(OH) ₄ ) as much as possible. . For example, Si(OC ₂ H ₅ ) ₄ as metal alkoxide
When using Si, a glass-like gel body is obtained that coats the inner surface of the tube, but its composition is Si.
Since it is composed of Si(OH) ₄ , which is a hydrolyzate of (OC ₂ H ₅ ) ₄ , or its low condensate, it has a much higher level of hydroxyl groups than the hydroxyl groups generated by alkali treatment on the SiO ₂ glass of conventional coating tubes. It itself has many hydroxyl groups. Therefore, it is considered that a larger amount of enzymes and the like can be immobilized compared to conventional immobilization glasses. Similarly for other metal alkoxides, since the obtained glass-like gel bodies are composed of metal hydroxide compounds and low condensates thereof, it is thought that large amounts of enzymes and the like can be immobilized. The glass-like gel coating tube thus obtained is subjected to the following process as it is or optionally dried. However, since the glass-like gel coating tube itself has a different surface activity from conventional glass coating tubes, it can also be used as a column for special separation and analysis, and it can also be treated at high temperatures (usually 300 to 500℃).
It can also be converted into a SiO ₂ -based glass coated tube. In particular, the method for producing SiO ₂ -based glass coated tubes is simpler than conventional methods and is extremely useful. Therefore, from this point of view, the present invention covers the inner surface of the tube with a solution containing one or more metal alkoxides,
The present invention also provides a method for producing a glass-coated tube, which comprises hydrolyzing a metal alkoxide to form a coating layer of a glass-like gel body, and heat-treating the coating layer. As the silane coupling agent to be reacted with the gel body, silane derivatives known in the art having functional groups such as amino groups, thiol groups, and epoxy groups can be used. Specifically, γ-aminopropyltriethoxysilane , γ-chloropropyltrimethoxysilane, vinyltriethoxysilane, γ-
Glycidoxypropyltrimethoxysilane, N-
β-(aminoethyl)-γ-aminopropyltrimethoxysilane and the like are used. Reactions with such silane coupling agents are carried out under conditions known in the art. For example, when γ-aminopropyltriethoxysilane is used, this coupling agent is dissolved in water to form a 1 to 10 wt% aqueous solution, preferably a 2 wt% aqueous solution, and after adjusting the pH to 3 to 5, this solution is poured into the tube. It is obtained by injecting the coupling agent into the solution and leaving it in contact for several hours under heating, followed by washing with water to remove unreacted coupling agents. Such a tube has more coupling groups on its inner surface than a conventional glass-coated tube. Peptide-containing compounds such as enzymes, antigens, and antibodies are immobilized on the inner surface of the tube treated in this manner by a known method. For example, when using a gel body-coated tube in which a large number of aminoalkyl groups are introduced into hydroxyl groups through ester bonds using γ-aminopropyltriethoxysilane as a coupling agent, geltaraldehyde is used for the aminoalkyl groups to form aldehyde groups. Introducing Schizuf base,
Immobilization can be carried out by bringing an enzyme or the like into contact with this to further form a Schiff base between the aldehyde group and the amino group of the enzyme, etc., and bonding.
In addition to this, an aminoalkyl group may be diazotized to introduce an aromatic amino group and an enzyme etc. may be immobilized thereon, or a carbodiimide may be used to directly form a peptide bond between an aminoalkyl group and an enzyme etc. Immobilization may be carried out and may be appropriately selected depending on the type of enzyme, etc. When using other coupling agents, enzymes and the like can be similarly immobilized directly or by appropriately converted coupling groups. Examples of enzymes for immobilization include, but are not limited to, glucose oxidase, uricase, urease, creatininase, CoA-synthetase, CoA-oxidase, cholesterol oxidase, cholesterol hydrolase, etc. Antigens and antibodies can also be fixed. As the tube used in this invention, tubes for columns in fields such as gas chromatographs and liquid chromatographs, various diagnostics, and synthesis can be applied, and thin tubes such as capillary columns can also be used. The material may be metal, glass, ceramics, plastic, etc., and glass is usually suitable. The thus obtained immobilized glass coating tube of the present invention has an increased amount of enzymes etc. immobilized compared to conventional immobilized glass coating tubes, and is useful as a bioreactor in various fields and is useful in chromatography. It is also useful as a column for graphs. Furthermore, according to the method for producing a coating tube of the present invention, the tube can be coated with a carrier without high-temperature heat treatment, and furthermore, there is no need for a step of introducing hydroxyl groups during immobilization, which is significantly improved compared to conventional methods. can reduce costs. Furthermore, since a solution is used, it is possible to easily coat the inner surfaces of thin tubes, etc., and extremely thin coatings are also possible. The obtained immobilized glass coated tube also has higher activity than conventional tubes, which is advantageous. This invention will be explained below using reference examples. Reference Example 1 (Preparation of gel-like material) A mixture (PH2) of 10 g of tetraethoxysilane Si (OC ₂ H ₅ ) _4, 10 ml of ethanol, 3.8 ml of water, and 1.4 ml of hydrochloric acid (IN) was heated at room temperature for about 1 hour until it became homogeneous. Stir and mix. This mixture was left in the air for about 4 days to promote hydrolysis and evaporation of water and ethanol.
It was dried at 120°C to obtain a Si(OH) ₄ gel. (Aminoalkylation) The resulting gel was crushed in a mortar and passed through a sieve to obtain beads with average particle diameters of 60/80, 80/120, and 120/200. A 5wt% γ-aminopropyltriethoxysilane aqueous solution was adjusted to pH 3.5 with 5N hydrochloric acid, and this solution
5 g of each of the above bead-like gels were added per ml, and the pH was adjusted to 3.5. Place this mixture in a four-necked flask equipped with a stirrer, thermometer, and Dimroth, and bring the temperature to 75°C in a water bath.
The reaction was carried out for 3 hours while maintaining the temperature at 0.degree. C. and vigorously stirring. After the reaction was completed, the beads were transferred to a suction membrane filter, and unreacted γ-aminopropyltriethoxysilane was removed with distilled water from step 1.
It was dried in a desiccator to obtain three types of aminoalkylated gels with different particle sizes. This aminoalkylated gel is stored in a desiccator. (Immobilization of enzyme) Each of the above aminoalkylated gels (beads) was mixed with difunctional glutaraldehyde (2.5wt%).
The mixture was immersed in a phosphate buffer solution (PH7.0), and reacted for about 30 minutes with stirring while reducing the pressure with an asbilator. Subsequently, the reaction was continued for about 30 minutes at normal pressure with stirring. The reaction temperature was 25°C. This was thoroughly washed with a phosphate buffer solution of pH 7.0 and dried. This treatment introduces Schiff base having aldehyde groups into the gel. The resulting gel was immersed in 1 mg/ml (glucose oxidase/PH7.0 phosphate buffer),
The reaction was first carried out at 25° C. with gentle stirring under reduced pressure for 30 minutes, and then the reaction was carried out under fixed conditions by gentle stirring under normal pressure for 60 minutes. Through this treatment, the amino groups of glucose oxidase participate in the reaction, and are further immobilized by forming a Schiff base with the aldehyde groups of the carrier (gel-like substance). In this way, each of the glucose oxidase-immobilized gels (glass) of the present invention was obtained. (Activity test) In order to compare the degree of immobilization of 200 meshes of the above-mentioned immobilized glass beads with the conventional one, an activity test was conducted by voltammetry. First, 1wt
% β-D glucose solution (PH7.0 phosphate buffer) was poured into a beaker containing 1 g of the immobilized glass beads of the present invention, and the mixture was stirred gently for 15 minutes to react. 9 ml was taken and 1 ml of 5% potassium iodide solution was added. Through this operation, I ^- becomes I ₂ with hydrogen peroxide generated by the enzymatic reaction.
Oxidized to (aq). By reducing this I ₂ by voltammetry and measuring the reduction wave, I ₂
The amount can be quantified, and the degree of immobilization of the enzyme in the immobilized glass beads, that is, the degree of activity, can be determined. Table 1 shows the results of voltammetry of immobilized glass beads obtained in the same manner as above using conventional glass as a carrier and immobilized glass beads of the present invention.
Shown below.

[Table] It can be seen that the immobilized glass coated inside the tube of the present invention clearly has higher enzyme activity than the conventional glass carrier.

Claims (1)

[Scope of Claims] 1. A glass-like gel body made of a metal hydroxide compound and/or a condensate thereof produced by hydrolysis of a metal alkoxide is used as an immobilization carrier to be coated on the inner surface of the tube. A glass coating tube for immobilizing a peptide-containing compound by immobilizing the peptide-containing compound on the silane coupling agent residue. 2. A solution containing one or more metal alkoxides is coated on the inner surface of the tube, and the metal alkoxide is hydrolyzed to form a glass-like gel coating layer, which is coated with a solution containing one or more metal alkoxides, and a solution containing one or more metal alkoxides selected from amino groups, thiol groups, or epoxy groups is added to the coating layer. 1. A method for producing an immobilized glass-coated tube, which comprises reacting a silane coupling agent having a functional group, and immobilizing a peptide-containing compound on the reactant.