WO2005033304A1 - Immobilization element coated with functional-substance-containing thin film and process for producing the same - Google Patents

Abstract

[PROBLEMS] To provide a functional substance immobilization element which can be used in drug candidate compound screening and clinical test or detection of environmental factor variation and can be used as a functional substance bioreactor and which can be easily produced and are available at low price, excelling in the variety of applicable physiologically active substance and the functional capability. [MEANS FOR SOLVING PROBLEMS] An immobilization element coated with a thin film wherein a functional substance is incorporated in a network structure of nanometer order; a porous immobilization element having pores of nanometer order; and a process for forming the thin film on the immobilization element.

Description

 Specification

 Immobilized element covered with a functional substance-containing thin film and method for producing the same

 The present invention relates to a fixing device covered with a functional substance-containing thin film and a method for producing the same. More specifically, the present invention relates to an immobilization device coated with a thin film containing a functional substance in a network structure in nanometer units, a porous immobilization device having pores in nanometer units, and The present invention relates to a method for forming a thin film on a fixed element.

 Background art

 [0002] Conventionally, screening of drug candidate conjugates, clinical tests for obtaining diagnostic data, and changes in environmental factors using functional substances such as enzymes, antibodies, or physiologically active substances have been performed. Detection and the like are performed, and useful compounds are produced using these functional substances as bioreactors.

 These functional substances have been used by immobilizing them on various carriers for efficient and economical repeated use or for improving the reaction efficiency and the detection sensitivity and efficiency. For example, a protein immobilization method widely used as a functional substance includes a carrier binding method in which a protein is bound to an insoluble carrier, and a crosslinking method in which an enzyme is reacted with a reagent having two or more functional groups. Inclusion methods include wrapping in a fine gel lattice or covering with a semipermeable polymer film.

 [0003] As the carrier binding method, there is a method of immobilizing trypsin using glutaraldehyde using aminoethylcellulose such as Glassmeyer and CK (Non-Patent Document 1). An immobilized enzyme preparation immobilized thereon (Patent Document 1) and a lipase complex immobilized on insoluble matrix (Patent Document 2) have been developed. Since the carrier binding method is an immobilization method using a covalent bond, the enzyme is not easily desorbed, but the reaction operation is complicated, relatively intense, and a protein that is a biologically active substance because of the treatment. However, there is a disadvantage that changes in the higher-order structure and a decrease in activity are likely to occur.

As the cross-linking method, there is a method in which a Schiff base is formed between enzymes by using dartartaldehyde of Quiocho, FA and the like to cross-link and immobilize carboxypeptidase A (Non-patent document). (2) For example, immobilized lipase produced by binding a coupling agent having a carboxylic acid ester bond to an inorganic carrier (Patent Document 3), and a substrate substrate having a bioactive agent immobilized on an organic film A flat biomolecule screening device (Patent Document 4) and the like have been developed. However, although the cross-linking method is simpler than the carrier-binding method, it has a drawback that the activity tends to decrease because the bioactive substance is carried out through a covalent bond.

 [0004] Furthermore, in the entrapment method, Bernfeld, P. et al. Add an acrylamide monomer, a cross-linking agent, a polymerization accelerator, a polymerization initiator, and the like to an enzyme solution of interest, and polymerize the solution. There is a method of capturing the information in the inside (Non-Patent Document 3). Unlike the carrier binding method and the cross-linking method, this method does not require the biologically active substance to be bonded to the carrier, and thus has the advantage that the activity does not easily decrease. Since there is no binding reaction, the strength tends to be low! /

 In addition, there is a method developed by Ellerby et al. In which a protein dissolved in a buffer solution is included in a silica matrix by a sol-gel method using alkoxysilane (Non-Patent Document 4). By this method, a bioactive substance can be fixed and applied as a bioreactor while retaining the higher-order structure and biological activity. After the sol-gel reaction, the gel is dried and crushed according to various uses. When the activity of immobilized protein and the like was reduced, and the treatment process was complicated! /

 [0005] In these immobilization methods, as described above, since a physiologically active substance needs to have a functional group suitable for immobilization, only a limited number can be immobilized. However, since a purification operation is required, there are drawbacks such as that the method is complicated and expensive, or that only a stable physiologically active substance can be immobilized.

[0006] In such a situation, in recent years, with the advancement of technology, it has functioned as a screening, clinical examination, detection of fluctuations in environmental factors, and a bioreactor for functional substances, with the use of a pharmaceutical compound. Sexual substances are being actively used. In addition, along with advances in combinatorial chemistry and the like, the number of compounds that can be drug candidates has increased significantly compared to compounds prepared by traditional methods and extracts of natural products, making them useful drugs. More efficient screening is needed to find compounds. In order to meet these demands, there is a need for a functional substance-fixing element having a variety of bioactive substances which are easy to manufacture and inexpensive and can be used, and which have high functions.

Patent Document 1: Japanese Patent Application Laid-Open No. 2000-83663

 Patent Document 2: Japanese Patent Publication No. 2002-539782

 Patent document 3: JP-A-9-313179

 Patent Document 4: Japanese Patent Publication No. 2002-520621

 Non-Patent Document l: Glassmeyer, C.K.et.al, Biochemistry, 1971, 10, 786.

 Non-Patent Document 2: Quiocho, F.A. et.al, Proc.Nat.Acad.Sci. 1964, 52, 833 Non-Patent Document 3: Bernfeld, P. et.al, Science, 142, 678, 1963

 Non-patent document 4: Ellerby et.al, Science, 1992, 255, 1113-1115

 Disclosure of the invention

 Problems to be solved by the invention

[0008] The present invention can be used for screening drug candidates, for clinical tests, or for detecting changes in environmental factors, and can also be used as a bioreactor for functional substances, and is simple and inexpensive to manufacture. It is an object of the present invention to provide a functional substance-immobilizing element having a wide variety of usable physiologically active substances and a high functionality.

 Means for solving the problem

[0009] As a result of conducting research to achieve the above object, the present inventors have found that a sol reaction solution containing an organometallic compound having a hydrolyzable functional group and a functional active substance is gelled on an immobilized element. It was found that by shading, a thin film having a network structure having voids of several nanometers (hereinafter abbreviated as nm) holding a physiologically active substance could be formed on the immobilized element.

 Furthermore, a physiologically active substance such as a metabolic enzyme which is unstable and easily loses its activity outside the cell is included in the micronome, and a sol reaction solution containing the micronome and an organometallic compound having a hydrolyzable functional group is prepared. By gelling on the immobilization device, it is possible to form a thin film having a network structure with several nm voids holding microsomes containing a physiologically active substance in a stable form on the immobilization device! ヽぅ Obtained knowledge.

[0010] Further, the present inventors have proposed an organometallic compound having a hydrolyzable functional group under a specific condition. By treating under the above conditions, it is possible to produce a porous fixed element having a porous structure having a through hole of several Wm that is permeable to a solvent, and having pores of several nm in diameter on the surface of the structure. Obtained knowledge. The present invention has been completed based on these findings.

 Therefore, the present invention provides an immobilized element, particularly a porous immobilized element, which is covered with a thin film having a network structure containing a functional substance.

[0011] Further, according to the present invention, the immobilization element has a porous structure having a through-hole of 0.1-10 O / zm, which is permeable to a solvent, and has a diameter of 11-100 nm on the surface of the structure. Disclosed is a fixing device which is a porous fixing device having pores and is coated with a thin film having a network structure containing a functional substance.

 Further, the present invention provides that the functional substance is at least one selected from the group consisting of a physiologically active substance such as a protein, a nucleic acid, a lipid, a sugar, a drug, a metal catalyst, and a compound having a specific shape. A coated immobilized element is provided.

[0012] The present invention also provides an immobilization element, particularly a porous immobilization element, which is covered with a network-like thin film containing microsomes containing a functional substance.

 Further, the present invention provides a porous immobilization element, wherein the immobilization element has a porous structure having several / zm through-holes permeable to a solvent and has pores having a diameter of several nm on the surface of the structure. The present invention provides an immobilization element covered with a network-like thin film containing microsomes containing a functional substance.

[0013] Further, the present invention provides an immobilization device, wherein the functional substance is coated with a thin film having a network structure including microsomes containing the functional substance, which is a metabolic enzyme such as P450 or Darc-transferase. A dani element is provided.

 In addition, the present invention provides a thin film having a network structure containing a functional substance, or a porous immobilization element covered with a thin film having a network structure containing microsomes containing a functional substance. Provide a column or microchip.

Further, the present invention is a method for coating a thin film having a network structure containing a functional substance on an immobilization element, comprising:

Organometallic compound having a hydrolyzable functional group, or contains a non-hydrolyzable organic functional group and a hydrolyzable functional group bonded via at least one metal 'carbon bond Hydrolyzing an organometallic compound; mixing the hydrolysis solution with a solution containing a functional substance to obtain a sol solution; and immersing the immobilizing element in the sol solution and drying. Accordingly, the present invention provides a method for coating the thin film, comprising forming a thin film on the surface of the fixing device.

 In addition, the present invention provides a porous immobilization film having a porous structure having a through hole of 0.1-10 O / zm that is permeable to a solvent, and having pores having a diameter of 11 lOO nm on the surface of the structure. Provide device.

 [0015] Further, the present invention is a method for producing the porous immobilized element, comprising:

 Hydrolyzes organometallic compounds having a hydrolyzable functional group, or an organometallic compound containing a non-hydrolyzable organic functional group and a hydrolyzable functional group bonded via at least one metal 'carbon bond Providing a solvent that causes phase separation in the course of gelation with the hydrolysis solution; and polymerizing the mixture by thermal polymerization or photopolymerization.

 As used herein, the term “fixing device” refers to a structure (support) that covers a thin film having a network structure containing a functional substance. Unless otherwise specified, when the term `` fixed ridge element '' is used alone, it means a structure that is not coated and is covered with a thin film having a network structure containing a functional substance. Described as “coated immobilized element”.

 The invention's effect

 [0016] According to the present invention, it can be used for screening of drug candidates, clinical examination, or detection of changes in environmental factors, and can also be used as a bioreactor for functional substances, which is simple and inexpensive to manufacture. A variety of physiologically active substances can be used, and a highly functional functional substance-fixing element is provided.

 Brief Description of Drawings

 FIG. 1 is an electron micrograph showing the structure of the porous immobilization element of the present invention. The space shown in the photograph shows a porous structure with voids of several meters permeable to the solvent.

FIG. 2 is an enlarged view further showing an extremely large number of depressions (nm pores) having a diameter of several nm on the surface of the porous structure. FIG. 3 is an enlarged view showing a network structure of a thin film formed on a porous immobilization element of the present invention, in which a surface of a depression (nm pore) of the porous immobilization element is This shows a state in which a thin film having a thickness of several nm is covered. The upper right enlarged portion A shows a state in which the thin film captures and fixes a functional substance in a network structure having voids of several nm.

 FIG. 4 is a diagram illustrating a sol-gel reaction at the time of forming a thin film of the present invention based on a polymerization reaction of alkoxysilane.

[FIG. 5] FIG. 5 shows that a functional substance is fixed to a network structure of a thin film in the course of polymerization of a monomer generated by hydrolysis of an alkoxysilane in a sol-gel reaction at the time of forming a thin film of the present invention. It is a figure showing a situation.

 FIG. 6 is a graph showing the activity of immobilized trypsin in Example 2.

 FIG. 7 is a graph showing the amount of metabolite produced by the action of P450 in Example 4.

 FIG. 8 is a conceptual diagram showing the relationship between pH conditions and the network structure of a gel.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0019] (immobilization element)

 The element for fixing a functional substance used in the present invention is not particularly limited as long as it can be covered with a thin film having a network structure containing the functional substance. Further, the shape can be selected without particular limitation according to the purpose of use. For example, standard or atypical particles such as tablet-like particles or bead-like particles having a size according to the purpose of use, a specified area on a flat or porous substrate, or a cylindrical shape formed on a substrate. An inner wall surface provided with a flat or porous portion of a concave portion such as the above can be used as a fixing device for coating the thin film. In addition, it can be used in a small space created using microfabrication technology.

Further, the material of the fixing device is not particularly limited as long as it can form a thin film having the network structure on the surface. For example, when the immobilizing element is a regular or irregular particle, silicon, silica, quartz, glass, porous glass, carbon, activated carbon, alumina, titer, tantalum oxychloride, germanium, silicon nitride, zeolite, And an organic polymer compound. When the fixing element is a specified region of the substrate or an inner wall of a concave portion formed in the substrate, it can be used in combination with glass, ceramics, a polymer, or various porous materials. Examples of the polymer include polystyrene, poly (tetra) fluoroethylene (PTFE), polyvinylidene difluoride, polycarbonate, polymethyl methacrylate, polyvinyl ethylene, polyethylene imine, polyvinyl phenol, and polyhydroxyethyl methacrylate. HHEMA), polydimethylsiloxane, polyacrylamide, polyimide, and their block copolymers. The fixing device according to the present invention can be manufactured by the above-mentioned conventional method according to the purpose of use.

It is preferable that the porous immobilization element of the present invention has voids through which the solvent easily passes, and fine irregularities that increase the surface area of the particles or walls constituting the voids. Particularly preferred is a porous structure having several m, for example 0.1-10 m, preferably 0.3-through holes, which penetrate the solvent, and having a diameter of several nm on the surface of the structure. For example, it is a porous fixing element having pores with a diameter of 11 15 Onm, preferably 11 10 nm. The porosity of the porous fixed element is preferably 40 to 95%, and more preferably 50 to 90%, although it depends on the functional substance used and the properties of the thin film to be coated. surface area ί or lg per 50- 1 that by the structure, 000m ^2, particularly preferably a force of some in the 100- 800m ^2! /ヽ.

 [0021] The porous fixing element has a porous structure having voids of several zms permeating the solvent as shown in the photograph of FIG. 1, and when further enlarged, as shown in FIG. The surface of the structure has a very large number of depressions (nm pores) several nm in diameter. Therefore, the surface area of the porous immobilization element is extremely large, and the reaction with the functional substance can be performed efficiently by coating the surface with a thin film containing the functional substance.

 The porous immobilizing element comprises an organometallic compound having a hydrolyzable functional group, and a non-hydrolyzable organic functional group bonded through Z or at least one metal'carbon bond. And a polymer obtained by hydrolyzing and polymerizing an organometallic compound having a functional group of

[0022] The organometallic compound includes an organometallic compound containing a metal selected from the group consisting of silicon, titanium, zirconium, and aluminum, and an organometallic compound containing silicon is particularly preferable. In the present invention, these can be used alone or in combination. Examples of the organometallic compound having a hydrolyzable functional group include a metal alkoxide represented by the following general formula (1). An example of an organometallic compound containing a non-hydrolyzable organic functional group and a hydrolyzable functional group bonded via at least one metal carbon bond is represented by the general formula (2)-(4 )). These metal alkoxides can be used alone or in combination.

 [0023] [Formula 1]

[0024] [Formula 2]

[0025]

[0026] [化 4]

[0026] [Formula 4]

In these formulas, R—R represents a methyl group, an ethyl group, a propyl group, an aryl group,

 14

 It represents a acryl group, an aryl methacryl group, an alkyl sulfol group, an aryl sulfol group, an aryl amino group, an alkyl amino group, or the like.

 The porous immobilization element of the present invention is preferably a polymer of tetramethoxysilane, a copolymer of tetramethoxysilane and methyltrimethoxysilane, or a polymer of tetraethoxysilane or 3-trimethoxysilylpropyl methacrylate. Preferred to make up. The porous fixing element can be manufactured as follows.

 [0028] The above-mentioned organometallic compound having a hydrolyzable functional group, or a non-hydrolyzable organic functional group and a hydrolyzable functional group bonded via at least one metal'carbon bond. (Hereinafter referred to as an organometallic compound). For the hydrolysis, acid catalysts such as hydrochloric acid, sulfuric acid, tosylic acid, and cation exchange resin, and basic catalysts such as ammonium, triethylamine, sodium hydroxide, sodium carbonate, sodium hydrogen carbonate, and anion exchange resin. Polymerization is carried out in water; alcohols such as methanol, ethanol, propanol and butanol; organic solvents such as acetonitrile, tetrahydrofuran and 1,4-dioxane; or a mixed solvent thereof. The reaction temperature is 0 to 120 ° C, preferably 0 to 80 ° C, and the reaction time is 1 minute to 200 hours, preferably 10 minutes to 24 hours.

Next, a solvent that causes phase separation in the course of gelation is mixed with a part of the hydrolysis solution. Any solvent that causes phase separation can be used without any particular limitation. Examples thereof include toluene, chloroform, and a polyethylene glycol solution. The mixture is polymerized by thermal polymerization or photopolymerization. At the time of photopolymerization, a radical generator, for example, Irgacure 1800 (Nippon Ciba Geigy Co., Ltd.) is coexisted. After the polymerization, the obtained reaction product is washed with an organic solvent such as water or methanol, and dried to obtain a porous fixed device. When forming the fixed element in a capillary or a column tube, a polymerization reaction is carried out in the capillary or the column tube. Further, the degree of polymerization in this case can be appropriately changed depending on the size of the porous fixing element.

 Finally, the solvent is vaporized to obtain a dried gel porous fixing element. The drying is preferably carried out at 30 to 80 ° C. for several hours to several tens of hours, and thereafter, it is preferably heated to about 200 to 800 ° C. to remove coexisting organic substances.

 The porous immobilization element of the present invention can be used by covering the entirety or part of a cavity or a column tube, or forming an array of microchips, and coating a functional substance. That is, the organic metal compound having a hydrolyzable functional group or the like is polymerized under appropriate conditions in the inside of the capillaries and the like, and has a porous structure having through holes of several meters that allow the solvent to pass through. In addition, a porous fixed element having pores with a diameter of several nm is formed on the surface of the structure, and then, the functional element is coated on the fixed element. Thus, biochemical or chemical reaction based on the functional substance can be performed, and the product can be easily separated, and the subsequent analysis or recovery process can be continuously performed.

 [0031] In the present invention, commercially available cavities for electrophoresis or gas chromatography can be used without any particular limitation. The dimensions of the capillaries are preferably 1/200 / 内径 πι, particularly 50-100 m, and the length is preferably 1 cm-10 m, particularly 10 cm-lm. If the length force is less than 1 cm, sufficient separation is not performed, and if it exceeds 10 m, it takes a long time for separation, which is not preferable. The column tube may be made of glass, stainless steel, plastic, or fluorine resin (for example, Teflon (registered trademark)) without any particular limitation.

Further, the inner wall surface of the concave portion such as a cylindrical shape formed on the substrate, which is provided with a flat or porous portion, that is, the inner wall of the assemble plate can be used as a fixing element for the thin film. The assay plate contains 6, 12, 24, 48, 96, 386, or 1536 pills formed on one plate, and the inner wall of these pills is used as an immobilization element. Can be. If necessary, the above-mentioned organometallic compound having a hydrolyzable functional group is polymerized under appropriate conditions on these inner wall surfaces, or separately prepared. The particles of the formed porous fixing element can be fixed and used as the porous fixing element.

 [0032] (Functional substance)

 As used herein, the term “functional substance” refers to a substance that causes or catalyzes a chemical, biochemical, or biological reaction, and includes not only physiologically active substances such as enzymes, hormones, and antibodies, but also , Containing a wide range of active substances. As long as it can be used by being fixed to the thin film having a network structure of the present invention, it can be used as the functional substance without any particular limitation.

 Examples of such functional substances include metabolic enzymes such as P450 and dalcuronyltransferase; proteins such as protein hydrolases such as trypsin and pepsin; nucleic acids, fats, sugars, physiologically active properties such as drugs, and metal catalysts. Synthetic functional materials, such as, fullerene, carbon nanotubes, polymorphic taxanes, catenane, and other specific shaped compounds. Examples of the drug include aspirin, morphine, tetrodotoxin and the like. In the present invention, various metabolic enzymes, which are particularly unstable and easily deactivated, can be used by immobilizing them on a thin film in a form included in a micronome.

 (Formation of Thin Film with Network Structure)

 First, a sol solution of an organometallic compound containing a functional substance is prepared. Hydrolyzing an organometallic compound having a hydrolyzable functional group, or an organometallic compound containing a non-hydrolyzable organic functional group and a hydrolyzable functional group bonded via at least one metal 'carbon bond Decompose.

 For the hydrolysis, acid catalysts such as hydrochloric acid, sulfuric acid, tosylic acid, and cation exchange resin, and basic catalysts such as ammonium, triethylamine, sodium hydroxide, sodium carbonate, sodium hydrogen carbonate, and anion exchange resin. Water; alcohols such as methanol, ethanol, propanol and butanol; organic solvents such as acetonitrile, tetrahydrofuran and 1,4-dioxane; or a mixed solvent thereof. The molar ratio between the organometallic compound and the solvent varies depending on the organometallic compound used, the solvent, and the like, but is preferably 3: 1 to 1:10, particularly preferably 1: 1 to 1: 4. The reaction temperature is 0 to 120 ° C, preferably 0 to 80 ° C, and the reaction time is 1 minute to 200 hours, preferably 10 minutes to 24 hours.

Next, the hydrolysis solution is mixed with a solution containing a functional substance. The mixture It is called a sol solution. When the functional substance is a biological substance such as an enzyme, it is preferably dissolved in a buffer solution. By appropriately optimizing the concentration of the functional substance, the pH of the solution, and the salt concentration in the case of a buffer, the gelation rate, the microstructure of the gel, and the like can be changed.

 Further, as shown in FIG. 8, the gel has a chain structure with large voids when the pH is on the acidic side, and has a network with many branches when the pH is on the basic side.

[0035] For example, when the functional substance force proteins force becomes physiologically active substance, the concentration in solution 10- ⁵ - IMolZl (mole Z l), preferably 10 ⁴ - and 10-MolZl, pH of the solution It is preferred to be 2-10, especially 418, and the salt concentration is preferably 0.001-1-1, particularly 0.01-0.2MolZl.

 Subsequently, before the sol solution gelates, the porous immobilization element is immersed in the sol solution, the excess of the porous immobilization element force is also removed, and the porous immobilization element is removed by drying. A thin film containing a functional substance can be applied on the surface of the quality fixing device. The higher the drying temperature, the faster the drying proceeds. However, when the functional substance is a physiologically active substance, it is 0 to 30 ° C, preferably 0 to 20 ° C to prevent denaturation or decrease in activity. ° C, more preferably 0 ° C-10 ° C.

[0036] The porous fixed element coated with a thin film having a network structure containing a functional substance can be taken out of the assay plate and reused for each measurement. Measurement and regeneration can be repeated.

 The sol-gel reaction at the time of forming the thin film will be described based on a polymerization reaction of alkoxysilane. As shown in FIG. 4, a desired gel is obtained by hydrolyzing a predetermined alkoxysilane and polycondensing a produced monomer. As shown in FIG. 5, the functional substance is fixed to the thin-film network structure in the process in which the monomer is polymerized to form a network structure.

[0037] When this is enlarged and drawn, as shown in Fig. 3, the surface of the depression (nm pore) of the porous immobilization element is covered with a thin film of 110, 10 nm and a thickness of OOOnm. As shown in FIG. 3A, the thin film captures and fixes the functional substance in a network structure having voids of 0.1 to 50 nm. In addition, the microsomes used in the present invention can purify the power of rat liver and the like by a conventional method. In addition, a micronome in which P450 and DARC-transferase are introduced is commercially available (Daiichi Pure Chemicals).

(Embodiment of Use of Fixed Device of the Present Invention)

 When using an attachment plate with a hole

 A porous fixing element coated with a thin film of a functional substance is put in or fixed in a well of an atsie plate, and a biochemical or chemical reaction is performed by the functional substance. In addition, a functional substance can be coated with a thin film using the inner wall of the well as a fixing element.

 As the assay plate, a plate having 6, 12, 24, 48, 96, 386, or 1536 beads on a single plate is commercially available. Is changed appropriately to make the size suitable for these wells. After performing a predetermined reaction, the activity can be measured using a plate reader or the like.

 Example

(Example 1)

 Production and performance test of pepsin immobilized Eich column

 On one end, a fused silica cavity with a 5 cm long porous immobilization element coated with a thin film containing pepsin (Wako Pure Chemicals) (75 μm id, lm length, Polymicro Technologies INC.).

The pepsin-fixed column used in this example was manufactured by the following procedure. To 750 μl of 3-trimethoxysilylpropyl methacrylate (MTMSP) (Tokyo Kasei), 225 μl of water and 22.5 μl of 1M HC1 were added, and the mixture was stirred at room temperature for 30 minutes. After adding 170 μl of toluene to 30 μl of the solution and stirring again for 30 minutes, 8.9 mg of Irgacure 18OO (Irgacurel 800 (Nippon Chino, Geigy One)) was added, and the mixture was further stirred for 2.5 hours or more. Filling the solution into a capillary with an inner diameter of 75 μm and a length of 40 cm, which is light-shielded with a polyimide coating except for the part where the fixed ridge element is to be made (one end of the capillary is about 5 cm), and performs UV irradiation for about 20 minutes, A fixed polymer element was prepared in a capillary by a photopolymerization reaction. For the UV irradiation, use a RPR-100 photochemical reactor (Ultraviolet Company, Branford, CT, U, A). After removing the unreacted solution with methanol, the mixture was allowed to stand in an oven at 110 ° C. and dried. [0040] Tetramethoxysilane (TMOS) (Tokyo Kasei Co., Ltd.) was added with 169 µl of water and 11 µl of 0.01% HC1 to 761 µl, and the mixture was stirred at room temperature for 20 minutes. After mixing 150 μl of 150 mM phosphate buffer (pH 5.5) 601 containing 25% (w / v) pepsin in 20 μl of the solution, the sol solution was immersed so that only the immobilizing element was immersed. It was introduced into the capillary with a syringe. After leaving at 4 ° C for 5 minutes, the mixed solution was expelled from the capillary force and allowed to stand at 4 ° C for 2 days or more to prepare a thin film containing pepsin on the immobilized element.

The electrophoresis apparatus ^{3 D} CE system manufactured by Agilent Technologies, the mass spectrometer using MSD mass spectrometer. In addition, a 500 mM formic acid solution (Wako Pure Chemical Industries) was used as the electrophoresis solution. A 15 kV potential difference was applied to both ends of the capillary at the time of analysis, and the liquid was sent at a pressure of 50 mbar from one end of the porous fixed element. went. The sample contains insulin chains (

 Sigma-Aldrich) lysozyme (Sigma-Aldrich) was used to inject a voltage of 2 OkV across the ends of the capillary for 20 seconds.

The injected sample was digested by a porous immobilizing element coated with a thin film containing pepsin, and the generated peptide fragments were separated by electrophoresis and directly introduced into a mass spectrometer for detection. The detected peptide fragment had undergone an enzyme-specific cleavage that would occur when the sample was digested with pepsin. The protein of the sample was identified by using Protein Prospector 4.0.5 (the University of California, San Francisco, http://prospector.ucsf.edu) which is a protein identification software using the generated peptide fragment. The activity of pepsin was maintained even when the detection step was performed 30 times in succession and analyzed.

(Example 2)

 Manufacture and use of porous immobilization device coated with a thin film containing trypsin

 Trypsin to fit the size of a 96-well Atsushi plate (Millipore)

 (Sigma-Aldrich) An immobilized element was prepared and used for the experiment.

The trypsin thin-film fixed device used in this example was manufactured by the following procedure. 2.5 ml of polyethylene glycol dissolved in 1 ml of tetramethoxysilane (TMOS) (Tokyo Kasei) was added with 2.5 ml of 0.01M acetic acid and stirred at 0 ° C for 45 minutes. The solution was left standing at 40 ° C for 1 晚 to carry out the polymerization reaction, washed with water, molded into the desired shape, and treated with 0.01M ammonia water at 120 ° C for 3 hours Thereafter, it was washed again with water and dried at 40 ° C. for 3 days. The polymer was heated at 600 ° C. for 2.5 hours to produce a porous fixed device.

 To 761 μl of tetramethoxysilane (TMOS) (Tokyo Kasei), 169 μl of water and 11 μl of 0.01% of HC1 were added, and the mixture was stirred at room temperature for 20 minutes. 20 μl of the solution was mixed with 5 OmM Tris-HCl buffer (pH 7.0) 1201 containing 5% (w / v) trypsin in 120 μl, followed by mixing the sol solution with the porous immobilization element for 5 minutes. The porous immobilized device was immersed, taken out, and allowed to stand at 4 ° C. for 2-3 days to form a trypsin-containing thin film on the surface of the porous immobilized device.

Measuring the activity of trypsin which is fixed to the fixed I匕素Ko is multilabel counter 1420 with ARVOsx (manufactured by Wallac), in a sample New ^alpha - Benzoiruarugi - down p-- trois - Lido (N - benzoy Bok arginine p- nitroaniiide (BAPNA) (Sigma)) and zocasein (azocasein, Sigma) were used. lmg / μ1 ΒΑΡΝΑ30 μ1 and 62.5 mM Tris / HCl buffer (pH 7.5) 1201 The enzyme reaction was performed by stirring at C for 15 minutes. After removing the porous immobilizing element, the enzyme activity was measured by measuring the change in absorption intensity (405 nm). In an experiment using azocasein, the activity was measured by a change in the absorption intensity at 355 nm using the same method.

 As shown in FIG. 6, even if the activity was measured 20 times or more, the activity of trypsin did not decrease, and the required activity was maintained even after 50 consecutive analyzes. When stored at 4 ° C, the activity of trypsin remained unchanged for more than 3 months.

 (Example 3)

 Production of porous immobilization device coated with a thin film containing microsomes containing P450 and examples of its use

First, a fixing device was manufactured in the same procedure as in Example 2. Next, 171 μl of water and 11 μl of 0.01% of HC1 were added to 761 μl of tetramethoxysilane (TMOS) (Tokyo Danisei), and the mixture was stirred at room temperature for 20 minutes. A microsomal (Daiichi Pure Chemicals) solution containing Ρ450 in 20 μl of the solution (protein concentration: 2.75 mgZml) 100 1 was added and stirred, and the porous immobilizing device was immersed in the mixed solution. After drying for 2 days or more, a P450-containing thin film was formed on the surface of the porous fixed device. The porous immobilization element has a through hole of 0.3 to 6 μm through which a solvent can pass, and further has a pore size of 110 nm to increase the surface area. It was expected to increase.

 On the other hand, the coating of the micronome involves the hydrolysis and polymerization of the alkoxysilane, the microsomes are included in the cross-linked structure under mild conditions, and the method is adapted to a high-throughput screening system using a means for immobilization. For the reaction, a 96-well microplate was used, and a porous immobilization element was manufactured so as to have a size that would fit in a well. A porous fixing element coated with a thin film containing P450 and a fluorescent substrate ethoxyresorufin solution were placed in a well of a 96-well microplate and reacted at 37 ° C. The reaction occurs as shown in the following chemical formula (5).

[0047]

[0048] P450 metabolic activity, remove the porous immobilization element, the generation amount of Rezorufuin raw form by using the plate reader was determined by fluorescence measurement (Ex.550nm, Em.590 nm) ₀ After the measurement By washing and drying the porous immobilizing element with a 20 mM phosphate buffer (pH 7.4), measurement could be repeated four times for four days.

 Until now, P450 was easily inactivated and could not be used in a fixed manner, and P450 power S had to be separated from P450 in order to prevent the measurement of S metabolic activity. On the other hand, by preparing and using a porous immobilizing element coated with a thin film containing P450 in the present invention, P450 can be used repeatedly, and it is necessary to separate metabolites to be measured from P450. Disappeared, and analysis became possible with a small amount of sample and reagent. That is, according to the present invention, analysis using P450 can be performed economically and in a short time.

(Example 4) Example in which a thin film containing microsomes containing P450 is formed directly on the bottom of a well of a 96-well microplate and oxidized.

 First, 11 μl of 0.04% HC1 and water 1691 were added to 761 μl of tetramethoxysilane (TMOS) (Tokyo Kasei) and stirred at room temperature for 20 minutes. After mixing and mixing 100 / ζ1 of a microsomal solution containing Ρ450 (protein concentration 2.75π ^ ιπι1) in the solution 201, each well of a 96-well microplate, the surface of which has been previously hydrophilically treated with polyvinyl acetate, was mixed. 10 μl each, and allowed to stand at 4 ° C. for 2 days or more to allow gelling to proceed, and a thin film containing P450 was fixed on the inner wall of the well.

 Using the immobilized P450, the metabolic reaction of the drug was measured in the same manner as in Example 3. FIG. 7 is a graph showing the relationship between the elapsed time and the amount of resolufin generated. Also in the case of the P450 fixed device manufactured by this method, measurement was possible repeatedly 13 times for 6 days.

Claims

The scope of the claims  [I] A fixed Ech element coated with a network-like thin film containing a functional substance.  [2] The coated stationary device according to claim 1, wherein the stationary device is a porous stationary device. [3] The immobilizing element has a porous structure having a through hole of 0.1 to 10 m that is permeable to a solvent, and a porous immobilizing element having pores of 11 lOO nm in diameter on the surface of the structure. Element The coated immobilization element of claim 1. [4] The coated immobilization element according to claim 3, wherein the immobilization element has a porous structure having a through-hole of 0.3 to 6 μm that is permeable to a solvent. [5] The coated immobilizer according to claim 1, wherein the functional substance is at least one selected from the group consisting of proteins, nucleic acids, lipids, sugars, metal catalysts, and compounds having a specific shape. Child.  [6] The functional substance is P450, darc-transferase, trypsin, pepsin, DNA, RNA, peptide nucleic acid, lipid, sugar, metal catalyst, fullerene, carbon nanotube, poly-mouth taxane, catenane. The coated fixed element according to claim 1, which is at least one selected from the group.  [7] The coated immobilized element according to claim 1, wherein the thin film having a network structure has an organometallic compound as a main component.  [8] An immobilization device coated with a network-like thin film containing microsomes containing a functional substance.  [9] The coated fixing device according to claim 8, wherein the fixing device is a porous fixing device.  [10] The immobilization element has a porous structure having a through hole of 0.1 to 10 m that is permeable to a solvent, and a porous immobilization film having pores of 11 lOO nm in diameter on the surface of the structure. The coated immobilized element according to claim 8, which is an element.  [II] The coated immobilization element according to claim 10, wherein the immobilization element has a porous structure having a through hole of 0.3 to 6 μm that is permeable to a solvent.  12. The coated immobilized element according to claim 8, wherein the functional substance is a metabolic enzyme. [13] The method according to claim 8, wherein the functional substance is P450 or dalcuronyltransferase. The coated fixed dangling element.  14. The coated immobilized element according to claim 8, wherein the thin film having a network structure has an organometallic compound as a main component.  [15] A capillaries in which a porous immobilization element coated with a thin film having a network structure containing a functional substance or a thin film having a network structure containing microsomes containing a functional substance is formed. Column tube or microchip.  [16] A method of coating a network-like thin film containing a functional substance on an immobilization element, comprising: an organometallic compound having a hydrolyzable functional group, or at least one metal'carbon bond Hydrolyzing an organometallic compound containing a bonded non-hydrolyzable organic functional group and a hydrolyzable functional group; mixing the hydrolyzed solution with a solution containing a functional substance to form a sol solution And immersing the immobilized element in the sol solution and drying to form a thin film on the surface of the immobilized element.  17. The method for coating a thin film according to claim 16, wherein the drying temperature is 0 ° C. to 120 ° C.  [18] A porous immobilizing element having a porous structure having a through hole of 0.1 to 10 m that is permeable to a solvent, and having pores having a diameter of 11 lOO nm on the surface of the structure. [19] The porous immobilization element according to claim 18, having a porous structure having a through-hole of 0.3 to 6 µm, which is permeable to a solvent. [20] A method for manufacturing a porous fixed element according to claim 18 or 19, wherein:  Hydrolyzes organometallic compounds having a hydrolyzable functional group, or an organometallic compound containing a non-hydrolyzable organic functional group and a hydrolyzable functional group bonded via at least one metal 'carbon bond The method comprising: mixing the hydrolysis solution with a solvent that causes phase separation in the course of gelation; and polymerizing the mixture by thermal polymerization or photopolymerization. 21. The production method according to claim 20, wherein a reaction temperature of the hydrolysis is 0 to 80 ° C., and a reaction time is 10 minutes to 24 hours. [22] The solvent which causes phase separation to be added to the hydrolysis solution is at least one selected from the group consisting of toluene, chloroform, and polyethylene glycol solution. Hf The production method according to claim 20.