JP4505581B2 - Substance immobilization method - Google Patents

Description

  The present invention relates to a substance immobilization method for immobilizing a desired substance such as a polypeptide, nucleic acid, lipid or the like on a substrate.

  In recent years, immunoplates for immunoassay in which antibodies or antigens are immobilized on plates, DNA chips in which nucleic acids are immobilized on chips, and the like are widely used. One method for immobilizing proteins and nucleic acids on a substrate is physical adsorption. For example, by leaving a hydrophobic substrate such as polystyrene in contact with an aqueous solution of protein or nucleic acid to be immobilized on the substrate, the protein or nucleic acid can be immobilized on the substrate by physical adsorption.

  However, the method using physical adsorption has a problem that the bond between the substrate and the immobilized substance is weak, and the stability of the substrate on which the substance is immobilized is insufficient. In addition, in order to prevent non-specific adsorption to regions not covered with the target protein or nucleic acid, proteins such as bovine serum albumin (BSA), casein, skim milk, etc. (when proteins are immobilized), salmon sperm DNA Blocking with DNA such as DNA (when DNA is immobilized), antibody or antigen that does not react immunologically (when antibody or antigen is immobilized) is performed (see Patent Document 1). However, the effect of preventing non-specific adsorption by such blocking is not always satisfactory, and there has been a problem of reduction in detection sensitivity due to non-specific adsorption.

  In addition, proteins and nucleic acids are also immobilized on a substrate by covalently bonding a functional group on the substrate and a functional group of a protein or nucleic acid to be immobilized (see Patent Document 2). However, in this method, when the functional group used is in or near the active site of the substance, the activity of the substance is lost due to immobilization. In addition, when there is no appropriate functional group, it cannot be immobilized by this method. Furthermore, as in the case of physical adsorption, non-specific adsorption is also prevented by blocking, but the prevention effect is not always satisfactory.

  On the other hand, it has also been proposed to immobilize a substance on a substrate using a photoreactive group (see Non-Patent Document 1). However, this method still has the problem of nonspecific adsorption. Therefore, a means for immobilizing a desired substance on a substrate and detecting it with high sensitivity while preventing non-specific adsorption has been demanded.

JP 11-337551 A JP 2001-337089 A Y. Ito and M.M. Nogawa, “Preparation of a protein micro-array using a photo-reactive polymer for a cell adhesion assay,” Biomaterials, 24, 3021-3026 (2003).

  An object of the present invention is to provide a substance immobilization method for detecting a substance immobilized on a substrate and / or a substance specifically reacting with an immobilized substance with high sensitivity.

Means for achieving the above object are as follows.
[1] A method for immobilizing a substance to be immobilized on a substrate,
A coating liquid containing a polymer having an effect of preventing non-specific adsorption of a substance to be immobilized and / or a substance specifically reacting with the substance to be immobilized on the surface of the substrate is coated on the substrate, and at least one of the surfaces of the substrate is coated. Forming a polymer layer on the part, then
A substance immobilization method comprising applying a mixture of a substance to be immobilized and a photocrosslinking agent having at least two photoreactive groups in one molecule on a substrate having the polymer layer, and irradiating with light.
[2] The substance immobilization method according to [1], wherein the coating liquid contains a photocrosslinking agent having at least two photoreactive groups in one molecule.
[3] The substance immobilization method according to [1] or [2], wherein the polymer has a photoreactive group and / or a group capable of covalent bonding or coordination bonding with the substrate surface.
[4] The substance immobilization method according to any one of [1] to [3], wherein the photoreactive group is an azide group.
[5] The substance immobilization method according to any one of [1] to [4], wherein the polymer is an electrically neutral water-soluble polymer as a whole molecule.
[6] The substance immobilization method according to [5], wherein the polymer is an ambipolar or nonionic polymer.
[7] The substance immobilization method according to [6], wherein the nonionic polymer is a vinyl polymer of polyethylene glycol (meth) acrylate.
[8] The method according to any one of [1] to [7], wherein the substance to be immobilized is at least one selected from the group consisting of polypeptides, nucleic acids, lipids, cells, and components thereof.

  According to the present invention, a desired substance can be immobilized on a substrate while suppressing nonspecific adsorption. According to the substrate on which the substance is immobilized by the method of the present invention, the substance to be immobilized and / or the substance that specifically reacts with the substance to be immobilized can be detected with high sensitivity.

Hereinafter, the present invention will be described in more detail.

The substance immobilization method of the present invention comprises:
A method for immobilizing a substance to be immobilized on a substrate,
A coating liquid containing a polymer having an effect of preventing non-specific adsorption of a substance to be immobilized and / or a substance specifically reacting with the substance to be immobilized on the surface of the substrate is coated on the substrate, and at least one of the surfaces of the substrate is coated. A polymer layer is formed on the part (hereinafter referred to as “first step”), and then
On the substrate having the polymer layer, a mixture of a substance to be immobilized and a photocrosslinking agent having at least two photoreactive groups in one molecule is applied and irradiated with light (hereinafter referred to as “second step”). A substance immobilization method.
It is.

First Step In the first step, a coating solution containing a polymer having a nonspecific adsorption preventing effect is applied onto a substrate, and a polymer layer is formed on at least a part of the substrate surface.
The polymer used in the first step has an effect of preventing nonspecific adsorption to the substrate surface of the substance to be immobilized and / or the substance specifically reacting with the substance to be immobilized. Here, “non-specific adsorption” means that the substance to be immobilized and / or the substance that specifically reacts with the substance to be immobilized is adsorbed to a region where the substance to be immobilized is not immobilized on the substrate surface. Say. The substance that specifically reacts with the substance to be immobilized can be, for example, an antibody when the substance to be immobilized is an antigen, and an antigen when the substance to be immobilized is an antibody. By immobilizing a substance on the substrate via such a polymer layer, nonspecific adsorption can be prevented and detection sensitivity can be increased. Furthermore, according to the method of the present invention, since the amount of the immobilized substance exposed on the outermost layer is large compared to the method of mixing the polymer, the immobilized substance and the photocrosslinking agent and applying the mixture onto the substrate, the S / N ratio is increased. In addition, a substance-immobilized substrate having excellent detection sensitivity can be obtained.

  Examples of the polymer having a non-specific adsorption preventing effect include water-soluble polymers that are electrically neutral as a whole molecule. “Electrically neutral as a whole molecule” means that it does not have a group that becomes ionized by ionization in an aqueous solution having a pH near neutrality (for example, pH 6 to 8), or even if it has a group that becomes a cation. Means that the total charge in one polymer molecule is substantially zero. In the present invention, the term “substantially” for the charge means that the total charge is 0 or small enough not to adversely affect the effect of the present invention even if it does not become 0.

  The polymer is preferably water-soluble. Here, “water-soluble” means, for example, that the solubility of the polymer in water (the number of grams dissolved in 100 g of water) is 5 or more. If a water-insoluble polymer is used, a non-aqueous solvent other than water or alcohol must be used. However, the non-aqueous solvent may denature the substance to be immobilized. Therefore, in the present invention, it is preferable to use a water-soluble polymer in order to prevent denaturation of the substance to be immobilized. Furthermore, the water-soluble polymer has an advantage of being excellent in non-specific adsorption suppressing effect.

  A water soluble polymer that is electrically neutral as a whole molecule can be a bipolar or nonionic polymer. Here, the term “bipolar” refers to a group that becomes a cation and a group that becomes a cation by ionization in an aqueous solution having a pH close to neutrality (for example, pH 6 to 8). Means substantially zero. Moreover, "nonionic" means having substantially no group ionized by ionization in an aqueous solution having a pH near neutrality (for example, pH 6 to 8). Here, “substantially free of ionized ionized groups” means that such a group is not included at all, or even if such a group is included, such a small amount that does not adversely affect the effect of the present invention ( For example, it means that the number of such groups is 1% or less of the carbon number). Among these, the nonionic polymer has an excellent non-specific adsorption preventing effect and has an advantage that it can be produced or obtained at a low cost.

  The number average molecular weight of the polymer is not particularly limited and is usually about 3.5 to 5 million. However, if the molecular weight of the polymer is excessively large, cross-linking between the polymers increases, and the substance to be immobilized and the substance to be immobilized are immobilized. Since reaction with a substance (such as a photocrosslinking agent) used for reaction with the substance may not easily occur, about 500 to several hundred thousand is preferable.

  Specific examples of the nonionic polymer that can be used in the present invention include the following polymers. However, the present invention is not limited to the following specific examples: polyalkylene glycols such as polyethylene glycol (PEG) and polypropylene glycol; vinyl alcohol, methyl vinyl ether, vinyl pyrrolidone, vinyl oxazolidone, vinyl methyl oxazolidone, 2- Vinylpyridine, 4-vinylpyridine, N-vinylsuccinimide, N-vinylformamide, N-vinyl-N-methylformamide, N-vinylacetamide, N-vinyl-N-methylacetamide, 2-hydroxyethyl methacrylate, polyethylene glycol Methacrylate, polyethylene glycol acrylate, acrylamide, methacrylamide, N, N-dimethylacrylamide, N-iso-propylacrylamide, diacetone acrylamide, methylol acrylamide, acryloylmo Ruphorin, acryloylpyrrolidine, acryloylpiperidine, styrene, chloromethylstyrene, bromomethylstyrene, vinyl acetate, methyl methacrylate, butyl acrylate, methyl cyanoacrylate, ethyl cyanoacrylate, n-propyl cyanoacrylate, iso-propyl cyanoacrylate, n-butyl Of monomer units such as cyanoacrylate, iso-butyl cyanoacrylate, tert-butyl cyanoacrylate, glycidyl methacrylate, ethyl vinyl ether, n-propyl vinyl ether, iso-propyl vinyl ether, n-butyl vinyl ether, iso-butyl vinyl ether, tert-butyl vinyl ether Nonionic vinyl polymers, which are used alone or in combination; gelatin, casein, collagen, gum arabic, key Sun tang gum, tragacanth gum, guar gum, pullulan, pectin, sodium alginate, hyaluronic acid, chitosan, chitin derivatives, carrageenan, starch (carboxymethyl starch, aldehyde starch), dextrin, cyclodextrin and other natural polymers, methyl cellulose, viscose, hydroxy Natural polymers such as water-soluble cellulose derivatives such as ethyl cellulose, hydroxyethyl methyl cellulose, carboxymethyl cellulose, and hydroxypropyl cellulose. It is also possible to use a commercially available product of a photocrosslinking water-soluble polymer based on these polymers, for example, “AWP” manufactured by Toyo Gosei Co., Ltd. based on polyvinyl alcohol. Among these, particularly preferred are polyethylene glycol polymers, and further vinyl polymers of polyethylene glycol (meth) acrylate. In the present invention, “(meth) acrylate” means acrylate or methacrylate. “(Meth) acryl” means acrylic or methacrylic.

Furthermore, as the polymer, a phosphorylcholine-containing bipolar polymer, specifically, a phosphorylcholine-containing bipolar polymer containing a unit having a structure represented by the following general formula [I] can also be used.

(In the general formula [I], X represents a polymerizable atomic group in a polymerized state.)

  The unit having the structure represented by the general formula [I] may be hereinafter referred to as “unit [I]” for convenience.

  The phosphorylcholine contained in the general formula [I] is a constituent component of the biological membrane. Despite the fact that biological membranes come into contact with various substances, non-specific adsorption hardly occurs. This is considered to be due to the non-specific adsorption preventing effect of phosphorylcholine. In the present invention, nonspecific adsorption can be effectively prevented by using a phosphorylcholine-containing polymer as the polymer.

  The unit [I] is a unit containing a phosphorylcholine group and represents a polymerizable atomic group in a polymerized state. X is preferably a vinyl monomer residue. As the unit [I], those represented by the following general formula [I ′] are preferable.

(However, X ′ represents a methacryloxy group, a methacrylamide group, an acryloxy group, an acrylamide group, a styryloxy group, or a styrylamide group in a state where the vinyl moiety is addition-polymerized, and R ¹ is a single bond or a carbon number of 1 to 10 alkylene groups (which may be substituted with 1 or 2 hydroxyl groups).)

  Preferred examples of such units include 2-methacryloyloxyethyl phosphorylcholine, 2-acryloyloxyethyl phosphorylcholine, N- (2-methacrylamide) ethyl phosphorylcholine, 4-methacryloyloxybutylphosphorylcholine, 6-methacryloyloxyhexyl phosphorylcholine, 10 -Units derived from methacryloyloxydecylsylphosphorylcholine, ω-methacryloyldioxyethylene phosphorylcholine or 4-styryloxybutylphosphorylcholine (that is, these units are polymerized). Among these, a unit derived from 2-methacryloyloxyethyl phosphorylcholine is particularly preferable.

  The polymer containing the unit [I] may be composed only of the unit [I], or a copolymer of the unit [I] and another polymerizable monomer that does not adversely affect the effects of the present invention. It may be. As the other polymerizable monomer, a vinyl-based monomer is preferable, and (meth) acrylic acid or a salt or ester thereof (preferably a lower alkyl (eg, having 1 to 6 carbon atoms) ester) is particularly preferable. In the case of a copolymer, the ratio of the unit [I] to the other polymerizable monomer is not particularly limited, but is preferably 1: 0.3 or less, particularly preferably 1: 0.1 or less in terms of molar ratio.

The coating solution containing the polymer preferably contains a photocrosslinking agent having at least two photoreactive groups in one molecule. In the present invention, the “photoreactive group” means a group that generates a radical when irradiated with light.
The photocrosslinking agent can form a covalent bond with an amino group, a carboxyl group, a carbon atom constituting an organic compound, or the like when a photoreactive group generates a radical upon irradiation with light. Thereby, the substrate and the polymer can be bonded via the photocrosslinking agent by irradiating light after applying the coating liquid containing the photocrosslinking agent on the substrate, and the nonspecific adsorption preventing effect is exerted on the substrate. A polymer layer can be formed. In the present invention, a photoreactive group and / or a group capable of covalent bond or coordinate bond with the substrate surface is introduced into the polymer without using the photocrosslinker or together with the photocrosslinker. Thus, it is possible to bond the substrate and the polymer by utilizing the group of the polymer.

When a photoreactive group is introduced into the polymer, the amount of introduction may be one or more per polymer molecule, and preferably two or more. However, if it is too much, non-specific adsorption may increase, so the amount introduced is preferably 10% or less of the number of carbons constituting the polymer (excluding side chain carbon), preferably 5% or less. More preferably it is. Preferred examples of the photoreactive group include, but are not limited to, an azide group (—N ₃ ). Specific examples of the photoreactive group that can be introduced into the polymer include a phenyl azide group, an acetyl group, and a benzoyl group, and a phenyl azide group is particularly preferable. A photoreactive group such as an azide group may be directly bonded to the polymer, but may be introduced into the polymer via an arbitrary spacer structure, and the latter is usually preferred because it is easier to produce. In the latter case, the spacer structure is not limited in any way. For example, an alkylene group having 1 to 10 carbon atoms (which may be substituted with one or two hydroxyl groups), a phenylene group (however, 1 To 3 alkyl groups having 1 to 4 carbon atoms or a hydroxyl group, which may be substituted).

  Introduction of the photoreactive group into the polymer can be easily performed based on a conventional method. For example, a polymer having a functional group and a diazide compound having a functional group that reacts with the functional group can be reacted to bond the azide group to the polymer. When polyethylene glycol, which is a preferred polymer, is used, polyethylene glycol having an amino group or a carboxyl group at both ends is commercially available. Thus, such a commercially available functional group-containing polyethylene glycol is reacted with an azide group-containing polymer. Can be introduced. In addition, in the case of a polymer formed by polymerization of a monomer such as a vinyl polymer, a light-reactive vinyl monomer is copolymerized with a vinyl monomer which is a main constituent unit of the vinyl polymer, thereby producing light. Reactive group-containing polymers can be produced. Specific examples of the photoreactive group-containing vinyl polymer obtained by this method include poly ((meth) acrylamide-photoreactive (meth) acrylic acid amide) copolymer, poly (glycidyl (meth) acrylate) -photoreaction. (Meth) acrylamide copolymer, (polyethylene glycol mono (meth) acrylate-photoreactive acrylic amide) copolymer, etc., and in particular, (polyethylene glycol mono (meth) acrylate-photoreactive acrylic) Acid amide) copolymers are preferred.

  In order to introduce a group capable of covalent bonding or coordination bonding with the substrate surface, the polymer preferably has a free carboxyl group. A free carboxyl group can be easily imparted by, for example, further copolymerizing a carboxyl group-containing vinyl monomer such as methacrylic acid or acrylic acid. In addition, it is preferable that content of a free carboxyl group shall be about 5 mol%-about 50 mol% on the basis of the total number of moles of all the units which comprise a polymer.

  When the substrate surface is made of gold, examples of the group that can be covalently bonded to the substrate surface include a thiol group. It is already known that a thiol group is covalently bonded to gold, and is described in, for example, Japan Nanonet Bulletin No. 58 (March 16, 2004).

  As a method for introducing a thiol group into the polymer, for example, a thiol group of a compound such as 2-mercaptoethylamine having both an amino group and a thiol group in one molecule is protected, and the amino group is released in the polymer. And a method of deprotecting the thiol group. The protection of the thiol group can be performed, for example, by converting a compound such as 2-mercaptoethylamine having an amino group and a thiol group in one molecule into a disulfide dimer. On the other hand, the deprotection can be performed by causing the disulfide dimer to act on a reducing agent that cleaves a disulfide bond such as dithiothreitol to cleave the disulfide bond to generate a free thiol. However, the method for introducing a thiol group is not limited to the above method, and the amount of thiol group introduced is preferably about 1 to 100 mol, particularly about 1 to 50 mol, per polymer molecule. The thiol group in the polymer is covalently bonded to gold simply by bringing the solution containing the polymer into contact with gold at room temperature.

  On the other hand, when the substrate surface is made of a metal, examples of the group capable of coordinating with the substrate surface include a carboxyl group and an amino group. As the carboxyl group, a free carboxyl group contained in the aforementioned polymer can be used as it is. An amino group can be easily introduced by bonding a diamine compound to the free carboxyl group using a crosslinking agent such as carbodiimide.

Examples of the photoreactive group possessed by the photocrosslinking agent include an azide group (—N ₃ ), an acetyl group, a benzoyl group, and a diazirine group. In particular, the azide group releases nitrogen molecules and generates nitrogen radicals when irradiated with light, and these nitrogen radicals bind not only to functional groups such as amino groups and carboxyl groups, but also to carbon atoms constituting organic compounds. It is preferable because it can form a covalent bond with most organic substances.

  The photocrosslinking agent is preferably water-soluble. “Water-soluble” for a photocrosslinking agent means that an aqueous solution having a concentration of 0.5 mM or more, preferably 2 mM or more can be provided.

  The photocrosslinking agent contains at least two photoreactive groups in one molecule. The number of photoreactive groups contained in one molecule of the crosslinking agent is, for example, 2 to 4, preferably 2 to 3. As the photocrosslinking agent, a diazide compound having two azide groups is preferable, and a water-soluble diazide compound is particularly preferable. Preferable examples of the photocrosslinking agent used in the present invention include diazide compounds represented by the following general formula [II].

In general formula [II], R represents a single bond or an arbitrary group. -R- is a structure for linking two phenyl azide groups, and is not particularly limited as long as the diazide compound has necessary water solubility. Examples of preferred —R— include a single bond (that is, two phenyl azide groups are directly linked), an alkylene group having 1 to 6 carbon atoms (including one or two carbon-carbon unsaturated bonds). 1 or 2 carbon atoms may be bonded to oxygen to form a carbonyl group (particularly preferably a methylene group), —O—, —SO ₂ —, —S—S. -, -S-, -R ² ... Y ... R ^3- (wherein ... represents a single bond or a double bond, Y represents a cycloalkylene group having 3 to 8 carbon atoms, R ² and R ³ is independently an alkylene group having 1 to 6 carbon atoms (which may contain one or two carbon-carbon unsaturated bonds, and the bond between the carbon atom at the base of the alkylene group and Y is doubled. 1 or 2 carbon atoms may form a carbonyl group by double bonds with oxygen. The cycloalkylene group may be substituted with one or two or more arbitrary substituents (when substituted, preferably among the carbon atoms constituting the cycloalkylene group, One or two double bonds with oxygen to form a carbonyl group and / or substituted with one or two alkyl groups having 1 to 6 carbon atoms), and Each benzene ring in the general formula [II] is one or more arbitrary substituents (preferably a hydrophilic group such as halogen, an alkoxyl group having 1 to 4 carbon atoms, a sulfonic acid or a salt thereof). May be substituted. Specific examples of preferred -R- include the following. _{-, - CH 2 -, -} O -, - SO 2 -, - S-S -, - S -, - CH = CH -, - CH = CH-CO -, - CH = CH-CO-CH = CH -, -CH = CH-,

  Specific examples of preferred diazide compounds include the following.

  The polymer and the photocrosslinking agent are known per se, can be produced by a known production method, and some are commercially available.

  The coating solution can be prepared by dissolving the polymer or the polymer and a photocrosslinking agent, for example, in a solvent. As the solvent, water, lower alcohol (preferably ethanol) mixed with water at an arbitrary ratio, and a mixture thereof can be used. Among these, water is preferably used as the solvent. Furthermore, a silane coupling agent may be included when a glass substrate is used, or a thiol compound may be included when a gold substrate is used.

  The concentration of the polymer and the photocrosslinking agent in the coating solution is not particularly limited, but the concentration of the polymer is, for example, 0.0001 to 10% by mass, preferably 0.001 to 1% by mass, and the concentration of the photocrosslinking agent is, for example, , 1 to 20% by weight, preferably 2 to 10% by weight, based on the polymer.

  In the present invention, the coating solution is applied onto a substrate. The application method is not particularly limited. For example, a spotting method using a micropipette or the like, a spotting method using a pin method, or a spotting method using a piezoelectric method can be used. The film thickness can be, for example, 0.01 to 10 μm, preferably 0.05 to 1 μm.

As the substrate, a metal substrate, for example, a substrate whose surface is made of gold is used when the polymer and the substrate are bonded by a covalent bond or a coordinate bond by a thiol group or an amino group introduced into the polymer as described above. It is preferable to do.
Further, when the polymer layer and the substrate are bonded by the above-mentioned photoreactive group, it is not particularly limited as long as at least the surface thereof is made of a substance that can bond to the photoreactive group. Examples include those made of organic substances such as widely used polystyrene, acrylic resin, polyvinyl chloride, polyethylene terephthalate, polycarbonate, and polypropylene. A glass plate coated with a silane coupling agent can also be used, and a SPR or QCM substrate in which the gold surface is thiol-treated can also be preferably used. Further, the form of the substrate is not limited at all, and a plate-like one such as a microarray substrate, a bead-like shape, a fiber-like shape, or the like can be used. Furthermore, holes and grooves provided in the plate, for example, wells of a microplate can be used. Of these, the present invention is particularly suitable for microarrays, SPR and QCM.

In the second step, the coating solution containing the polymer is applied onto the substrate, preferably dried to form a polymer layer on at least a part of the substrate surface, and then on the substrate having the polymer layer. A mixture of a substance to be immobilized and a photocrosslinking agent having at least two photoreactive groups in one molecule is applied and irradiated with light. The mixture is preferably applied on the polymer layer on the substrate. By this light irradiation, radicalization of the photoreactive group contained in the photocrosslinking agent occurs, and the polymer layer and the substance to be immobilized can be bonded via the photocrosslinking agent, resulting in immobilization. The substance can be immobilized on the substrate. In addition, when the substrate and the polymer are bonded by the photoreactive group as described above, the coating liquid is applied in the first step, preferably light irradiation is performed after drying, and then the coating is performed in the second step. The mixture of the immobilizing substance and the cross-linking agent may be applied, or the light of the photo-crosslinking agent and the polymer contained in the coating liquid is not irradiated in the first step but is irradiated in the second step. Reactive groups may be radicalized. The details of the photocrosslinking agent used in the second step are as described above.

  The substance immobilized by the method of the present invention is not particularly limited, but includes polypeptides (including glycoproteins and lipoproteins), nucleic acids, lipids, and cells (animal cells, plant cells, microbial cells, etc.) and components thereof. (Including organelles such as nuclei and mitochondria, and membranes such as cell membranes and unit membranes). In the present invention, an azide group that can be used as a photoreactive group is a nitrogen radical that is released when irradiated with light and a nitrogen radical is generated. This nitrogen radical is not only a functional group such as an amino group or a carboxyl group, Since it is possible to bond to carbon atoms constituting the organic compound, most organic substances can be immobilized.

  The mixture of the substance to be immobilized and the photocrosslinking agent can be applied, for example, on a substrate having a polymer layer in a solution after being dissolved in a solvent. As the solvent, water, lower alcohol (preferably ethanol) mixed with water at an arbitrary ratio, and a mixture thereof can be used. Among these, water is preferably used as the solvent.

  The concentration of the substance to be immobilized and the photocrosslinking agent in the solution is not particularly limited, but the concentration of the substance to be immobilized is, for example, 0.01 to 10% by mass, preferably 0.05 to 1% by mass. The concentration of can be, for example, 0.1 to 20% by mass, preferably 1 to 10% by mass with respect to the substance to be immobilized.

The method for applying the mixture is not particularly limited, and the above-described application method and the like can be used. After application, preferably the applied solution is dried and then irradiated with light. The light may be any light as long as the photoreactive group to be used can generate radicals. In particular, when an azide group is used as the photoreactive group, ultraviolet light (for example, a wavelength of 300 to 400 nm) is preferable. The irradiation time can be, for example, 1 to 15 minutes. The dose of the irradiated light is not particularly limited, but is usually about 1 mW to 100 mW per 1 cm ² . The photoreactive group contained in the photocrosslinking agent (or the photoreactive group if the polymer has a photoreactive group) generates radicals by this light irradiation, and the substrate and the polymer layer, and the polymer layer and the immobilization are fixed. The chemical substance can be bound via a photocrosslinking agent. Moreover, light irradiation in the case of performing light irradiation after the first step and before the second step to bond the polymer layer and the substrate is also as described above. Thereby, a desired substance can be immobilized on the substrate via the polymer layer.

  In the present invention, microspotting may be used as a method for applying the above-described coating solution and mixture (solution). Micro spotting is a technique in which a liquid is applied to a very narrow area on a substrate. This method is commonly used for producing a DNA chip and the like, and an apparatus for that purpose is also commercially available, and can be easily performed using a commercially available apparatus. In the present invention, a coating solution containing a polymer or a polymer and a photocrosslinking agent is coated on the entire surface of the substrate, and a mixture (solution) containing the substance to be immobilized and the photocrosslinking agent is microspotted thereon, and then the whole surface of the substrate is coated. It may be irradiated with light. Further, the above-described coating solution may be microspotted, and a mixture (solution) of the substance to be immobilized and the photocrosslinking agent may be microspotted thereon, and then the entire surface of the substrate may be irradiated with light.

  In the present invention, it is also possible to selectively perform exposure through a photomask. In the case where a photomask is used, the photoreactive group does not bind to the substrate and the substance to be immobilized in a portion where the light is not irradiated. Therefore, the photocrosslinking agent and the substance to be immobilized are removed by washing. Therefore, by performing selective exposure through a photomask or the like, the substance can be fixed in an arbitrary pattern. Therefore, selective exposure can be very advantageous because the substance can be immobilized in any of various shapes such as a microarray.

In the present invention, it is preferable that after fixing a desired substance as described above, the substrate is washed by a known method to remove unreacted components and the like. In this way, a substrate on which a desired substance is immobilized can be obtained while suppressing nonspecific adsorption.
In the present invention, since the coupling reaction is performed using radicals generated by the photoreactive group, the photocrosslinking agent does not bind to a specific site of the substance to be immobilized, but binds to a random site. Therefore, there may be molecules that lose their activity due to the active site being bound, but there are many molecules that bind at sites that do not affect the active site, so the overall effect is small. it is conceivable that. According to the present invention, since a suitable substituent is conventionally located at or near the active site, even a substance that has been difficult to immobilize by a covalent bond is not lost as a whole. It can be immobilized on the substrate by covalent bonding.

Hereinafter, the present invention will be specifically described based on examples. However, the present invention is not limited to the following examples.

[Example 1]
1. Preparation of Water-Soluble Polymer 4.5 g of polyethylene glycol monomethacrylate (polyethylene glycol part molecular weight 350) was dissolved in 50 ml of ethyl acetate, and 14.03 mg of AIBN was added as an initiator, and reacted at 60 ° C. for 6 hours. After removing the solvent with an evaporator, the reaction solution was dissolved in water and subjected to ultrafiltration to remove unreacted monomers. Polymer formation was confirmed by gel filtration chromatography (GPC).

2. Preparation and Immobilization of Coating Solution Sodium 4,4′-diazidostilbene-2,2′-disulfonate (commercial product) was used as a photocrosslinking agent. It mixed with water so that the density | concentration of a photocrosslinking agent might be 0.0125 mass%, and the density | concentration of the said water-soluble polymer might be 0.25 mass%. The obtained coating solution was applied on an acrylic resin substrate in an area of 10 × 10 mm ² and dried (film thickness: about 0.2 μm). Next, 0.5 μL of an aqueous solution mixed so as to be 0.125% by mass of BSA and 0.00156% by mass of the photocrosslinking agent was spotted. After drying, BSA was immobilized by UV irradiation (black light (wavelength 300 to 400 nm) for 7 minutes).

3. Measurement After washing with PBS (0.1% by mass Tween 20 (registered trademark)), the mixture was reacted with anti-BSA rabbit IgG (commercially available) (100 ng / mL) at room temperature for 20 minutes. After washing with PBS (0.1% by mass Tween 20 (registered trademark)), it was reacted with a horseradish peroxidase (HRP) labeled secondary antibody (commercially available) for 20 minutes at room temperature. After washing with PBS (0.1% by mass Tween 20 (registered trademark)), a chemiluminescent reagent was added, and the luminescence intensity was measured.

4). Results The measured emission intensity is shown in Table 1 below. The lower part of Table 1 shows the result of spotting only BSA as a comparative example.

[Example 2]
1. Preparation of water-soluble polymer The same treatment as in Example 1 was performed.

2. Preparation and Immobilization of Coating Solution Sodium 4,4′-diazidostilbene-2,2′-disulfonate (commercial product) was used as a photocrosslinking agent. Mixing was performed so that the concentration of the photocrosslinking agent was 0.003125 mass% and the concentration of the water-soluble polymer was 0.0625 mass%. The obtained coating solution was treated with a metal core for SPR manufactured by Biacore, Inc. with mercaptoethanol, and then 20 μL was coated on an area of 7 × 7 mm ² and dried (film thickness: about 0.2 μm). Subsequently, 3 μL of an aqueous solution in which 0.125% by mass of BSA and a photocrosslinking agent were mixed so as to have the concentrations shown in Table 2 was spotted. After drying, BSA was immobilized by UV irradiation (black light for 7 minutes).

3. Measurement After washing with PBS (0.1% by mass Tween 20 (registered trademark)), SPR measurement was performed using anti-BSA rabbit IgG (commercially available) (20 μg / mL).

4). Results The measured results are shown in Table 2 below. The lower part of Table 2 shows, as a comparative example, the result of spotting only BSA and the result of spotting by mixing 1: 1 the coating solution and BSA used in Example 2.

  The method of the present invention can be suitably used for the production of an immunoassay plate on which an antibody or an antigen-binding fragment thereof or an antigen is immobilized, the production of a nucleic acid chip or a microarray on which DNA or RNA is immobilized on a substrate, etc. However, the present invention is not limited to these, and can also be applied to, for example, immobilization of whole cells or components thereof.

Claims (8)

A method for immobilizing a substance to be immobilized on a substrate,
A coating liquid containing a polymer having an effect of preventing non-specific adsorption of a substance to be immobilized and / or a substance specifically reacting with the substance to be immobilized on the surface of the substrate is coated on the substrate, and at least one of the surfaces of the substrate is coated. Forming a polymer layer on the part, then
A substance immobilization method comprising applying a mixture of a substance to be immobilized and a photocrosslinking agent having at least two photoreactive groups in one molecule on a substrate having the polymer layer, and irradiating with light. The material immobilization method according to claim 1, wherein the coating liquid contains a photocrosslinking agent having at least two photoreactive groups in one molecule. The material immobilization method according to claim 1, wherein the polymer has a photoreactive group and / or a group capable of covalent bonding or coordination bonding with the substrate surface. The method for immobilizing a substance according to claim 1, wherein the photoreactive group is an azide group. The substance immobilization method according to claim 1, wherein the polymer is a water-soluble polymer that is electrically neutral as a whole molecule. The material immobilization method according to claim 5, wherein the polymer is an ambipolar or nonionic polymer. The material immobilization method according to claim 6, wherein the nonionic polymer is a vinyl polymer of polyethylene glycol (meth) acrylate. The method according to claim 1, wherein the substance to be immobilized is at least one selected from the group consisting of a polypeptide, a nucleic acid, a lipid, a cell, and a component thereof.