JP2010101661A - Method for immobilizing substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for immobilizing a substance for improvement of sensitivity, in particular, higher sensitivity immobilizing method for a low molecular substance to be immobilized. <P>SOLUTION: A method for immobilizing a substance includes at least applying onto a substrate a non-specific adsorption inhibitor comprised of a water-soluble polymer which is electrically neutral as a whole molecular, and then spotting an immobilizing agent having at least a photo-reactive group, an amino group, and a room temperature reactive functional group in one molecule, and a substance to be immobilized. The prior photo-immobilizing method with a photo-crosslinking agent having at least two photo-reactive groups in one molecule performs random immobilization, and has a problem in sensitivity to, in particular, a low molecular substance to be immobilized. The method for immobilizing a substance is a photo-alignment immobilizing method, superior in characteristics with a higher sensitivity, and effective to, in particular, a low molecular substance to be immobilized. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for immobilizing a desired substance such as a protein, peptide, nucleic acid or the like.

  Conventionally, an immunoplate for immunoassay in which an antibody or antigen is immobilized on a plate, a DNA chip in which a nucleic acid is immobilized on a chip, and the like are widely used. Conventionally, physical adsorption has been widely used as one method for immobilizing proteins and nucleic acids on a substrate. That is, when a hydrophobic substrate such as polystyrene and an aqueous solution of protein or nucleic acid to be immobilized on the substrate are left in contact with each other, the protein or nucleic acid is 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 areas 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 Although blocking with DNA such as DNA (when immobilizing DNA) is performed, the effect of preventing nonspecific adsorption due to blocking is not always satisfactory.

  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. However, even in this method, as in the case of physical adsorption, since the functional group is present on the entire surface of the substrate, non-specific adsorption is prevented by blocking, but the prevention effect is not always satisfactory.

  On the other hand, it is also known that a substance is immobilized on a substrate using a photoreactive group (Patent Documents 1 to 3 and Non-Patent Document 1). However, in this method, no special consideration is given to prevent nonspecific adsorption.

Thereafter, various substances to be immobilized on the substrate can be immobilized by covalent bonds, and are excellent in the effect of preventing nonspecific adsorption, a substance immobilizing method using the same, and the substance immobilizing method Although the invention of the substance-immobilized substrate using the material has been made, further improvement in sensitivity is an eternal problem. In particular, increasing sensitivity in immobilizing peptides with small molecules is an important issue.
JP 11-337551 A JP 2001-337089 A Table 2005/111618 Y. Ito and M. Nogawa, "Preparation of a protein micro-array using a photo-reactive polymer for a cell adhesion assay," Biomaterials, 24, 3021-3026 (2003)

  Therefore, an object of the present invention is to provide a substance immobilization method for increasing sensitivity, in particular, a high-sensitivity immobilization method for a low-molecule immobilization product.

  As a result of diligent research, the inventors of the present application applied a nonspecific adsorption-preventing material consisting of an electrically neutral water-soluble polymer as a whole molecule on a substrate, and then at least a photoreactive group and an amino acid in one molecule. The present invention was completed by finding that the sensitivity can be improved by spotting a fixing agent having a functional group reactive with a group at room temperature and a substance to be immobilized.

  That is, in the present invention, a non-specific adsorption preventing material composed of an electrically neutral water-soluble polymer as a whole molecule is coated on a substrate, and then at least a photoreactive group, an amino group, and room temperature reactivity in one molecule. A material immobilization method comprising at least a fixing agent having a functional group and a substance to be immobilized is spotted. The conventional photofixation method using a photocrosslinking agent having at least two photoreactive groups in one molecule is random fixation, and the sensitivity is a problem particularly with respect to a low-molecule immobilization product. The substance immobilization method is a photo-alignment immobilization method, has high sensitivity and excellent characteristics, and is particularly effective for a low-molecule immobilization object.

  According to the present invention, a substance to be immobilized can be immobilized by covalent bonding, and a stable immobilized substrate can be obtained. Further, when the substance is immobilized using the method of the present invention, nonspecific adsorption is effectively prevented. Furthermore, a substance-immobilized substrate with high sensitivity and excellent S / N ratio can be obtained because of the orientation fixation.

  Hereinafter, the best mode for carrying out the present invention will be described in detail based on examples.

  The substrate is not particularly limited, and examples thereof include those made of organic substances such as polystyrene, acrylic resin, polyvinyl chloride, polyethylene terephthalate, polycarbonate, and polypropylene, which are widely used in microplates and the like. A glass plate or the like can also be used, and it is also preferably used for a substrate for SPR or QCM in which the gold surface is thiol-treated. 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.

  Examples of water-soluble polymers that can be used as non-specific adsorption inhibitors include ambipolar polymers such as phosphorylcholine-containing polymers and nonionic polymers. Nonionic polymers include polyalkylene glycols such as polyethylene glycol (PEG) and polypropylene glycol; vinyl alcohol, methyl vinyl ether, vinyl pyrrolidone, vinyl oxazolidone, vinyl methyl oxazolidone, 2-vinyl pyridine, 4-vinyl pyridine, N-vinyl. Succinimide, 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, diacetoneacrylamide, methylolacrylamide, acryloylmorpholine, acryloylpi Loridine, acryloylpiperidine, styrene, chloromethylstyrene, bromomethylstyrene, vinyl acetate, methyl methacrylate, butyl acrylate, methyl cyanoacrylate, ethyl cyanoacrylate, n-propyl cyanoacrylate, iso-propyl cyanoacrylate, n-butyl cyanoacrylate, Single or mixed monomer units such as 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-based polymers containing bismuth as a constituent; gelatin, casein, collagen, gum arabic, xanthan gum, tragacanth gum, gum Argam, pullulan, pectin, sodium alginate, hyaluronic acid, chitosan, chitin derivatives, carrageenan, starch (carboxymethyl starch, aldehyde starch), dextrin, cyclodextrin and other natural polymers, methylcellulose, viscose, hydroxyethylcellulose, hydroxyethyl Natural polymers such as water-soluble cellulose derivatives such as methyl cellulose, carboxymethyl cellulose, and hydroxypropyl cellulose can be exemplified, but are not limited thereto. 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.

  The water-soluble polymer used in the substance fixing agent of the present invention is a water-soluble polymer that is electrically neutral as a whole molecule. Here, “electrically neutral as a whole molecule” means that it does not have or does not have a group that becomes ionized by ionization in an aqueous solution having a pH near pH (pH 6 to 8). It has an ion and an anion, and it means that the total charge becomes substantially zero. Here, “substantially” means that the total charge becomes 0 or is small enough not to adversely affect the effect of the present invention even if it does not become 0.

  The water solubility of the water-soluble polymer used in the present invention (the number of grams dissolved in 100 g of water) is preferably 5 or more.

  “Nonionic” means having substantially no group that becomes ionized by ionization in an aqueous solution having a pH close to neutrality (pH 6 to 8). Here, “substantially” means that such a group is not contained at all, or even if such a group is contained, it is so small that the effect of the present invention is not adversely affected (for example, the number of such groups is the number of carbon atoms). 1% or less).

  The molecular weight (average molecular weight, the same applies hereinafter) of the nonionic water-soluble polymer is not particularly limited and is usually about 1000 to 50 million. However, if the molecular weight of the nonionic water-soluble polymer becomes too large, the water solubility is limited. Will arise. Moreover, since immobilization cannot be performed efficiently when the molecular weight is small, about 10,000 to 50 million is preferable. The concentration at the time of using the water-soluble polymer is not particularly limited.

  When used as a nonspecific adsorption preventing material, a photoreactive group may be introduced into the water-soluble polymer, but a photocrosslinking agent having at least two photoreactive groups in one molecule is used as the whole molecule. It is convenient and desirable to use it by adding it to an electrically neutral water-soluble polymer.

  The addition amount of the photocrosslinking agent having at least two photoreactive groups in one molecule is not particularly limited, but is preferably 0.1 to 50% by weight based on the water-soluble polymer, More preferably, it is 1-30 weight%, More preferably, it is 1-20 weight%.

Preferred examples of the photoreactive group possessed by the photocrosslinking agent include an azide group (—N 3), but are not limited thereto. 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 formulas [I] and [II].

上記式中のｎが１〜1000、好ましくは１〜500である。分子量が大きくなりすぎると光架橋効率が悪くなる。 In general formula [I], R represents a single bond or an arbitrary group. Since -R- has a structure that only connects two phenyl azide groups, it is not particularly limited as long as the diazide compound has the necessary water solubility (described later). 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-, -SO2-, -SS-,- S-, -R2 ... Y ... R3- (where ... indicates a single bond or double bond, Y is a cycloalkylene group having 3 to 8 carbon atoms, R2 and R3 are carbon atoms independently of each other. An alkylene group of 1 to 6 (which may contain one or two carbon-carbon unsaturated bonds, and the bond between the carbon atom at the base end of the alkylene group and Y may be a double bond) 1 or 2 carbon atoms may be bonded to oxygen to form a carbonyl group), and the cycloalkylene group is 1 or May be substituted with one or more arbitrary substituents (when substituted, one or two of the carbon atoms constituting the cycloalkylene group are preferably double-bonded to 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 [I] has one or It may be substituted with two 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). Specific examples of preferred —R— include the following. -CH2-, -O-, -SO2-, -SS-, -S-, -CH = CH-, -CH = CH-CO-, -CH = CH-CO-CH = CH-, -CH = CH -.

N in the above formula is 1-1000, preferably 1-500. If the molecular weight is too large, the photocrosslinking efficiency is deteriorated.

Specific examples of preferred diazide compounds include the following.

  Among the photocrosslinking agents used in the present invention, particularly preferred are water-soluble ones. “Water-soluble” for the 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 concentration at the time of using the photocrosslinking agent is not particularly limited.

  In addition, the nonspecific adsorption | suction prevention material which concerns on this invention is used including appropriate solvents, such as buffer solutions, such as water and phosphate buffered saline (PBS), in the case of the use. This is to facilitate application to the substrate. At this time, the concentration of the water-soluble polymer is not particularly limited, but is preferably 0.005% by weight to 20% by weight, and preferably 0.04% by weight to 10% by weight with respect to the entire solution (suspension). % Is more preferable. As described above, the concentration of the photocrosslinking agent in this case is preferably 0.1 to 50% by weight, more preferably 1 to 30% by weight, and still more preferably 1 to 20% by weight with respect to the water-soluble polymer. It is.

  Moreover, the substance fixing agent according to the present invention may contain a surfactant such as polyoxymonolaurate (Tween 20 (registered trademark)). In particular, by including both the buffer solution and the surfactant, an effect of reducing the background value is obtained as a synergistic effect, which is particularly useful when detecting using serum or the like. The addition amount of the surfactant is preferably 0.1 to 20% with respect to the entire solution (suspension). Furthermore, an organic solvent may be included. As this organic solvent, a lower alcohol (preferably ethanol) or the like mixed with water at an arbitrary ratio can be used.

  As the immobilizing agent having a photoreactive group, an amino group and a room temperature reactive group in one molecule used together with the substance to be immobilized, the following are preferable. That is, the photoreactive group is preferably an azido group or a diazirine group, and the room temperature reactive group is preferably an active ester such as an epoxy group or a succinimide ester. Specifically, N- [4- [3- (trifluoromethyl) -3H-diazirine-3-yl] phenyl] oxirane, N- [4- [3- (trifluoromethyl) -3H-diazirine-3 -yl] benzoxyl] succinimide, 5-azido-2-nitrobenzoic acid N-hydroxysuccinimide ester and the like are preferably used.

  When used in combination with a substance to be immobilized, the amount of the immobilizing agent having a photoreactive group, amino group, and room temperature reactive group in one molecule is not particularly limited. On the other hand, 0.001 to 100% by weight is preferable, and 0.01 to 10% by weight is particularly preferable. The concentration of the immobilizing agent having a photoreactive group, an amino group, and a room temperature reactive group in one molecule is not particularly limited.

  The immobilizing agent having a photoreactive group, an amino group, and a room temperature reactive group in one molecule according to the present invention is applied including a solvent. As the solvent, water, lower alcohol mixed with water at an arbitrary ratio, dimethylformamide (DMF), and a mixture thereof can be used.

  The photocrosslinking agent, the water-soluble polymer, and the immobilizing agent having a photoreactive group, an amino group, and a room temperature reactive group in one molecule are known per se. These can be produced by known production methods, and some are commercially available.

  The substance immobilized by the present invention is not particularly limited, and examples thereof include proteins having amino groups, peptides, and nucleic acids.

  The azide group or diazirine group used as the photoreactive group of the present invention is a nitrogen radical or a carbon radical that is released when irradiated with light, and a nitrogen radical or a carbon radical is generated. It is possible to bond not only to functional groups such as, but also to carbon atoms constituting organic compounds, so that it can crosslink with most organic substances. That is, the photocrosslinking agent contained in the nonspecific adsorption inhibitor effectively crosslinks the substrate and the water-soluble polymer of the nonspecific adsorption inhibitor, and the immobilizing agent according to the present invention effectively nonspecifically adsorbs. Crosslinks with the inhibitor.

  On the other hand, the epoxy group or active ester in the immobilizing agent having a photoreactive group, an amino group, and a room temperature reactive group in one molecule selectively reacts with the amino group of the substance to be immobilized, on the substrate. This makes it possible to fix the orientation of the material to be immobilized.

  According to the present invention, immobilization of a desired substance on a substrate can be performed as follows. First, a non-specific adsorption inhibitor is coated on a substrate, and a mixture of a substance to be immobilized thereon and a fixing agent having a photoreactive group, an amino group, and a room temperature reactive group in one molecule is spotted. Then, after the reaction at room temperature, the entire surface of the substrate may be irradiated with light, or a fixing agent having a photoreactive group, an amino group, and a room temperature reactive group in one molecule on the nonspecific adsorption preventing material is firstly prepared. After spotting and light irradiation, the substance to be immobilized may be spotted thereon and then reacted at room temperature.

  Although there is no restriction | limiting in particular in the film thickness of a nonspecific adsorption | suction prevention material, Usually, 0.01-10 micrometers, Preferably it is 0.05-1 micrometer. The concentration of the substance to be immobilized is not particularly limited, but is usually 0.01 to 10%, preferably 0.05 to 1%.

The light to be irradiated is light that allows the photoreactive group to be used to generate radicals. When an azide group or a diazirine group is used as the photoreactive group, ultraviolet rays of 300 to 400 nm are preferable. The dose of the irradiated light is not particularly limited, but is usually about 1 mW to 100 mW per 1 cm ² .

  By irradiating light to the non-specific adsorption preventing material, the photoreactive group generates a radical, and the substrate and the water-soluble polymer are covalently bonded. On top of that, a mixture of a substance to be immobilized and a fixing agent having a photoreactive group, an amino group, and a room temperature reactive group in one molecule is spotted, and a photoreactive group is contained in one molecule by reacting at room temperature. And a fixing agent having an amino group and a room temperature reactive group and a substance to be immobilized are covalently bonded, and then irradiated with light, whereby a photoreactive group, an amino group, and a room temperature reactive group are contained in one molecule. The photoreactive group of the immobilizing agent having a radical generates a radical and is covalently bonded to the water-soluble polymer. As a result, the substance to be immobilized on the substrate is oriented and fixed. In addition, an immobilizing agent having a photoreactive group, an amino group, and a room temperature reactive group in one molecule is first spotted on a non-specific adsorption preventing material, and after irradiation with light, a substance to be immobilized is spotted thereon. And may be allowed to react at room temperature. In particular, the two-step spot method is effective when a buffer solution or the like is included in the solution of the substance to be immobilized. In the normal one-step spot method, the sensitivity decreases when a buffer solution or the like is included, but the sensitivity does not decrease in the two-step spot method. In the method of the present invention, the substance to be immobilized is oriented and fixed because it binds to the room temperature reactive group of the immobilizing agent having a photoreactive group, an amino group, and a room temperature reactive group in one molecule. It will be. Therefore, the sensitivity can be increased as compared with a random immobilization method in which a substance to be immobilized is not bonded to a specific site that is immobilized with a radical by a conventional photoreactive group.

  The method of the present invention can be suitably used for preparation of immunoassay plates on which antibodies or antigens are immobilized, nucleic acid chips on which DNA or RNA is immobilized on a substrate, microarrays on which peptides or proteins are immobilized, and the like. However, it is not limited to these.

Hereinafter, the present invention will be described more specifically based on experimental examples. However, the present invention is not limited to the following experimental examples.
<Experimental Example 1 and Comparative Example 1>
1. Preparation of water-soluble polymer 26.3 g of polyethylene glycol monomethacrylate (polyethylene glycol part molecular weight 350) was dissolved in 200 ml of ethyl acetate, 61.6 mg of AIBN was added as an initiator, and the mixture was reacted for 6 hours under reflux. 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. Formation and immobilization of non-specific adsorption inhibitor
Sodium 4,4′-diazidostilbene-2,2′-disulfonate (commercially available) was used as a photocrosslinking agent. Mixing was performed so that the concentration of the photocrosslinking agent was 0.0125% and the concentration of the water-soluble polymer was 0.25%. The obtained aqueous solution was injected onto an acrylic resin substrate in an area of 10 × 10 mm ² , dried (film thickness: about 0.2 μm), and then irradiated with UV (black light for 7 minutes). Next, peptide (amino acid sequence H-CIKHIATNAVLFFGRCVSP-NH2) 0.1% and immobilizing agent N- [4- [3- (trifluoromethyl) -3H-diazirine-3-yl] phenyl] oxirane (commercially available) 0.01% 20 nL of the DMF-water (1: 1) solution thus mixed was spotted. After reacting at 37 ° C. for 2 hours, the peptide was immobilized by UV irradiation (black light for 7 minutes).

  On the other hand, as a comparative example, 20 nL of an aqueous solution mixed so that the peptide (amino acid sequence H-CIKHIATNAVLFFGRCVSP-NH2) 0.1% and the photocrosslinking agent 4,4′-diazidostilbene-2,2′-sodium disulfonate 0.01% Spotted. After drying, the peptide was immobilized by UV irradiation (black light for 7 minutes).

3. Measurement
After washing with PBS (0.1% Tween20 (registered trademark)), it was reacted with allergic dog serum at room temperature for 20 minutes. After washing with PBS (0.1% Tween20 (registered trademark)), it was reacted with a horseradish peroxidase (HRP) -labeled secondary antibody (commercially available) at room temperature for 20 minutes. After washing with PBS (0.1% Tween20 (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 results of Comparative Example 1.

<Experimental Examples 2 and 3>
1. Preparation of water-soluble polymer The same treatment as in Experimental Example 1 was performed.

2. Formation and immobilization of non-specific adsorption inhibitor <Experimental example 2>
Sodium 4,4′-diazidostilbene-2,2′-disulfonate (commercially available) was used as a photocrosslinking agent. Mixing was performed so that the concentration of the photocrosslinking agent was 0.15% and the concentration of the water-soluble polymer was 3%. The obtained aqueous solution was spin-coated on a high impact polystyrene resin substrate, dried (film thickness: about 0.1 μm), and then irradiated with UV (black light for 7 minutes). Next, 0.2 μL of 0.001% DMF-water (1: 1) solution of the fixing agent 5-azido-2-nitrobenzoic acid N-hydroxysuccinimide ester (commercial product) was spotted, dried, and then irradiated with UV ( (Black light for 7 minutes). Thereafter, 0.2 μL of a peptide (amino acid sequence H-CIKHIATNAVLFFGRCVSP-NH2) 0.1% aqueous solution was spotted and reacted at 37 ° C. for 2 hours to immobilize the peptide.
<Experimental example 3>
All were carried out in the same manner except that the 0.1% peptide aqueous solution of Experimental Example 2 was used as a PBS solution.

3. Measurement
After washing with PBS (0.1% Tween20 (registered trademark)), the mixture was reacted with an anti-OVA (ovalbumin) antibody (commercially available) at room temperature for 20 minutes. After washing with PBS (0.1% Tween20 (registered trademark)), it was reacted with a horseradish peroxidase (HRP) -labeled secondary antibody (commercially available) at room temperature for 20 minutes. After washing with PBS (0.1% Tween20 (registered trademark)), a chemiluminescent reagent was added, and the luminescence intensity was measured.

4). Results The measured results are shown in Table 2 below.

  In addition, this invention is not limited to the Example mentioned above, A various change can be added in the range which does not deviate from the summary of this invention.

  According to the present invention, a substance to be immobilized can be immobilized by covalent bonding, and a stable immobilized substrate can be obtained. Further, when the substance is immobilized using the method of the present invention, nonspecific adsorption is effectively prevented. Furthermore, a substance-immobilized substrate with high sensitivity and excellent S / N ratio can be obtained because of the orientation fixation.

Claims (9)

  A non-specific adsorption inhibitor made of a water-soluble polymer that is electrically neutral as a whole molecule is applied onto a substrate, and then fixed at least with a photoreactive group, an amino group, and a room temperature-reactive functional group in one molecule. A substance immobilization method comprising spotting the agent and the substance to be immobilized.   A non-specific adsorption inhibitor made of a water-soluble polymer that is electrically neutral as a whole molecule is applied onto a substrate, and then fixed at least with a photoreactive group, an amino group, and a room temperature-reactive functional group in one molecule. The method for immobilizing a substance according to claim 1, wherein the substance immobilizing agent and the mixture of the substance to be immobilized are spotted.   A non-specific adsorption inhibitor made of a water-soluble polymer that is electrically neutral as a whole molecule is applied onto a substrate, and then fixed at least with a photoreactive group, an amino group, and a room temperature-reactive functional group in one molecule. 2. The method for immobilizing a substance according to claim 1, comprising spotting the agent and then spotting the substance to be immobilized thereon.   The non-specific adsorption preventing material comprises a photocrosslinking agent having at least two photoreactive groups in one molecule and a water-soluble polymer that is electrically neutral as a whole molecule. 3. The method for immobilizing a substance according to 3.   5. The method for immobilizing a substance according to claim 1, wherein the photoreactive group is an azide group or a diazirine group.   The method for immobilizing a substance according to any one of claims 1 to 5, wherein the functional group reactive with the amino group at room temperature is an epoxy group or a succinimide ester.   The method for immobilizing a substance according to any one of claims 1 to 6, wherein the water-soluble polymer is a nonionic polymer.   The material immobilization method according to claim 7, wherein the nonionic polymer is a vinyl polymer of polyethylene glycol mono (meth) acrylate.   The substance immobilization method according to any one of claims 1 to 8, wherein the substance to be immobilized is selected from the group consisting of peptides, nucleic acids, and proteins having an amino group.