JP2014102194A - High polymer and carrier having surface covered with high polymer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a high polymer having excellent ability immobilizing a sugar chain, etc., high adhesion to a carrier, small non-specific absorption of protein, etc., and physical and chemical stability, and to provide a carrier having the high polymer immobilized on the surface and a composite carrier having a sugar chain, etc., immobilized with the high polymer.SOLUTION: A high polymer obtained by co-polymerizing an ethylene unsaturated polymeric monomer having a hydrophobic group and an ethylene unsaturated polymeric monomer having a functional group immobilizing a sugar chain, etc., or a high polymer obtained by co-polymerizing the monomers and an ethylene unsaturated polymeric monomer having a hydrophilic group has excellent ability immobilizing the sugar chain, etc., high adhesion to a carrier, small non-specific absorption of an object to be detected and physical and chemical stability such as hard dissolution in a cleaning process and the like. A carrier covered with the high polymer can be used for a biochip, etc..

Description

  The present invention relates to a polymer substance used for immobilizing sugar chains and the like, and a carrier whose surface is coated with the polymer substance.

  In the field of biochemistry, in recent years, sugar chain molecules have attracted attention as the third chain following nucleic acids and proteins. In particular, research on cell differentiation, canceration, immune reaction, fertilization, etc. has been conducted, and attempts to create new medicines and medical materials are continuing.

  In addition, sugar chains are receptors for many toxins, viruses and bacteria, and are also attracting attention as cancer markers. In this field as well, new drugs and medical materials are also being created. Attempts continue.

  However, while the importance of research is being recognized on sugar chains, the promotion of research is significantly delayed compared to nucleic acids and proteins, which are the first and second chains, due to their complex structure and diversity.

  For the purpose of promoting this research, various methods for purifying sugar chains have been developed. In addition, sugar chains are often confirmed to function as ligands to cell receptors rather than to function alone, and thus various sugar chains are immobilized for analysis of receptors for sugar chains. Development of the base material for this is also performed (patent document 1, patent document 2).

  In Patent Document 1, a trifunctional spacer is used to bind a sugar chain by first binding a functional group to a sugar chain, a second functional group to a solid phase carrier, and a third functional group to a chromophore. A method of making an array is disclosed. In the Examples, it is described that a blocking operation using bovine serum albumin is performed in order to prevent nonspecific adsorption after sugar chain fixation. However, it is complicated to perform a blocking operation after sugar chain fixation.

  On the other hand, Patent Document 2 discloses a method of immobilizing a sugar chain to a base material via a spacer, and it is shown that nonspecific adsorption is suppressed by using a hydrophilic compound for the spacer. Yes. However, in this method, it is necessary to introduce a halogenated acetyl group into the solid phase carrier in immobilizing the sugar chain to the base material, and it is relatively easy when the base material is glass. Poor versatility. In addition, as described above, sugar chains are also used in research on many receptors for toxins, viruses, bacteria, and the like. Therefore, incineration is preferable for disposal, and it is disadvantageous that the material is glass.

  In this way, in combination with the appropriate method for immobilizing the sugar chain and the delay in the development of the base material, the sugar in which the sugar chain useful for the analysis of the interaction between the detection target substance and the sugar chain is immobilized. Currently, the development of chain arrays is also delayed.

JP 2004-115616 A JP 2006-078418 A

  The present invention is a polymer substance that has excellent ability to immobilize sugar chains or sugar chain-containing substances, has high adhesion to a carrier, has little non-specific adsorption of proteins, etc., and is physically and chemically stable The purpose is to provide. Another object of the present invention is to provide a carrier having the polymer substance immobilized on the surface thereof. Furthermore, an object of the present invention is to provide a composite carrier in which a sugar chain or a sugar chain-containing substance is immobilized via the polymer substance.

  As a result of diligent studies to solve the above problems, the present inventors have found that an ethylenically unsaturated polymerizable monomer having a hydrophobic group and an ethylenically unsaturated polymerizable monomer having a functional group for immobilizing a sugar chain or the like. Polymeric substances obtained by copolymerizing these monomers and polymeric substances obtained by copolymerizing these monomers with ethylenically unsaturated polymerizable monomers having a hydrophilic group are excellent in the ability to immobilize sugar chains and the like. The present inventors have found that adhesion to a carrier is high and nonspecific adsorption of a detection target substance is small. Furthermore, it was found to be physically and chemically stable, such as being difficult to dissolve in the washing process. From such characteristics, the present inventors have found that the carrier coated with the polymer substance can exhibit excellent performance when used for biochips, etc., and have completed the present invention. .

  More specifically, the present invention provides the following.

  (1) (a) an ethylenically unsaturated polymerizable monomer having a hydrophobic group, and (b) an ethylenically unsaturated polymerizable monomer having a functional group that immobilizes a sugar chain or a sugar chain-containing substance. The resulting polymer material.

  (2) The polymer substance according to (1), wherein the ethylenically unsaturated polymerizable monomer having a hydrophobic group is a monomer represented by the following general formula [1].

(Wherein R1 represents a hydrogen atom or a methyl group, and R2 represents an alkyl group having 1 to 20 carbon atoms.)
(3) The polymer substance according to (1) or (2), wherein the ethylenically unsaturated polymerizable monomer having a hydrophobic group is n-butyl methacrylate.

  (4) The ethylenically unsaturated polymerizable monomer having a functional group for immobilizing the sugar chain or the sugar chain-containing substance is a monomer represented by the following general formula [2]: (1) to (3) The polymer substance according to any one of the above.

(Wherein R 3 represents a hydrogen atom or a methyl group, Y represents an alkylene group or an alkylene glycol residue having 1 to 10 carbon atoms, W represents a sugar chain or a functional group for immobilizing a sugar chain-containing substance. Q Represents an integer of 1 to 20. When q is an integer of 2 or more and 20 or less, the repeated Ys may be the same or different.
(5) The polymer substance according to (4), wherein the functional group for immobilizing the sugar chain or the sugar chain-containing substance is p-nitrophenyl ester or N-hydroxysuccinimide ester.

  (6) The polymer material according to any one of (1) to (5), wherein in the polymer material, (c) an ethylenically unsaturated polymerizable monomer having a hydrophilic group is copolymerized. .

  (7) The polymer substance according to (6), wherein the ethylenically unsaturated polymerizable monomer having a hydrophilic group is a monomer represented by the following general formula [3].

(Wherein R4 represents a hydrogen atom or a methyl group, R5 represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms, X represents an alkylene glycol residue having 1 to 10 carbon atoms, and p represents 1 to 100) (Indicates an integer. When p is an integer of 2 or more, the repeated Xs may be the same or different.)
(8) The polymer substance according to (7), wherein the ethylenically unsaturated polymerizable monomer having a hydrophilic group is methoxypolyethylene glycol acrylate or methoxypolyethylene glycol methacrylate.

  (9) The polymer substance according to (8), wherein an average number of ethylene glycol residues in the methoxypolyethylene glycol acrylate or methoxypolyethylene glycol methacrylate is 3 to 100.

  (10) The polymer substance according to (6), wherein the ethylenically unsaturated polymerizable monomer having a hydrophilic group is a monomer represented by the following general formula [4].

(Wherein R6 represents a hydrogen atom or a methyl group, and Z represents an alkylene group having 1 to 100 carbon atoms.)
(11) The polymer substance according to (10), wherein the ethylenically unsaturated polymerizable monomer having a hydrophilic group is 2-hydroxyethyl (meth) acrylate.

  (12) A carrier whose surface is coated with the polymer substance described in any one of (1) to (11).

  (13) A carrier obtained by immobilizing a sugar chain or a sugar chain-containing substance on the carrier according to (12).

  (14) The carrier according to (12) or (13), wherein the substrate is made of plastic.

  (15) The carrier according to (14), wherein the plastic is a saturated cyclic polyolefin.

  ADVANTAGE OF THE INVENTION According to this invention, the high molecular substance which is excellent in the ability to fix | immobilize sugar chains etc., has high adhesiveness with a support | carrier, has few nonspecific adsorption | suctions, such as protein, and is hard to melt | dissolve in a washing process can be provided. Further, a carrier whose surface is coated with the polymer substance can be provided. Furthermore, by immobilizing sugar chains or the like on the carrier, it is possible to provide a composite carrier that can selectively capture substances to which they specifically bind. In particular, when a carrier is used as a substrate, it is possible to provide a biochip substrate having a high SN ratio.

<Polymer material>
The polymer substance of the present invention comprises (a) an ethylenically unsaturated polymerizable monomer having a hydrophobic group, and (b) an ethylenically unsaturated polymerizable monomer having a functional group that immobilizes a sugar chain or a sugar chain-containing substance. It is obtained by copolymerizing

  In this polymer substance, (a) a unit derived from an ethylenically unsaturated polymerizable monomer having a hydrophobic group (hereinafter simply referred to as a “unit having a hydrophobic group”) is polymerized by hydrophobic interaction. And (b) an ethylenically unsaturated polymerisable having a functional group that immobilizes a sugar chain or a sugar chain-containing substance. A unit derived from a monomer (hereinafter simply referred to as “unit having a functional group for immobilizing a sugar chain or the like”) plays a role of immobilizing a sugar chain or a sugar chain-containing substance in the functional group.

-Ethylenically unsaturated polymerizable monomer having a hydrophobic group-
The “ethylenically unsaturated polymerizable monomer having a hydrophobic group” in the present invention is not particularly limited in structure, but preferably has a structure in which a hydrophobic group is bonded to a (meth) acrylic group residue. Although a hydrophobic group is not specifically limited, An alkyl group and aromatics are mentioned.

  One preferred embodiment of the ethylenically unsaturated polymerizable monomer having a hydrophobic group is a compound represented by the general formula [1].

  In the formula, R1 represents a hydrogen atom or a methyl group. R2 represents an alkyl group having 1 to 20 carbon atoms. The structure of the alkyl group is not particularly limited, and may be linear, branched, or cyclic.

  Carbon number of an alkyl group becomes like this. Preferably it is 1-10, More preferably, it is 1-6, More preferably, it is 3-6, Most preferably, it is 4.

  Specific examples of the ethylenically unsaturated polymerizable monomer having a hydrophobic group include iso-butyl (meth) acrylate, sec-butyl (meth) acrylate, t-butyl (meth) acrylate, n-neopentyl (meth) acrylate, iso-neopentyl (meth) acrylate, sec-neopentyl (meth) acrylate, neopentyl (meth) acrylate, n-hexyl (meth) acrylate, iso-hexyl (meth) acrylate, heptyl (meth) acrylate, n-octyl (meth) Acrylate, iso-octyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, n-nonyl (meth) acrylate, iso-nonyl (meth) acrylate, n-decyl (meth) acrylate, iso-decyl (meth) acrylate, n-dodecyl (meth) acryl Rate, iso-dodecyl (meth) acrylate, n-tridecyl (meth) acrylate, iso-tridecyl (meth) acrylate, n-tetradecyl (meth) acrylate, iso-tetradecyl (meth) acrylate, n-pentadecyl (meth) acrylate, iso-pentadecyl (meth) acrylate, n-hexadecyl (meth) acrylate, iso-hexadecyl (meth) acrylate, n-octadecyl (meth) acrylate, iso-octadecyl (meth) acrylate, cyclohexyl (meth) acrylate, isobonyl (meth) Acrylate, phenoxy (meth) acrylate, phenoxyethyl (meth) acrylate, 2-ethylphenoxy (meth) acrylate, 2-ethylthiophenyl (meth) acrylate, phenyl (meth) acrylate , Benzyl (meth) acrylate, 2-phenylethyl (meth) acrylate, 3-phenylpropyl (meth) acrylate, 4-phenylbutyl (meth) acrylate, 2--2-methylphenylethyl (meth) acrylate, 2-3 -Methylphenylethyl (meth) acrylate, 2-4-methylphenylethyl (meth) acrylate, 2- (4-propylphenyl) ethyl (meth) acrylate, 2- (4- (1-methylethyl) phenyl) ethyl ( (Meth) acrylate, 2- (4-methoxyphenyl) ethyl (meth) acrylate, 2- (4-cyclohexylphenyl) ethyl (meth) acrylate, 2- (2-chlorophenyl) ethyl (meth) acrylate, 2- ( 3-chlorophenyl) ethyl (meth) acrylate, 2- (4 Chlorophenyl) ethyl (meth) acrylate, 2- (4-bromophenyl) ethyl (meth) acrylate, 2- (3-phenylphenyl) ethyl (meth) acrylate and 2- (4-benzylphenyl) ethyl (meth) acrylate, Although n-butyl methacrylate etc. are mentioned, n-butyl methacrylate is preferable from the viewpoint of the balance between ease of coating and ease of polymerization.

  By including a unit having a hydrophobic group in the polymer substance, the wettability is improved even on a hydrophobic substrate such as plastic, so that it can be applied evenly. Further, since the hydrophobicity increases, it is possible to prevent the polymer substance from being eluted during the assay.

  The composition ratio of the unit having a hydrophobic group contained in the polymer substance is preferably 20 to 99 mol%, more preferably 30 to 95 mol%, and most preferably 50 to 95 mol%. If the composition ratio is below the lower limit, unevenness may occur when applied, or the polymer substance may be eluted during the assay. On the other hand, when the value exceeds the upper limit, the number of units having a functional group for immobilizing sugar chains and the like is decreased, which may reduce the signal and increase nonspecific adsorption.

-Ethylenically unsaturated polymerizable monomers having functional groups that immobilize sugar chains, etc.-
The “ethylenically unsaturated polymerizable monomer having a functional group for immobilizing a sugar chain or a sugar chain-containing substance” in the present invention is not particularly limited in structure, but a sugar chain or the like is immobilized on a (meth) acrylic group residue. It is preferable that the functional group is bonded to the structure. The functional group is not particularly limited, but a functional group that reacts with a reducing end of a sugar chain, such as an amino group, a hydrazide group, or an oxylamino group, or a functional group or substance that reacts with a sugar chain or a sugar chain-containing substance. Groups are preferred. Specific examples include aldehyde groups, active ester groups, epoxy groups, amino groups, isocyanate groups, isothiocyanate groups, hydroxyl groups, maleimide groups, azide groups, sulfonate groups, acceptor groups, biotin, and the like.

  Of these, amino groups, hydrazide groups, and oxylamino groups are preferred because many of the sugar chains have a reducing end. Further, when immobilizing a sugar chain having an amino group or a sugar chain-containing substance, an aldehyde group, an active ester group, and an epoxy group are preferable, and the active ester group is the most in view of the balance between reactivity with amino groups and storage stability. preferable.

  In addition, it is preferable to protect beforehand the functional group which acts as an inhibitor of radical polymerization, such as a primary amine, or the functional group where the reaction easily proceeds by heating, such as isocyanate. The kind of the protecting group is not particularly limited as long as the functional group is protected. A tert-butoxycarbonyl group (BOC group) or a benzyloxycarbonyl group (Z group) is used as a functional group having a primary amine such as an amino group, an oxylamino group, or a hydrazide group. Among them, a tert-butoxycarbonyl group (BOC group) is preferable because of easy deprotection. In addition, isocyanates can be protected with pyrazole or pyrazole derivatives.

  Deprotection conditions may be appropriately selected depending on the protecting group. For example, deprotection of a tert-butoxycarbonyl group (BOC group) may be performed by applying a strong acid such as trifluoroacetic acid, hydrochloric acid, hydrochloric acid-dioxane solution, hydrochloric acid-ethyl acetate solution, or the like. Although the timing of deprotection is arbitrary, since it is advantageous to react with a sugar chain or a sugar chain-containing substance in a more active state, it is preferably performed immediately before the reaction with them.

  The “active ester group” used in the present invention is an ester group having a highly acidic electron-withdrawing group in one of the substituents of the ester group and activated for nucleophilic reaction, ie, the reaction activity. As meaning a high ester group, it is commonly used in the fields of various chemical syntheses such as polymer chemistry and peptide synthesis. In fact, phenol esters, thiophenol esters, N-hydroxyamine esters, esters of heterocyclic hydroxy compounds, and the like are known as active ester groups having much higher activity than alkyl esters and the like.

  Examples of such active ester groups include p-nitrophenyl active ester groups, N-hydroxysuccinimide active ester groups, succinimide active ester groups, phthalimide active ester groups, 5-norbornene-2, and 3-dicarboxyl. Although an imide active ester group etc. are mentioned, a p-nitrophenyl active ester group or an N-hydroxysuccinimide active ester group is preferable, and a p-nitrophenyl active ester group is most preferable.

  The ethylenically unsaturated polymerizable monomer having a functional group for immobilizing a sugar chain or a sugar chain-containing substance used in the present invention is not particularly limited in structure, but is represented by the following general formula [2] (meta ) A compound in which an acrylic group and a functional group that immobilizes a sugar chain or a sugar chain-containing substance are bonded via a chain of an alkylene glycol residue having 1 to 10 carbon atoms or an alkylene group is preferable.

  In the formula, R3 represents a hydrogen atom or a methyl group. Moreover, carbon number of the alkylene glycol residue Y is 1-10, Preferably it is 1-6, More preferably, it is 2-4, More preferably, it is 2-3, Most preferably. The repeating number q of the alkylene glycol residue Y is an integer of 1 to 20, preferably an integer of 2 to 18, more preferably an integer of 3 to 16, and most preferably an integer of 3 to 14. When the number of repetitions is 2 or more and 20 or less, the carbon number of the alkylene glycol residue to be repeated may be the same or different.

  The composition ratio of the ethylenically unsaturated polymerizable monomer having a functional group that immobilizes the sugar chain or the sugar chain-containing substance contained in the polymer substance is preferably 1 to 50 mol%, more preferably 1 to 30 mol%, and most preferably Is 1 to 20 mol%. If the composition ratio falls below the lower limit, the signal may be reduced. On the other hand, in a polymer substance having a composition ratio in which the monomer exceeds the upper limit, sugar chains can be immobilized at a higher density, but a huge molecule (for example, lectin) is used as a detection target substance compared to sugar chains. In some cases, sugar chains that do not participate in binding to the detection target substance in the polymer substance increase. Therefore, even if the composition ratio of the monomer is increased, the signal may not increase. On the other hand, if the upper limit is exceeded, the number of units having a relatively hydrophobic group decreases, which may cause unevenness when applied or the polymer substance may be eluted during the assay.

− Ethylenically unsaturated polymerizable monomer having hydrophilic group −
In the polymer substance of the present invention, (c) an ethylenically unsaturated polymerizable monomer having a hydrophilic group may be copolymerized. In this case, (c) the ethylenically unsaturated polymerizable monomer having a hydrophilic group plays a role of suppressing the non-specific adsorption of the detection target substance to the polymer substance.

  The “ethylenically unsaturated polymerizable monomer having a hydrophilic group” in the present invention is not particularly limited in structure, but a structure in which a hydrophilic group is bonded to a (meth) acrylic group residue is preferable.

  One preferred embodiment of the ethylenically unsaturated polymerizable monomer having a hydrophilic group is a compound represented by the general formula [3].

  In the formula, R4 represents a hydrogen atom or a methyl group, and R5 represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms. Moreover, carbon number of alkylene glycol residue X is 1-10, Preferably it is 1-6, More preferably, it is 2-4, More preferably, it is 2-3, Most preferably. The repeating number p of the alkylene glycol residue X is an integer of 1 to 100, preferably an integer of 2 to 100, more preferably an integer of 2 to 95, and most preferably an integer of 4 to 90. When the number of repeats is 2 or more and 100 or less, the carbon number of the alkylene glycol residue X to be repeated may be the same or different.

  Specific examples include methoxypolyethylene glycol (meth) acrylate, (meth) acrylate having polypropylene glycol as a side chain, methoxydiethylene glycol (meth) acrylate, ethoxydiethylene glycol (meth) acrylate, ethoxypolyethylene glycol (meth) acrylate, and the like. However, methoxypolyethylene glycol methacrylate is preferable from the viewpoint of availability.

  Another preferred embodiment of the ethylenically unsaturated polymerizable monomer having a hydrophilic group is a compound represented by the general formula [4].

  In the formula, R6 represents a hydrogen atom or a methyl group. Z represents an alkylene group having 1 to 100 carbon atoms. Carbon number of an alkylene group becomes like this. Preferably it is 1-10, More preferably, it is 1-6, More preferably, it is 2-4, Most preferably, it is 2.

  Specific examples of the ethylenically unsaturated polymerizable monomer having a hydrophilic group include 3-hydroxypropyl (meth) acrylate, 4-hydroxybutyl (meth) acrylate, 2-hydroxyethyl (meth) acrylate, and the like. However, 2-hydroxyethyl (meth) acrylate is preferable from the viewpoint of suppressing nonspecific adsorption.

  The composition ratio of the ethylenically unsaturated polymerizable monomer having a hydrophilic group contained in the polymer substance is preferably 0 to 50 mol%, more preferably 0 to 40 mol%, and most preferably 0 to 30 mol%. When the composition ratio exceeds the upper limit, the number of units having functional groups that immobilize sugar chains and the like is relatively reduced, so that the signal is reduced or the number of units having relatively hydrophobic groups is reduced. In some cases, unevenness may occur or the polymer substance may be eluted during the assay.

  The chemical structure of the polymer substance of the present invention may be in any form such as random, block or graft as long as the above monomers are copolymerized.

  Highly obtained by copolymerizing (a) an ethylenically unsaturated polymerizable monomer having a hydrophobic group, and (b) an ethylenically unsaturated polymerizable monomer having a functional group for immobilizing a sugar chain or a sugar chain-containing substance. A particularly preferable embodiment of the molecular substance is a polymer substance having the following chemical structure.

  Here, the repeating number q of the alkylene glycol residue and x and y which are the composition ratio of each unit are as described above, but preferably q is 3 to 14 units on average and the composition ratio x is 80 to 95 mol. %, And the composition ratio y is 5 to 20 mol%.

  (A) an ethylenically unsaturated polymerizable monomer having a hydrophobic group, (b) an ethylenically unsaturated polymerizable monomer having a functional group that immobilizes a sugar chain or a sugar chain-containing substance, and (c) a hydrophilic group. A particularly preferable embodiment of the polymer material obtained by copolymerizing the ethylenically unsaturated polymerizable monomer is two polymer materials having the following chemical structures.

  Here, p and q, which are the repeating number of alkylene glycol residues, and x, y, and z, which are the composition ratio of each unit, are as described above, and preferably q is an average of 3 to 14 units, The composition ratio x is 10 to 30 mol%, the composition ratio y is 5 to 20 mol%, and the composition ratio z is 50 to 85 mol%.

  Here, q, which is the number of repeating alkylene glycol residues, and x, y, and z, which are the composition ratios of each unit, are as described above, but preferably q is an average of 3 to 14 units, and the composition ratio x is 10 to 30 mol%, the composition ratio y is 50 to 85 mol%, and the composition ratio z is 5 to 20 mol%.

  The molecular weight of the polymer substance of the present invention is preferably 5,000 or more and more preferably 10,000 or more because the polymer substance and the unreacted ethylenically unsaturated polymerizable monomer can be easily separated and purified.

<Synthesis of polymer materials>
The method for synthesizing the polymer substance of the present invention is not particularly limited. From the viewpoint of ease of synthesis, at least (a) an ethylenically unsaturated polymerizable monomer having a hydrophobic group, and (b) a sugar chain or It is preferable to radically polymerize a mixture containing an ethylenically unsaturated polymerizable monomer having a functional group for immobilizing a sugar chain-containing substance in a solvent in the presence of a polymerization initiator. The mixture for radical polymerization can further contain (c) an ethylenically unsaturated polymerizable monomer having a hydrophilic group.

  Any solvent may be used as long as each ethylenically unsaturated polymerizable monomer can be dissolved. Examples thereof include methanol, ethanol, t-butyl alcohol, benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, methyl ethyl ketone, and acetone. be able to. These solvents are used alone or in combination of two or more. When the polymer substance is applied to a plastic substrate, ethanol, methanol, 2-butanone, and a mixed solvent thereof are preferable because they do not denature the substrate.

  The polymerization initiator may be any ordinary radical initiator, such as 2,2′-azobisisobutylnitrile (hereinafter referred to as “AIBN”), 1,1′-azobis (cyclohexane-1-carbonitrile), and the like. Examples thereof include organic peroxides such as azo compounds, benzoyl peroxide, and lauryl peroxide.

<Coating of carrier surface with polymer substance>
By coating the carrier surface with the polymer substance of the present invention, it is possible to easily impart the property of immobilizing a specific sugar chain or sugar chain-containing substance. When the polymer substance includes a unit having a hydrophilic group, it is possible to impart a property of suppressing nonspecific adsorption of protein or the like to the carrier.

  For coating the surface of the polymer material, for example, a polymer solution in which the polymer material is dissolved in an organic solvent so as to have a concentration of 0.05 to 10% by weight is prepared, and then a known method such as dipping or spraying is used. After coating on the surface of the carrier, drying is performed at room temperature or under heating.

  As the organic solvent, a single solvent such as ethanol, methanol, t-butyl alcohol, benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, acetone, methyl ethyl ketone, or a mixed solvent thereof is used. Among these, ethanol, methanol, 2-butanone, and a mixed solvent thereof are preferable because they do not denature the plastic carrier and can be easily dried.

<Carrier whose surface is coated with a polymer substance>
Glass, plastic, metal or the like can be used as the material for the carrier used in the present invention. From the viewpoint of easy surface treatment and mass productivity, plastic is preferable, and thermoplastic resin is more preferable.

  The shape of the carrier is not particularly limited, but when used as a biochip, for example, a glass substrate or a multiwell plate is preferably used.

  When the carrier is used as a biochip, the thermoplastic resin preferably has a small amount of fluorescence generation. For example, linear polyolefins such as polyethylene and polypropylene, cyclic polyolefins, fluorine-containing resins, etc. are preferably used. It is more preferable to use a saturated cyclic polyolefin that is particularly excellent in chemical resistance, low fluorescence, and moldability. Here, the saturated cyclic polyolefin refers to a saturated polymer obtained by hydrogenating a polymer having a cyclic olefin structure or a copolymer of a cyclic olefin and an α-olefin.

  In order to enhance the adhesion between the carrier surface and the polymer substance to be coated, it is preferable to activate the substrate surface. As a means for activation, there are a plasma treatment method under conditions such as an oxygen atmosphere, an argon atmosphere, a nitrogen atmosphere, and an air atmosphere, and a treatment method using an excimer laser such as ArF or KrF. A plasma treatment method is preferred.

  Various sugar chains or sugar chain-containing substances can be immobilized using the carrier of the present invention. The sugar chain or sugar chain-containing substance to be immobilized is a monosaccharide, a sugar chain of two or more sugars, a sugar amino acid, a glycopeptide, a glycoprotein, a glycolipid, a glycosaminoglycan, a glycosylphosphatidylinositol, a peptidoglycan, a lipopolysaccharide, and the like There are derivatives of When the functional group that immobilizes the sugar chain or the sugar chain-containing substance in the polymer material coated on the surface of the carrier is a functional group that reacts with the reducing end of the sugar, the sugar having the reducing end is adjusted to pH 4.0 to 6. It can be fixed by dissolving in 0 buffer solution and allowing the solution to stand on a carrier at room temperature to 100 ° C. for 10 minutes to 24 hours.

  On the other hand, when the functional group that immobilizes the sugar chain or the sugar chain-containing substance is a group that reacts with an amino group such as an active ester group, an amino group is required for the sugar chain or the sugar chain-containing substance. An amino group-containing sugar chain, sugar amino acid, glycopeptide or glycoprotein is dissolved in a buffer solution having a pH of 7.0 to 10.0, and the solution is allowed to stand on a carrier at room temperature to 100 ° C. for 10 minutes to 24 hours. However, in the case of a sugar chain having no amino group, it is necessary to introduce it. In this case, the amino group is preferably introduced on the reducing end side.

<Carrier with immobilized sugar chains and the like>
The carrier obtained by immobilizing the sugar chain or the sugar chain-containing substance thus obtained has a sufficient amount of sugar chain or sugar chain-containing substance firmly immobilized by chemical bonding, and further has less non-specific adsorption of proteins and the like. The substance that specifically binds to the sugar chain or the sugar chain-containing substance can be selectively captured. In particular, when the carrier is a substrate, it can be suitably used as a biochip.

  In the present invention, when the sugar chain or the sugar chain-containing substance is immobilized on the biochip substrate, a method of spotting a liquid in which the sugar chain or the sugar chain-containing substance is dissolved or dispersed is preferable. After standing, the sugar chain or sugar chain-containing substance is chemically bonded to the functional group on the substrate, and the sugar chain or sugar chain-containing substance is immobilized on the surface. For example, when a sugar chain having an amino group is used, the above-described method can be applied.

  After the sugar chain or sugar chain-containing substance is immobilized on the biochip substrate, it is preferable to inactivate functional groups on the substrate surface other than the part where the sugar chain or sugar chain-containing substance is immobilized. When the functional group of the substrate is an active ester group or an aldehyde group, it is preferable to use an alkali compound or a compound having a primary amino group.

  As the alkali compound, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogen carbonate, disodium hydrogen phosphate, calcium hydroxide, magnesium hydroxide, sodium borate, lithium hydroxide, potassium phosphate, etc. are preferably used. Can do.

  Examples of the compound having a primary amino group include methylamine, ethylamine, butylamine, glycine, 9-aminoacazine, aminobutanol, 4-aminobutyric acid, aminocaprylic acid, aminoethanol, 5-amino2,3-dihydro-1,4 -Pentanol, aminoethanethiol hydrochloride, aminoethanethiolsulfuric acid, 2- (2-aminoethylamino) ethanol, 2-aminoethyl dihydrogen phosphate, aminoethyl hydrogensulfate, 4- (2-aminoethyl) morpholine, 5-aminofluorescein, 6-aminohexanoic acid, aminohexyl cellulose, p-aminohippuric acid, 2-amino-2-hydroxymethyl-1,3-propanediol, 5-aminoisophthalic acid, aminomethane, aminophenol, 2 -Aminooctane, 2-aminooctanoic acid, 1 Amino 2-propanol, 3-amino-1-propanol, 3-aminopropene, 3-aminopropionitrile, aminopyridine, 11-aminoundecanoic acid, aminosalicylic acid, aminoquinoline, 4-aminophthalonitrile, 3-aminophthalimide , P-aminopropiophenone, aminophenylacetic acid, aminonaphthalene and the like are preferable, and aminoethanol and glycine are most preferable.

  Further, in the inactivation treatment step, it is often possible to simultaneously wash and remove sugar chains or sugar chain-containing substances that have not been bonded to the functional group of the substrate. If it cannot be removed, it is preferable to remove the substrate by washing it before the inactivation treatment. For example, it can be removed by immersing the substrate in ultrapure water, a surfactant-containing buffer solution or the like for 1 to 5 minutes and washing it.

  The biochip obtained by immobilizing sugar chains or sugar chain-containing substances in this way can be used in many analysis systems including immunodiagnostic systems, sugar chain microarray systems, and microfluidic devices.

[Synthesis of p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate (MEONP)]
0.01 mol of polyethylene glycol monomethacrylate (Blenmer PE-200 (n = 4), manufactured by NOF Corporation) was dissolved in 20 mL of chloroform and then cooled to −30 ° C. While maintaining at −30 ° C., 0.01 mol of p-nitrophenyl chloroformate (manufactured by Aldrich), 0.01 mol of triethylamine (manufactured by Wako Pure Chemical Industries, Ltd.) and 20 mL of chloroform were added to this solution. The homogeneous solution was slowly added dropwise. After reacting at −30 ° C. for 1 hour, the solution was further stirred for 2 hours. Thereafter, salts were removed from the reaction solution by filtration, and the solvent was distilled off to obtain p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate (MEONP). The obtained monomer was measured by 1H-NMR in deuterated chloroform solvent, and it was confirmed that an average of 4.5 units of ethylene glycol residue was contained.

[Synthesis Example 1 of Polymeric Substance]
N-Butyl methacrylate (hereinafter referred to as “BMA”; manufactured by Kanto Chemical Co., Inc.), p-nitrophenyloxycarbonyl-4.5 polyethylene glycol methacrylate (hereinafter referred to as “MEONP”) as 2-butanone It was dissolved to prepare a monomer mixed solution. The total monomer concentration is 1.2 mol / L, and the respective molar ratios are 90:10 in the order of BMA and MEONP. Further, 2,2-azobisisobutyronitrile (hereinafter referred to as “AIBN”, manufactured by Wako Pure Chemical Industries, Ltd.) was added to 0.024 mol / L and stirred until uniform. . Then, after making it react at 60 degreeC by argon gas atmosphere for 4 hours, the reaction solution was dripped in ethanol and precipitation was collected. The obtained polymer substance was measured by 1H-NMR in deuterated chloroform solvent, and the composition ratio of this polymer substance was calculated. Table 1 shows the results.

[Synthesis example 2 of polymer substance]
Polyethylene glycol methyl ether methacrylate (also known as methoxy polyethylene glycol methacrylate, hereinafter referred to as “PEGMA”. Number average molecular weight Mn = about 470, manufactured by Shin-Nakamura Chemical Co., Ltd.), BMA, and MEONP are dissolved in 2-butanone and mixed with monomers. A solution was made. The total monomer concentration is 0.85 mol / L, and the respective molar ratios are 22:68:10 in the order of PEGMA, BMA, and MEONP. AIBN was further added so that it might become 0.017 mol / L, and it stirred until it became uniform. Then, after making it react at 60 degreeC under argon gas atmosphere for 4 hours, the reaction solution was dripped in the mixed solvent of hexane and acetone, and precipitation was collected. The obtained polymer substance was measured by 1H-NMR in a heavy ethanol solvent, and the composition ratio of the polymer substance was calculated. Table 1 shows the results.

[Synthesis example 3 of polymer substance]
Hydroxyethyl acrylate (hereinafter referred to as “HEA”; manufactured by Wako Pure Chemical Industries, Ltd.), BMA and MEONP were dissolved in a mixed solvent of 2-butanone and ethanol to prepare a monomer mixed solution. The total monomer concentration is 1.2 mol / L, and the respective molar ratios are 22:68:10 in the order of PEGMA, BMA, and MEONP. AIBN was further added so that it might become 0.024 mol / L, and it stirred until it became uniform. Then, after making it react at 60 degreeC under argon gas atmosphere for 4 hours, the reaction solution was dripped in the mixed solvent of hexane and acetone, and precipitation was collected. The obtained polymer substance was measured by 1H-NMR in a heavy ethanol solvent, and the composition ratio of the polymer substance was calculated. Table 1 shows the results.

[Fabrication of immobilized substrate]
For the production of the microarray, a glass substrate made of cyclic polyolefin resin in the form of a glass slide (manufactured by Sumitomo Bakelite Co., Ltd.) was used. Then, this plastics substrate was immersed in a 1.0 wt% ethanol solution of the polymer material obtained in the polymer material synthesis example, thereby introducing a layer containing the polymer material on the substrate surface.

[Immobilization of sugar chains]
Two types of sugar chains (ELICITYL, GM3-aminopropyl (product number: GLY090AP, hereinafter referred to as “GM3”), Blood group A trioseose-aminoproyl (product number: GLY034AP, hereinafter referred to as “A”)) 1 to 50 μM, spotted on the substrate, and allowed to stand at room temperature for 1 hour to immobilize sugar chains on the substrate.
<Solution 1> 300 mM phosphate buffer (pH 8.5) + 0.005% Tween 20
[Deactivation treatment of active ester]
The substrate was immersed in the solution 2 for 1 hour, washed with pure water, and dried.
<Solution 2> 100 mM borate buffer (pH 8.5) + 0.01% Tween 20, 25 mM Ethanolamine

[Detection of lectins]
Biotin-labeled AAL lectin (J-Oil Mills, J201), which is a biotin-labeled AAL lectin that specifically binds to fucose, was prepared at 10 ug / mL with the following solution.

Solution: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl, 1 mM CaCl ₂ , MnCl ₂ , MgCl ₂ , 0.05% Tween 20
The solution was developed on the substrate and reacted at room temperature for 2 hours.

  After the reaction, the substrate was washed with the following solution and further washed with pure water to dry the substrate.

Solution: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl, 1 mM CaCl ₂ , MnCl ₂ , MgCl ₂
Cy3-Streptavidin (GE Healthcare, PA43001) was prepared to 0.5 μg / mL with the following solution and allowed to react on the substrate at room temperature for 1 hour.

Solution: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl, 1 mM CaCl ₂ , MnCl ₂ , MgCl ₂ , 0.05% Tween 20
After the reaction, the substrate was washed with the following solution and further washed with pure water to dry the substrate.

Solution: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl, 1 mM CaCl ₂ , MnCl ₂ , MgCl ₂
The measurement was performed by measuring the fluorescence intensity of Cy3 using a microarray reader, ScanArrayLite (manufactured by PerkinElmer). (Laser = 90, PMT = 50)
Table 2 shows the results. The sugar chain “A” containing fucose was specifically detected.

  According to the present invention, it has become possible to produce a high-performance polymer substance for immobilizing sugar chains and a carrier coated with the polymer substance. Furthermore, a biochip can be easily produced by using a composite carrier in which a sugar chain or a sugar chain-containing substance is fixed to the sugar chain fixing carrier. The biochip thus produced can be used in many analysis systems including immunodiagnostic systems, sugar chain microarray systems, and microfluidic devices.

Claims (15)

Highly obtained by copolymerizing (a) an ethylenically unsaturated polymerizable monomer having a hydrophobic group, and (b) an ethylenically unsaturated polymerizable monomer having a functional group for immobilizing a sugar chain or a sugar chain-containing substance. Molecular material. The polymer substance according to claim 1, wherein the ethylenically unsaturated polymerizable monomer having a hydrophobic group is a monomer represented by the following general formula [1].
(Wherein R1 represents a hydrogen atom or a methyl group, and R2 represents an alkyl group having 1 to 20 carbon atoms.)   The polymer material according to claim 1 or 2, wherein the ethylenically unsaturated polymerizable monomer having a hydrophobic group is n-butyl methacrylate. The ethylenically unsaturated polymerizable monomer having a functional group that immobilizes the sugar chain or the sugar chain-containing substance is a monomer represented by the following general formula [2]. The polymeric material described.
(Wherein R 3 represents a hydrogen atom or a methyl group, Y represents an alkylene group or an alkylene glycol residue having 1 to 10 carbon atoms, W represents a sugar chain or a functional group for immobilizing a sugar chain-containing substance. Q Represents an integer of 1 to 20. When q is an integer of 2 or more and 20 or less, the repeated Ys may be the same or different.   The polymer substance according to claim 4, wherein the functional group that immobilizes the sugar chain or the sugar chain-containing substance is p-nitrophenyl ester or N-hydroxysuccinimide ester.   The polymer material according to claim 1, wherein (c) an ethylenically unsaturated polymerizable monomer having a hydrophilic group is copolymerized. The polymer material according to claim 6, wherein the ethylenically unsaturated polymerizable monomer having a hydrophilic group is a monomer represented by the following general formula [3].
(Wherein R4 represents a hydrogen atom or a methyl group, R5 represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms, X represents an alkylene glycol residue having 1 to 10 carbon atoms, and p represents 1 to 100) (Indicates an integer. When p is an integer of 2 or more, the repeated Xs may be the same or different.)   The polymer material according to claim 7, wherein the ethylenically unsaturated polymerizable monomer having a hydrophilic group is methoxypolyethylene glycol acrylate or methoxypolyethylene glycol methacrylate.   The polymer substance according to claim 8, wherein an average number of ethylene glycol residues in the methoxypolyethylene glycol acrylate or methoxypolyethylene glycol methacrylate is 3 to 100. The polymer substance according to claim 6, wherein the ethylenically unsaturated polymerizable monomer having a hydrophilic group is a monomer represented by the following general formula [4].
(Wherein R6 represents a hydrogen atom or a methyl group, and Z represents an alkylene group having 1 to 100 carbon atoms.)   The polymer substance according to claim 10, wherein the ethylenically unsaturated polymerizable monomer having a hydrophilic group is 2-hydroxyethyl (meth) acrylate.   A carrier having a surface coated with the polymer material according to claim 1.   A carrier obtained by immobilizing a sugar chain or a sugar chain-containing substance on the carrier according to claim 12.   The carrier according to claim 12 or 13, wherein the substrate is made of plastic.   The carrier according to claim 14, wherein the plastic is a saturated cyclic polyolefin.