WO2015008834A1 - Silane compound containing active ester group and material produced using same - Google Patents

Abstract

[Problem] To provide: a silane compound containing an active ester group; and a material produced using the silane compound. [Solution] A silane compound represented by formula (1). [In formula (1), R¹ represents a specific alkyl group; R² represents a specific alkoxy group, a halogen atom or a combination thereof; a represents an integer of 0 to 2; X represents a hydrogen atom, a phenyl group or a specific alkyl group; Y represents a specific alkylene group; Z represents a monovalent organic group represented by formula (1-1) or formula (1-2); R³ to R⁵ independently represent a hydrogen atom or a specific alkyl group; T¹ represents a specific hydrocarbon ring or an aromatic ring; and b represents an integer of 4 or more, wherein the maximum value of b is the maximum number of substituents that T¹ can have.]

Description

Silane compound containing active ester group and material using the same

The present invention relates to a silane compound containing an active ester group (also referred to herein as “silane coupling agent”), and in particular, a novel silane coupling agent that can be easily synthesized, and The present invention relates to a material using the silane coupling agent.

The technology for immobilizing biomaterials such as nucleic acids, antibodies, and proteins on inorganic material substrates such as glass and silicon wafers is useful in the fields of cell culture, cell / protein separation / detection, genome analysis, and proteome analysis. When a biomaterial is immobilized on an inorganic material substrate, a silane coupling agent is used as a crosslinking agent.
For this reason, various silane coupling agents have been proposed so far (Patent Documents 1 to 3).

JP 2005-225789 A JP 2007-186472 A JP 2006-143715 A

Since the silane compounds described in Patent Documents 1 to 3 are synthesized through a plurality of reaction steps, there is a problem that the synthesis is complicated and is not suitable for industrial production.
Therefore, a silane compound that can be easily synthesized using a compound that is easily available in the market is desired.
Then, this invention aims at provision of the novel silane compound which can be synthesize | combined simply. In addition, a silane-supported product in which proteins, cells, compounds, etc. are supported on the silane compound, a monolayer or multimolecular layer-forming composition containing the silane compound, the surface using the composition or the silane-supported material. An object of the present invention is to provide a substrate having a modified high function. These substrates can be expected to be applied as, for example, cell scaffolding materials or cells / proteins / compound separation / detection materials.

That is, this invention relates to the silane compound represented by Formula (1) as a 1st viewpoint.

[In Formula (1),
R ¹ represents an optionally substituted linear or branched alkyl group having 1 to 10 carbon atoms,
R ² represents an optionally substituted linear or branched alkoxy group having 1 to 6 carbon atoms, a halogen atom or a combination thereof,
a is an integer of 0 to 2,
X represents a hydrogen atom, a phenyl group, or a linear or branched alkyl group having 1 to 6 carbon atoms which may be substituted;
Y represents a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted;
Z is a monovalent organic group represented by formula (1-1) or formula (1-2),
R ^{3 to} R ⁵ each independently represents a hydrogen atom or an optionally substituted linear or branched alkyl group having 1 to 10 carbon atoms;
T ¹ represents a hydrocarbon ring or aromatic ring having 4 to 10 carbon atoms,
b is an integer of 4 or more, and the maximum value is the maximum number of substituents that T ¹ can take. ]
As a second aspect, the present invention relates to the silane compound according to the first aspect, wherein Z is a monovalent organic group represented by the formula (1-1), and R ³ and R ⁴ are hydrogen atoms.
As a third aspect, the present invention relates to a silane-supported product in which the silane compound described in the first or second aspect is supported on a supported material.
As a fourth aspect, the present invention relates to the silane-supported article according to the third aspect, wherein the supported substance is a protein, a cell, a compound, or a combination thereof.
As a fifth aspect, the present invention relates to the silane carrier according to the fourth aspect, wherein the protein is an antibody, a disease marker, a cell growth factor, a cell adhesion factor, or a combination thereof.
As a sixth aspect, the present invention relates to the silane support according to the fourth aspect, wherein the compound is a peptide, an amino acid, a pharmaceutical, a physiologically active substance, or a combination thereof.
As a seventh aspect, the present invention relates to a composition for forming a monomolecular layer or a multimolecular layer, comprising the silane compound described in the first aspect or the second aspect and an organic solvent.
As an eighth aspect, the present invention relates to the monomolecular layer or multimolecular layer forming composition according to the seventh aspect, further comprising water and / or an organic acid.
As a ninth aspect, the present invention relates to a substrate surface-modified with the silane compound described in the first aspect or the second aspect.
As a tenth aspect, the present invention relates to the base according to the ninth aspect, in which a supported substance is supported on the surface-modified surface of the base.
As an eleventh aspect, the present invention relates to the substrate according to the tenth aspect, in which the supported substance is a protein, a cell, a compound, or a combination thereof.
As a twelfth aspect, the present invention relates to the substrate according to the eleventh aspect, wherein the protein is an antibody, a disease marker, a cell growth factor, a cell adhesion factor, or a combination thereof.
As a thirteenth aspect, the present invention relates to the substrate according to the eleventh aspect, wherein the compound is a peptide, an amino acid, a pharmaceutical, a physiologically active substance, or a combination thereof.
As a fourteenth aspect, the present invention relates to the base according to any one of the ninth to thirteenth aspects, wherein the base is a flat substrate, a nonwoven fabric, or fine particles.
As a fifteenth aspect, the present invention relates to the substrate according to the fourteenth aspect, wherein the fine particles are silica-based fine particles, plastic-based fine particles, metal fine particles, or magnetic fine particles.
As a sixteenth aspect, the present invention relates to the substrate according to the fourteenth aspect or the fifteenth aspect, wherein the fine particles have a diameter of 0.001 μm to 1000 μm.
As a seventeenth aspect, the present invention relates to a cell scaffold material produced using the substrate according to any one of the ninth to sixteenth aspects.
As an eighteenth aspect, the present invention relates to a cell, protein, or compound separation / detection material produced using the substrate according to any one of the ninth to sixteenth aspects.
As a nineteenth aspect, a step of applying the monomolecular layer or multimolecular layer forming composition according to the seventh aspect or the eighth aspect to a substrate, a step of drying the substrate, a step of cleaning the substrate, and the substrate The manufacturing method of the base | substrate as described in any one of the 9th viewpoint thru | or 16th viewpoint including the process of drying.

The silane compound of the present invention can be easily synthesized using a compound that is readily available on the market. For example, the target silane compound can be obtained in only one step.
The silane compound of the present invention is useful as a cell scaffold material, a cell / protein separation material, a cell / protein detection material, and the like.
The cell / protein / compound separation / detection material of the present invention can selectively separate / detect cells / proteins or compounds.

FIG. 1 is an optical micrograph of HepG2 cells on a glass substrate treated in Example 1 in a cell adhesion test. FIG. 2 is an optical micrograph of HepG2 cells on the glass substrate treated in Example 2 in the cell adhesion test. FIG. 3 is an optical micrograph of HepG2 cells on the glass substrate treated in Example 3 in the cell adhesion test.

[Silane compound]
The present invention is a silane compound represented by the formula (1).

[In Formula (1),
R ¹ represents an optionally substituted linear or branched alkyl group having 1 to 10 carbon atoms,
R ² represents an optionally substituted linear or branched alkoxy group having 1 to 6 carbon atoms, a halogen atom or a combination thereof,
a is an integer of 0 to 2,
X represents a hydrogen atom, a phenyl group, or a linear or branched alkyl group having 1 to 6 carbon atoms which may be substituted;
Y represents a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted;
Z is a monovalent organic group represented by formula (1-1) or formula (1-2),
R ^{3 to} R ⁵ each independently represents a hydrogen atom or an optionally substituted linear or branched alkyl group having 1 to 10 carbon atoms;
T ¹ represents a hydrocarbon ring or aromatic ring having 4 to 10 carbon atoms,
b is an integer of 4 or more, and the maximum value is the maximum number of substituents that T ¹ can take. ]

Examples of the linear or branched alkyl group having 1 to 10 carbon atoms include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, and tertiary butyl group.
Examples of the linear or branched alkoxy group having 1 to 6 carbon atoms include methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, isobutoxy group, tertiary butoxy group, and hexyloxy group.
Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
Examples of the linear or branched alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an isobutyl group, and a tertiary butyl group.
Examples of the linear or branched alkylene group having 1 to 10 carbon atoms include methylene group, ethylene group, propylene group, isopropylene group, isobutylene group, and n-octylene group.

Examples of the hydrocarbon ring having 4 to 10 carbon atoms include a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, a norbornane ring, and a norbornene ring.
Examples of the aromatic ring having 4 to 10 carbon atoms include a benzene ring and a naphthalene ring.

The linear or branched alkyl group having 1 to 10 carbon atoms, the linear or branched alkoxy group having 1 to 6 carbon atoms, the linear or branched alkyl group having 1 to 6 carbon atoms, and the number of carbon atoms 1 The linear or branched alkylene group of 1 to 10 is an arbitrary substituent such as a hydroxy group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a cyano group, a phenyl group, a methoxycarbonyl group, an ethoxycarbonyl group, and a butoxycarbonyl group. It may be substituted with a group or the like.

Among these, a silane compound in which Z is a monovalent organic group represented by the formula (1-1) and R ³ and R ⁴ are hydrogen atoms is preferable from the viewpoint of easier synthesis.

[Method for producing silane compound]
The silane compound of the present invention can be synthesized, for example, by reacting the compound represented by the formula (2) and the compound represented by the formula (3) in an aprotic solvent in the presence of a base. .

[In the formulas (1) to (3), R ¹ , R ² , a, X, Y, and Z have the same definitions as those described in the above formula (1). ]

Examples of the compound represented by the formula (2) include acrylate N-succinimidyl methacrylate, N-succinimidyl methacrylate, 2-ethyl acrylate N-succinimidyl, 2-benzyl acrylate N-succinimidyl and 2-trifluoromethylacrylic acid. N-succinimidyl, N-hydroxy-5-norbornene-2,3-dicarboxyimidyl acrylate, N-hydroxy-5-norbornene-2,3-dicarboxyimidyl methacrylate, N-hydroxy 2-ethyl acrylate -5-norbornene-2,3-dicarboxyimidyl, 2-benzylacrylic acid N-hydroxy-5-norbornene-2,3-dicarboxyimidyl, 2-trifluoromethylacrylic acid N-hydroxy-5-norbornene -2,3-dicarboxyimidyl , N-hydroxyphthalimidyl acrylate, N-hydroxyphthalimidyl methacrylate, N-hydroxyphthalimidyl 2-ethylacrylate, N-hydroxyphthalimidyl 2-benzylacrylate, and 2-trifluoromethylacrylic Examples include acid N-hydroxyphthalimidyl.

Examples of the compound represented by the formula (3) include (3-mercaptopropyl) trimethoxysilane, (3-mercaptopropyl) triethoxysilane, 3-mercaptopropyl (dimethoxy) methylsilane, and 3-mercaptopropyl (diethoxy) methylsilane. 3-mercaptopropyl (methoxy) dimethylsilane, 3-mercaptopropyl (ethoxy) dimethylsilane, (3-mercaptooctyl) trimethoxysilane, (3-mercaptooctyl) triethoxysilane, 3-mercaptooctyl (dimethoxy) methylsilane, Examples include 3-mercaptooctyl (diethoxy) methylsilane, 3-mercaptooctyl (methoxy) dimethylsilane, and 3-mercaptooctyl (ethoxy) dimethylsilane.

The amount of the compound represented by the formula (3) is 0.1 to 20 times, preferably 0.5 to 10 times the molar equivalent of 1 mole equivalent of the compound represented by the formula (2). Equivalent, more preferably 1 to 5 times molar equivalent.

The solvent is not particularly limited as long as it is aprotic, but is preferably a polar solvent. Examples of aprotic polar solvents include ethers such as diethyl ether, tetrahydrofuran (THF), 1,3-dimethoxyethane, 1,4-dioxane; N, N-dimethylformamide (DMF), N, N-dimethyla Amides such as cetamide and hexamethylphosphoric triamide (HMPT); dichloromethane, dimethyl sulfoxide (DMSO), acetonitrile and the like. These solvents can be used alone or in combination of two or more.

Examples of the base include tertiary amines such as triethylamine and diisopropylethylamine, among which triethylamine is preferable.
The amount of the base used is 0.001 to 20 times molar equivalent, preferably 0.005 to 5 times molar equivalent, more preferably 0.01 to 1 molar equivalent of the compound represented by the formula (2). To 1-fold molar equivalent.

As reaction conditions, the reaction time is appropriately selected from 0.01 to 100 hours and the reaction temperature is from 0 to 100 ° C. Preferably, the reaction time is 0.1 to 10 hours and the reaction temperature is 0 to 30 ° C.

[Silane-supported product in which a silane compound is supported on a supported material]
The present invention is a silane-supported product in which the silane compound is supported on a supported material.
Examples of the method for supporting the silane compound on the supported material include known methods, such as a method in which the silane compound is treated with a solvent in which the supported material is dissolved and dispersed, followed by drying or heat treatment. .

Examples of the supported substance include proteins, cells, and compounds.
Examples of the protein include carcinoembryonic antigen, squamous cell carcinoma-related antigen, cytokeratin 19 fragment, sialylated sugar chain antigen KL-6, natriuretic peptide, troponin, myoglobin, and other disease markers; interleukin-1 (IL-1 ), Interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6) ), Interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11) ), Interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL- 14), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interferon-α (IFN-α), interferon-β (IFN-β) , Interferon-γ (IFN-γ), granulocyte colony stimulating factor (G-CSF), monocyte colony stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF) , Flk2 / flt3 ligand (FL), leukemia cell inhibitory factor (LIF), oncostatin M (OM), erythropoietin (EPO), thrombopoietin (TPO), transforming growth factor-α (TGF-α), transforming growth factor -Β (TGF-β), macrophage inflammatory protein-1α (MIP-1α), Skin cell growth factor (EGF), fibroblast growth factor-1, 2, 3, 4, 5, 6, 7, 8, or 9 (FGF-1, 2, 3, 4, 5, 6, 7, 8 9), nerve cell growth factor (NGF), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), protease nexin I, protease nexin II, platelet derived growth factor (PDGF), cholinergic differentiation factor (CDF), chemokine, Notch ligand (such as Delta1), Wnt protein, angiopoietin-like protein 2, 3, 5 or 7 (Angpt2, 3, 5, 7), insulin-like growth factor (IGF), insulin-like growth factor binding protein (IGFBP), pleiotrophin (Pleiotrophin), insulin, growth factors such as growth hormone; collagen I to XIX, fib Nectin, vitronectin, laminin-1 to 12, nitogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocholine, desmoglein, various integrins, E- Cell adhesion factors such as selectin, P-selectin, L-selectin, immunoglobulin superfamily, matrigel, poly-D-lysine and poly-L-lysine; and various antibodies such as IgG, IgM, IgA, IgD and IgE Can be mentioned.

Examples of the cells include fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monocytes, bone cells, bone marrow cells, pericytes, dendritic cells, keratinocytes, fat Cells, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatocytes, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglia, astrocytes , Cardiac cells, esophageal cells, muscle cells (eg smooth muscle cells or skeletal muscle cells), pancreatic beta cells, melanocytes, hematopoietic progenitor cells, mononuclear cells, embryonic stem cells (ES cells), embryonic tumor cells, embryos Reproductive stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, reproductive stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells And various cell lines (for example, HCT116, Huh7, HEK293 (human embryonic kidney cell), HeLa (human cervical cancer cell line), HepG2 (human hepatoma cell line), UT7 / TPO (human leukemia cell line), CHO ( Chinese hamster ovary cell line), MDCK, MDBK, BHK, C-33A, HT-29, AE-1, 3D9, Ns0 / 1, Jurkat, NIH3T3, PC12, S2, Sf9, Sf21, High Five, Vero), etc. Can be mentioned.

Examples of the compound include angiotensin I to IV, bradykinin, fibrinopeptide, natriuretic peptide, urodilatin, guanylin, endothelin 1 to 3, salusin, urotensin, oxytocin, neurophysin, vasopressin, adrenocorticotropic hormone, melanocyte stimulating hormone , Endorphins, lipotropins, urocortin 1 to 3, luteinizing hormone releasing hormone, growth hormone releasing hormone, somatostatin, cortisatin, prolactin releasing peptide, metastin, tachykinin, substance P, neurokinin, endokinin, neurotensin, neuromedin, zenin, ghrelin , Obestatin, melanin-concentrating hormone, orexin, neuropeptide, dynorphin, neoendorpy , Endomorphin, nociceptin, pyroglutamylated RF amide peptide, galanin, gastrin, cholecystokinin, secretin, relaxin, glucagon, glicentin, adrenomedullin, amylin, calcitonin, parathyroid hormone, defensin, thymosin and other peptides; alanine, arginine, Asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, cystine, hydroxyproline, hydroxylysine, dihydroxyphenylalanine, thyroxine, Phosphoserine, desmosine, β-alanine, sarcosine, ornithine, creatine , Γ-aminobutyric acid, theanine, kainic acid, domoic acid, ibotenic acid and other amino acids; ampicillin, streptomycin, amphetamine, mescaline, oseltamivir phosphate, metformin, diphenhydramine, ephedrine hydrochloride, amantadine hydrochloride, procaine hydrochloride, chlorpromazine, Pharmaceuticals such as ethyl aminobenzoate; sugars such as D-glucosamine, D-galactosamine, neuraminic acid, hyaluronic acid, chondroitin sulfate, heparan sulfate, heparin; and dopamine, serotonin, noradrenaline, adrenaline, 3- (3,4-dichlorophenyl) ) -1,1-dimethylurea (DCMU), physiologically active substances such as atrazine, linuron and simazine.

[Composition for forming a monomolecular layer or a multimolecular layer]
The present invention is a composition for forming a monomolecular layer or a multimolecular layer comprising a silane compound represented by the above formula (1) and an organic solvent.
In this specification and the like, “monolayer” refers to a state in which silane compounds bonded to a substrate are arranged in a single layer. On the other hand, the “multimolecular layer” refers to a state in which another silane compound is bonded to the silane compound bonded to the substrate and several layers of the silane compound are stacked.

The organic solvent used in the composition for forming a monomolecular layer or a multimolecular layer of the present invention is not particularly limited as long as the silane compound represented by the above formula (1) can be dissolved. Ether, ester, carbonization Examples thereof include hydrogen, ketone, aldehyde, or higher alcohol, such as methyl cellosolve acetate, ethyl cellosolve acetate, propylene glycol, propylene glycol monomethyl ether, propylene glycol monoethyl ether, methyl isobutyl carbinol, propylene glycol monobutyl ether, propylene glycol monomethyl. Ether acetate, propylene glycol monoether ether acetate, propylene glycol monopropyl ether acetate, propylene glycol monobutyl ether acetate, 2 Ethyl hydroxypropionate, ethyl 2-hydroxy-2-methylpropionate, ethyl ethoxyacetate, ethyl hydroxyacetate, methyl 2-hydroxy-3-methylbutanoate, methyl 3-methoxypropionate, ethyl 3-methoxypropionate, 3- Ethyl ethoxypropionate, methyl 3-ethoxypropionate, methyl pyruvate, ethyl pyruvate, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monopropyl ether, ethylene glycol monobutyl ether, ethylene glycol monomethyl ether acetate, ethylene glycol Monoethyl ether acetate, ethylene glycol monopropyl ether acetate, ethylene glycol monobutyl ether acetate , Diethylene glycol dimethyl ether, diethylene glycol diethyl ether, diethylene glycol dipropyl ether, diethylene glycol dibutyl ether, propylene glycol monomethyl ether, propylene glycol dimethyl ether, propylene glycol diethyl ether, propylene glycol dipropyl ether, propylene glycol dibutyl ether, ethyl lactate, propyl lactate, lactic acid Isopropyl, butyl lactate, isobutyl lactate, methyl formate, ethyl formate, propyl formate, isopropyl formate, butyl formate, isobutyl formate, amyl formate, isoamyl formate, methyl acetate, ethyl acetate, amyl acetate, isoamyl acetate, hexyl acetate, methyl propionate , Ethyl propionate, propyl propionate, Isopropyl lopionate, butyl propionate, isobutyl propionate, methyl butyrate, ethyl butyrate, propyl butyrate, isopropyl butyrate, butyl butyrate, isobutyl butyrate, ethyl hydroxyacetate, ethyl 2-hydroxy-2-methylpropionate, 3-methoxy-2 -Methyl methyl propionate, methyl 2-hydroxy-3-methylbutyrate, ethyl methoxyacetate, ethyl ethoxyacetate, methyl 3-methoxypropionate, ethyl 3-ethoxypropionate, ethyl 3-methoxypropionate, 3-methoxybutyl acetate , 3-methoxypropyl acetate, 3-methyl-3-methoxybutyl acetate, 3-methyl-3-methoxybutyl propionate, 3-methyl-3-methoxybutyl butyrate, methyl acetoacetate, toluene, xylene Methyl ethyl ketone, methyl propyl ketone, methyl butyl ketone, 2-heptanone, 3-heptanone, 4-heptanone, cyclopentanone, cyclohexanone, N, N-dimethylformamide, N-methylacetamide, N, N-dimethylacetamide, N-methyl Examples include pyrrolidone and γ-butyrolactone. These solvents can be used alone or in combination of two or more.
In particular, propylene glycol monomethyl ether can be preferably used.

Moreover, the density | concentration which dissolves the silane compound represented by the said Formula (1) in the said organic solvent is arbitrary, but with respect to the total mass (total mass) of the silane compound represented by the said Formula (1), and an organic solvent. The concentration of the silane compound represented by the formula (1) is 0.001 to 90% by mass, preferably 0.002 to 80% by mass, and more preferably 0.005 to 70% by mass.

In addition, the monomolecular layer or multimolecular layer forming composition of the present invention may contain water and / or an organic acid.
Examples of the water include purified water, purified water, hard water, soft water, natural water, deep ocean water, electrolytic alkali ion water, electrolytic acid ion water, ion water, and cluster water.
Examples of the organic acid include acetic acid, propionic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, oxalic acid, maleic acid, methylmalonic acid, adipic acid, sebacic acid, gallic acid , Butyric acid, meritic acid, arachidonic acid, mikimic acid, 2-ethylhexanoic acid, oleic acid, stearic acid, linoleic acid, linolenic acid, salicylic acid, benzoic acid, p-aminobenzoic acid, p-toluenesulfonic acid, benzenesulfonic acid Monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, trifluoroacetic acid, formic acid, malonic acid, sulfonic acid, phthalic acid, fumaric acid, citric acid, and tartaric acid.

The concentration of water contained in the composition is arbitrary, but the concentration of water is 0.001 to the total mass of the organic solvent (the total mass when two or more organic solvents are used). It is 99 mass%, Preferably it is 0.002 to 80 mass%, More preferably, it is 0.005 to 50 mass%.
The concentration of the organic acid contained in the composition is arbitrary, but the concentration of the organic acid relative to the total mass of the organic solvent (the total mass when two or more organic solvents are used) is 0.00. It is 001 to 50% by mass, preferably 0.002 to 30% by mass, and more preferably 0.005 to 10% by mass.

[Surface-modified substrate, and substrate having a supported substance supported on the surface-modified surface of the substrate]
The present invention is a substrate whose surface is modified with the silane compound.
The substrate surface-modified with the silane compound of the present invention can be obtained by silanizing the substrate surface using a silane compound.
The substrate is not particularly limited as long as the surface is silanized by the silane compound of the present invention, but glass, silica, silicon wafer, alumina, talc, clay, aluminum, iron, mica, titanium oxide, Examples thereof include quartz, a substrate, a nonwoven fabric, and fine particles. The shape of the substrate is not particularly limited, and may be a plate shape, a film shape, a three-dimensional molded body, or the like.
Among these, a flat substrate, a nonwoven fabric, and fine particles are preferable. The fine particles are preferably silica-based fine particles, plastic-based fine particles, metal fine particles, and magnetic fine particles. The diameter of the fine particles is usually 0.001 μm to 1000 μm, preferably 0.01 μm to 500 μm.

Examples of the silanization method include known methods. The substrate is treated with a solvent in which the silane compound of the present invention is dissolved and then subjected to heat treatment. The substrate is treated with an alkaline solution, and then the silane of the present invention is treated. Examples thereof include a method of treating with an alcohol solution of the compound, followed by a heat treatment, and a method of vibrating and stirring the substrate in an organic solvent in which the silane compound of the present invention is dissolved at reflux or at room temperature.

The present invention also provides a substrate in which a supported substance is supported on the surface-modified surface of the substrate.
Examples of the method for supporting the supported substance on the surface-modified surface of the substrate include a method in which the surface is treated with a solvent in which the supported substance is dissolved and dispersed, followed by drying or heat treatment.
As the supported material, the supported materials listed in paragraphs [0021] to [0023] can be used.

[Materials for cell scaffolds]
The present invention is a cell scaffold material produced using the above-mentioned substrate.
The “material for cell scaffold” means a material in which various cell functions such as cell adhesion, proliferation, differentiation, activation, migration, migration, and morphological change are expressed and promoted by contact of the cell with the material. .
Base materials for producing cell scaffolding materials include hydroxyapatite, β-TCP (tricalcium phosphate), ceramics such as α-TCP, glass, polyvinyl chloride, cellulose polymers such as ethyl cellulose and acetyl cellulose, Polystyrene, polymethyl methacrylate, polycarbonate, polysulfone, polyurethane, polyester, polyamide, polypropylene, polyethylene, polybutadiene, poly (ethylene-vinyl acetate) copolymer, poly (butadiene-styrene) copolymer, poly (butadiene-acrylonitrile) copolymer, poly (ethylene) -Ethyl acrylate) copolymer, poly (ethylene-methacrylate) copolymer, polychloroprene, styrene resin, chlorosulfonated polyethylene, Examples thereof include plastics such as ethylene vinyl acetate and acrylic block copolymers. In addition, these base materials may be made of any one of the above substances, or may be made of a composite containing a plurality of kinds.
As the culture equipment holding the cell scaffold material, petri dishes, flasks, plastic bags, Teflon (registered trademark) bags, dishes, petri dishes, tissue culture dishes, multi dishes, micro plates, Examples include a microwell plate, a multiplate, a multiwell plate, a chamber slide, a cell culture flask, a spinner flask, a tube, a tray, a culture bag, and a roller bottle.
The form of the cell scaffold material is not particularly limited, and examples thereof include a sponge, a mesh, and a non-woven cloth-like molded article. However, the material may be porous so that cells can be uniformly seeded. preferable.
The shape of the cell scaffold material is not particularly limited, and any shape such as a membrane shape, a spherical shape, a disk shape, a particle shape, or a block shape can be used.

Cell seeding on the cell scaffolding material can be performed using a known method, and a suspension obtained by suspending cells in a liquid such as a buffer solution, physiological saline, a culture solution, or a collagen solution. The cell scaffolding material can be immersed in the suspension, or the suspension can be injected into the cell scaffolding material. Moreover, you may seed | inoculate on drawing pressure or pressurization conditions as needed. The number of cells to be seeded (seeding density) can be adjusted by the cell concentration and the injection amount of the suspension, and it is preferable to adjust appropriately according to the characteristics of the type of cells used and the material for the cell scaffold. Culture conditions, culture apparatus, type of medium, type of scaffold material, content, type of additive, content of additive, content of additive, culture period, culture temperature, and the like when culturing cells are appropriately selected by the party.

[Cell, protein or compound separation / detection material]
The present invention is a cell / protein / compound separation / detection material produced using the substrate.
“Materials for cell / protein separation / detection” refers to a sample or antibody carrying a measurement target, which selectively separates target cells / proteins from biological tissue, body fluid, bone marrow fluid, blood, cell culture fluid, etc. Means material.
The shape of the cell / protein separation / detection material is not particularly limited, and any shape such as a flat substrate shape, a filter shape, or a fine particle shape can be used. The form of the filter may take any form such as a membrane, a sphere, a container, a cassette, a bag, a tube, and a column. The material of the filter is polyolefin such as polypropylene, polyethylene, high density polyethylene, low density polyethylene, polyester, vinyl chloride, polyvinyl alcohol, vinylidene chloride, rayon, vinylon, polystyrene, acrylic (polymethyl methacrylate, polyhydroxyethyl methacrylate, acrylonitrile, Acrylic, acrylate, etc.), nylon, polyurethane, polyimide, aramid, polyamide, cupra, kevlar, carbon, polyacrylate, phenol, tetron, pulp, hemp, cellulose, kenaf, chitin, chitosan, glass, cotton, etc. A material selected from one type is used.

Separation of cells / proteins can be performed using known methods, and materials for cell / protein separation are added to suspensions of cells / proteins such as biological tissues, body fluids, bone marrow fluids, blood, cell culture fluids, etc. Alternatively, the suspension can be added or injected into the cell / protein separation material. In the case of a filter-like cell / protein separation material, the suspension can be permeated. The captured cells / proteins can be washed or recovered using an appropriate buffer, physiological saline, medium, or the like. At the time of collection, the cells / proteins can be peeled from the cell / protein separation material by using various chelating agents, various surfactants, ultrasonic waves and enzymes. When the cell / protein separating material is a magnetic fine particle, the cell / protein separating material retaining the cell / protein can be recovered by magnetic force. Further, if necessary, the cells captured by the cell / protein separation material can be differentiated or proliferated into specific cells by culturing them under appropriate conditions.

The separated cells / proteins can be detected using a known method. The cell / protein to be detected may be held in the cell / protein separation material or may be peeled from the material. For example, when the detection target is a cell, the cell captured by the cell / protein separation material can be observed using a standard microscope in this field. At this time, the separated cells may be stained with a specific antibody. Cells can be detected by ELISA or flow cytometry using specific antibodies that recognize cell surface markers. Alternatively, cells can be detected by extracting DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) from the separated cells and analyzing them by Southern blotting, Northern blotting, RT-PCR, or the like. When measuring the number of cells, colony formation method, crystal violet method, thymidine incorporation method, trypan blue staining method, 3- (4,5-Dimethylthyl-2-yl) -2,5-Diphenyltetrazalum Bromide (MTT) A staining method, a WST-1 (registered trademark) staining method, a WST-8 (registered trademark) staining method, a flow cytometry method, a method using an automatic cell number measuring apparatus, or the like can be used.
For example, when the detection target is a protein or peptide, the target protein or peptide is detected by a method using a mass spectrometer, a method using an antigen-antibody reaction such as Western blotting, dot blotting, ELISA, or flow cytometry. be able to. In addition, proteins can be separated by various electrophoresis methods and detected by Coomassie brilliant blue staining or silver staining. As a protein or peptide quantification method, an ultraviolet absorption method, Bradford method, Raleigh method, phenol reagent method, bicinchoninic acid method (BCA method) or the like can be used.
Moreover, the protein or peptide to be detected can be labeled in advance. Examples of methods for labeling proteins or peptides include fluorescent labels, enzyme labels, biotin labels, polyethylene glycol (PEG) labels, and the like, and any method can be used. In the case of the fluorescent labeling method, substances such as Cy3, Cy5, FITC (fluorescein isothiocyanate) and rhodamine can be used. In the case of the enzyme labeling method, substances such as peroxidase, alkaline phosphatase, acid phosphatase, and glucose oxidase can be used.

[Manufacturing method of substrate]
The method for producing a substrate of the present invention comprises a step of applying the monomolecular layer or multimolecular layer forming composition to a substrate, a step of drying the substrate, a step of washing the substrate, and a step of drying the substrate. Including.
Examples of a method for applying the monomolecular layer or multimolecular layer forming composition to a substrate include, for example, a cast coating method, a spin coating method, and a blade coating method on the substrate. Dip coating method, roll coating method, bar coating method, die coating method, ink jet method, printing method (such as relief printing, intaglio printing, planographic printing, and screen printing).
Moreover, as a method of drying a board | substrate, the well-known drying method using a hotplate, oven, etc. is mentioned.
Furthermore, as a method for cleaning the substrate, a known cleaning method using water, an organic solvent or the like can be used.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

[Synthesis Example 1]

A 500 mL four-necked flask equipped with a magnetic stirrer was charged with N-succinimidyl acrylate (12.50 g, 0.0739 mol), (3-mercaptopropyl) trimethoxylane (15.24 g, 0.0776 mol), triethylamine (0 .37 g, 0.0037 mol) and acetonitrile (250 g) were charged, and the mixture was stirred at 5 to 10 ° C. for 4 hours. Thereafter, the solvent in the reaction solution and the remaining triethylamine were distilled off under reduced pressure using an evaporator to obtain Compound 1 (yield: 27.00 g, yield: 100%).
1H-NMR (400MHz) in CDCl 3: 0.73-0.79ppm (m, 2H), 1.66-1.76ppm (m, 2H), 2.59ppm (t, = 7.3 Hz, 2H), 2.76-2.95ppm (m, 8H ), 3.57ppm (s, 9H)

[Examples 1 to 3]
A solution was prepared by stirring and mixing 0.1 g of Compound 1, 0.5 g of water, and 9.5 g of PGME (propylene glycol monomethyl ether). Then, it filtered using the polyethylene micro filter with the hole diameter of 0.03 micrometer, and prepared the composition for monomolecular layer or multimolecular layer formation.
The prepared composition was applied onto a glass substrate using a spin coater, and baked on a hot plate at 180 ° C. for 1 minute. Then, it was immersed in propylene glycol monomethyl ether for 1 minute, spin-dried, and then dried at 100 ° C. for 30 seconds to form a monomolecular layer or a multimolecular layer on the glass substrate.
The obtained glass substrate was immersed in the following reagents for 4 hours and then washed with water.
Example 1: No immersion Example 2: Poly-L-lysine FITC Labeled reagent (Aldrich) 0.1 wt% aqueous solution Example 3: Collagen Type FITC Conjugate from bovine skin (Aldrich) 0.1 wt% aqueous solution

[Comparative Examples 1 to 3]
The glass substrate was immersed in the following reagents for 4 hours and then washed with water.
Comparative Example 1: No immersion Comparative Example 2: Poly-L-lysine FITC Labeled Reagent (Aldrich) 0.1 wt% aqueous solution Comparative Example 3: Collagen Type FITC Conjugate from bovine skin (Aldrich) 0.1 wt% aqueous solution

[Poly-L-lysine and collagen loading confirmation]
Each glass substrate treated in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3 is placed on a 24-hole flat bottom microplate (manufactured by Corning), and PBS (phosphate buffered physiological) 1 mL of saline solution (Sigma Aldrich) was added. The fluorescence intensity (relative fluorescence unit, RFU) of each glass substrate at an excitation wavelength of 490 nm and a fluorescence wavelength of 530 nm was measured using a 2104 EnVision (registered trademark) multi-label counter (manufactured by PerkinElmer Japan Co., Ltd.). By this measurement, adsorption of FITC-labeled poly-L-lysine and collagen can be confirmed. The results are shown in Table 1 below.

As a result of the measurement of fluorescence intensity, it was shown that poly-L-lysine and collagen labeled with FITC by the treatment of Examples 2 and 3 were more adsorbed on the glass substrate than in the comparative example.

[Cell adhesion test]
(Preparation of cell suspension)
As a cell used for the test, a human liver cancer cell line HepG2 (DS Pharma Biomedical) was used. The culture medium used was a DMEM medium (manufactured by Wako Pure Chemical Industries, Ltd.) containing 10% (v / v) FBS (fetal bovine serum (manufactured by Biological Industries)). The cells were statically cultured for 2 days or longer in a petri dish (10 mL of medium) having a diameter of 10 cm while maintaining a 5% carbon dioxide concentration in a 37 ° C. CO 2 incubator. Subsequently, the cells were washed with 5 mL of PBS, 1 mL of trypsin-EDTA solution (manufactured by Invitrogen) was added, the cells were detached, and suspended in 10 mL of the above medium. After centrifuging this suspension (manufactured by Kubota Corporation, model number 5900, 1500 rpm / 3 minutes, room temperature), the supernatant was removed, and the above medium was added to prepare a cell suspension.

(Adhesion of cells to glass substrate)
The above HepG2 cell suspension prepared by arranging each glass substrate treated in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2 and Comparative Example 3 on a 24-hole flat bottom microplate (manufactured by Corning). 1 mL of each turbid solution was added so that it might become 2.5 * 10 < ⁵ > cells / well. Thereafter, the sample was allowed to stand in a CO 2 incubator at 37 ° C. for 24 hours while maintaining a 5% carbon dioxide concentration.

(Measurement of cell attachment number)
After 24 hours, each glass substrate treated in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2 and Comparative Example 3 in which the above cell adhesion test was performed was subjected to an optical microscope (CK30, manufactured by OLYMPUS). -F100, magnification of 100) was used to observe the state of cell attachment. Subsequently, each of these glass substrates was transferred to another 24-hole flat bottom microplate (Corning) and washed with 1 mL of PBS. After removing PBS, 500 μL of trypsin-EDTA solution (Invitrogen) was added. After 5 to 10 minutes, 500 μL of DMEM medium (manufactured by Wako Pure Chemical Industries, Ltd.) containing 10% (v / v) FBS was added, and the detached cells were transferred to a 1.5 mL micro test tube (manufactured by Eppendorf). did. After centrifugation (manufactured by Tommy Seiko Co., Ltd., model number: MX-307, 300 G / 3 min, room temperature), the supernatant was removed, and DMEM medium containing 10% (v / v) FBS (Wako Pure Chemical Industries, Ltd.) 100 μL was added to prepare a cell suspension. After adding an equal amount of trypan blue staining solution to this suspension, the number of viable cells (cell adhesion number) was measured with a blood cell counter (manufactured by Elma Sales Co., Ltd.).
The number of cells adhered to each glass substrate treated in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3 was compared after 24 hours of culture. The results are shown in Table 2 below. Moreover, the observation result in the optical microscope regarding Example 1, Example 2, and Example 3 is shown to FIG.

As a result of the cell adhesion test, the number of HepG2 cells adhered increased in each glass substrate treated in Examples 2 and 3 with respect to the glass substrate treated in Example 1 after 24 hours of culture. In particular, in Example 2, the number of cells after 24 hours was larger than the number of seeded cells, suggesting that cell proliferation was promoted after adhesion. Moreover, also by observation with a microscope, it was confirmed that the number of HepG2 cells attached to each glass substrate treated in Examples 2 and 3 increased with respect to the glass substrate treated in Example 1.

Claims (19)

1. Silane compound represented by Formula (1).
   

   

   [In Formula (1),
   R ¹ represents an optionally substituted linear or branched alkyl group having 1 to 10 carbon atoms,
   R ² represents an optionally substituted linear or branched alkoxy group having 1 to 6 carbon atoms, a halogen atom or a combination thereof,
   a is an integer of 0 to 2,
   X represents a hydrogen atom, a phenyl group, or a linear or branched alkyl group having 1 to 6 carbon atoms which may be substituted;
   Y represents a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted;
   Z is a monovalent organic group represented by formula (1-1) or formula (1-2),
   R ^{3 to} R ⁵ each independently represents a hydrogen atom or an optionally substituted linear or branched alkyl group having 1 to 10 carbon atoms;
   T ¹ represents a hydrocarbon ring or aromatic ring having 4 to 10 carbon atoms,
   b is an integer of 4 or more, and the maximum value is the maximum number of substituents that T ¹ can take. ]
2. The silane compound according to claim 1, wherein Z is a monovalent organic group represented by formula (1-1), and R ³ and R ⁴ are hydrogen atoms.
3. A silane-supported product in which the silane compound according to claim 1 or 2 is supported on a supported material.
4. The silane-supported product according to claim 3, wherein the supported material is a protein, a cell, a compound, or a combination thereof.
5. The silane carrier according to claim 4, wherein the protein is an antibody, a disease marker, a cell growth factor, a cell adhesion factor, or a combination thereof.
6. The silane carrier according to claim 4, wherein the compound is a peptide, an amino acid, a pharmaceutical, a physiologically active substance, or a combination thereof.
7. A composition for forming a monolayer or a multi-layer comprising the silane compound according to claim 1 or 2 and an organic solvent.
8. The composition for forming a monomolecular layer or a multimolecular layer according to claim 7, further comprising water and / or an organic acid.
9. A substrate surface-modified with the silane compound according to claim 1 or 2.
10. The substrate according to claim 9, wherein a supported substance is supported on the surface-modified surface of the substrate.
11. The substrate according to claim 10, wherein the supported substance is a protein, a cell, a compound, or a combination thereof.
12. The substrate according to claim 11, wherein the protein is an antibody, a disease marker, a cell growth factor, a cell adhesion factor, or a combination thereof.
13. The substrate according to claim 11, wherein the compound is a peptide, an amino acid, a pharmaceutical, a physiologically active substance, or a combination thereof.
14. The substrate according to any one of claims 9 to 13, wherein the substrate is a flat substrate, a nonwoven fabric, or fine particles.
15. 15. The substrate according to claim 14, wherein the fine particles are silica-based fine particles, plastic-based fine particles, metal fine particles, or magnetic fine particles.
16. The substrate according to claim 14 or 15, wherein the fine particles have a diameter of 0.001 µm to 1000 µm.
17. A cell scaffold material produced using the substrate according to any one of claims 9 to 16.
18. A cell, protein or compound separation / detection material produced using the substrate according to any one of claims 9 to 16.
19. A step of applying the monomolecular layer or multimolecular layer forming composition according to claim 7 or 8 to a substrate, a step of drying the substrate, a step of washing the substrate, and a step of drying the substrate The manufacturing method of the base | substrate of any one of Claim 9 thru | or 16 containing.

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