JP4640150B2 - Biochip and method of use thereof - Google Patents

Description

  The present invention relates to a biochip for screening low molecular weight compounds having specific selectivity with a protein.

A method for screening a low molecular weight compound that specifically binds to a specific protein in a cell and adjusts its function and explores the function of the protein from the phenotype represented by the low molecular weight compound has attracted attention. Various screening methods have been established to obtain low molecular weight ligands that can act in combination with specific proteins. Among them, the small molecule developed by Macbeath and Schreiber et al. It is a microarray. (Non-patent document 1) In this method, a synthesized compound is immobilized on a single glass slide substrate in units of thousands to tens of thousands, and this array is treated with a fluorescently-labeled protein, whereby a protein and a small molecule are treated. The interaction between multiple samples can be detected simultaneously.
In such biochip signal detection, non-specific adsorption of a substance to be detected on a substrate causes a reduction in the signal-to-noise ratio, thereby reducing the detection accuracy.
For this reason, a biochip with a small amount of nonspecific adsorption of protein is required.
J. Am. Chem. Soc., 121 7967-7968 (1999)

  An object of the present invention is to provide a biochip substrate with high detection accuracy by suppressing nonspecific adsorption of a substance to be detected.

The present invention
(1) A biochip in which a low molecular weight compound is immobilized on the surface of a solid phase substrate, the monomer having an alkylene glycol residue, a monomer having a crosslinkable functional group, and an active ester group on the surface of the substrate A biochip characterized in that a polymer substance obtained by polymerizing a monomer containing the polymer is formed on a substrate surface ;
(2) The biochip according to (1), wherein the monomer having an alkylene glycol residue is a monomer represented by the following general formula [1]:

(Wherein R1 represents a hydrogen atom or a methyl group, R2 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) Represents an integer, and the repeated Xs may be the same or a chain of different alkylene glycol residues.
(3) The biochip according to (2), wherein the monomer having an alkylene glycol residue is methoxypolyethylene glycol (meth) acrylate,
(4) The biochip according to (3), wherein an average chain of ethylene glycol of the methoxypolyethylene glycol (meth) acrylate is 3 to 100,
( 5 ) The monomer having a crosslinkable functional group is represented by the following general formula [2] (wherein R3 represents a hydrogen atom or a methyl group, and Z represents an alkyl group having 1 to 20 carbon atoms, provided that Z may be omitted, and at least one of A1, A2, and A3 is a hydrolyzable group, and the other is an alkyl group.) The biochip according to ( 1 ),

( 6 ) In the general formula [2], at least one of A1, A2, and A3 is an alkoxyl group ( 5 ),
( 7 ) The biochip according to any one of (1) to ( 6 ) , wherein the high-molecular substance immobilizes a low-molecular compound via the active ester group ,
( 8 ) The monomer having an active ester group is represented by the following general formula [3] (wherein R2 represents a hydrogen atom or a methyl group, Y represents an alkyleneoxy group or an alkyl group having 1 to 10 carbon atoms. W Represents an active ester group, q represents an integer of 1 to 20. When q is an integer of 2 or more and 20 or less, repeated Ys may be the same or a chain of different alkyleneoxy groups. ( 7 ) The biochip according to ( 7 ),

( 9 ) The biochip according to ( 8 ), wherein the monomer having an active ester group is p-nitrophenyloxycarbonyl-4,5-ethylene glycol methacrylate or N-hydroxysuccinimide-4,5-ethylene glycol methacrylate.
( 10 ) The biochip according to any one of (1) to ( 9 ), wherein the solid phase substrate is made of plastic,
( 11 ) The biochip according to ( 10 ), wherein the plastic is a saturated cyclic polyolefin,
( 12 ) The biochip according to any one of (1) to ( 9 ), wherein the solid phase substrate is made of glass,
( 13 ) The biochip as set forth in any one of (1) to ( 12 ), wherein the solid phase substrate is provided with a flow path.
( 14 ) A method for using a biochip, characterized by reacting a bio-derived substance with the biochip according to any one of (1) to ( 11 ),
( 15 ) After using the biochip according to any one of ( 7 ) to ( 9 ), the biochip is reacted, and then the biochip is used,
It is.

  According to the biochip of the present invention, non-specific adsorption of a detection target substance can be suppressed, and a microarray with high detection accuracy can be obtained.

  The biochip substrate of the present invention is characterized by having a polymer substance obtained by polymerizing a monomer having an alkylene glycol residue on the substrate surface. A polymer substance obtained by polymerizing a monomer having an alkylene glycol residue is a polymer that suppresses nonspecific adsorption of a physiologically active substance, and an alkylene glycol residue prevents nonspecific adsorption of a physiologically active substance. Play a role to suppress.

  The monomer having an alkylene glycol residue used in the present invention is not particularly limited in structure, but is a (meth) acryl group represented by the general formula [1] and an alkylene glycol residue X having 1 to 10 carbon atoms. A compound consisting of a chain is preferred.

Carbon number of the alkylene glycol residue X in Formula [1] is 1-10, More preferably, it is 1-6, More preferably, it is 2-4, More preferably, it is 2-3, Preferably it is 2. The repeating number p of the alkylene glycol residue X is an integer of 1 to 100, more preferably an integer of 2 to 100, still more preferably an integer of 2 to 95, and most preferably an integer of 20 to 90. is there. When the number of repetitions 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.
Examples of such a monomer include monosubstituted hydroxyl groups such as methoxypolyethylene glycol (meth) acrylate, 2-hydroxyethyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate, and 2-hydroxybutyl (meth) acrylate. (Meth) acrylates of esters, glycerol mono (meth) acrylate, (meth) acrylate having polypropylene glycol as a side chain, 2-methoxyethyl (meth) acrylate, 2-ethoxyethyl (meth) acrylate, methoxydiethylene glycol (meth) Acrylate, ethoxydiethylene glycol (meth) acrylate, ethoxypolyethylene glycol (meth) acrylate, etc., but methoxypolyethylene glycol methacrylate is preferred because of its availability. Arbitrariness.

The monomer having a crosslinkable functional group used in the present invention is not particularly limited as long as the reaction of the crosslinkable functional group does not proceed during the synthesis of the polymer compound. It is preferable that it is a monomer represented by this.
As the crosslinkable functional group, for example, a functional group that generates a silanol group by hydrolysis, a glycidyl group, or the like is used, but a functional group that generates a silanol group by hydrolysis is preferable because it can be crosslinked at a lower temperature.

  The monomer having a functional group that generates a silanol group by hydrolysis has a (meth) acryl group and a functional group that generates a silanol group by hydrolysis bonded directly or via an alkyl chain having 1 to 20 carbon atoms. A compound. The functional group that generates a silanol group by hydrolysis is a group that readily undergoes hydrolysis and forms a silanol group when contacted with water. For example, a halogenated silyl group, an alkoxysilyl group, a phenoxysilyl group, an acetoxysilyl group Etc. Among these, an alkoxysilyl group is preferable because it easily generates a silanol group. Examples of the monomer containing an alkoxysilyl group include 3- (meth) acryloxypropenyltrimethoxysilane, 3- (meth) acryloxypropylbis (trimethylsiloxy) methylsilane, and 3- (meth) acryloxypropyldimethyl. Methoxysilane, 3- (meth) acryloxypropyldimethylethoxysilane, 3- (meth) acryloxypropylmethyldimethoxysilane, 3- (meth) acryloxypropylmethyldiethoxysilane, 3- (meth) acryloxypropyltrimethoxy Silane, 3- (meth) acryloxypropyltriethoxysilane, 3- (meth) acryloxypropyltris (methoxyethoxy) silane, 8- (meth) acryloxyoctanyltrimethoxysilane, 11- (meth) acryloxyundenyl bird It can be exemplified Tokishishiran like of (meth) acryloxy alkyl silane compound. Among these, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-methacryloxypropyldimethylmethoxysilane, and 3-methacryloxypropyldimethylethoxysilane are copolymerizable with a monomer having a phosphorylcholine group. Is preferable from the viewpoints of being excellent and easy to obtain. These monomers having an alkoxysilyl group are used alone or in combination of two or more.

The monomer having an active ester group used in the present invention is not particularly limited in structure, but the (meth) acryl group represented by the general formula [3] and the active ester group are alkyleneoxy groups having 1 to 10 carbon atoms. It is preferable that it is a compound couple | bonded through the chain or alkyl group of these. The repeating number of the alkyleneoxy group Y is an integer of 1 to 20, and when the repeating number is 2 or more and 20 or less, the carbon number of the alkyleneoxy group to be repeated may be the same or different.
The “active ester group” used in the present invention means an ester group having an electron-withdrawing group with high acidity on the alcohol side of the ester group to activate the nucleophilic reaction, that is, an ester group with high reaction activity. As such, it is commonly used in various chemical synthesis fields such as polymer chemistry and peptide synthesis. In practice, phenol esters, thiophenol esters, N-hydroxyamine esters, esters of heterocyclic hydroxy compounds, etc. 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 ester group, N-hydroxysuccinimide ester group, succinimide ester group, phthalimide ester group, 5-norbornene-2,3-dicarboximide ester group, The p-nitrophenyl ester group or the N-hydroxysuccinimide ester group is preferable because the physiologically active substance can be immobilized at a lower pH.

  The method for synthesizing the polymer compound of the present invention is not particularly limited, but has a monomer having an alkylene glycol residue, a monomer having an active ester group, and a crosslinkable group for ease of synthesis. The mixture containing the monomer is preferably radically polymerized in a solvent in the presence of a polymerization initiator.

  Any solvent may be used as long as each monomer can be dissolved, and examples thereof include methanol, ethanol, t-butyl alcohol, benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, and chloroform. These solvents are used alone or in combination of two or more. When the polymer compound is applied to a plastic substrate, ethanol and methanol are preferable because they do not denature the substrate.

The polymerization initiator may be any ordinary radical initiator, for example, 2,
Azo compounds such as 2′-azobisisobutylnitrile (hereinafter referred to as “AIBN”), 1,1′-azobis (cyclohexane-1-carbonitrile), organic peroxides such as benzoyl peroxide and lauryl peroxide be able to.

  As long as the chemical structure of the polymer compound of the present invention is such that each monomer having an alkylene glycol residue, an active ester group and a crosslinkable functional group is copolymerized, the bonding method is random, block, graft Any form may be used.

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

  The polymer compound of the present invention can be easily imparted with the property of immobilizing a low-molecular compound by suppressing nonspecific adsorption of a physiologically active substance by coating the surface of the substrate with the polymer compound. is there.

  For coating the base material surface with a polymer compound, for example, a polymer solution in which a polymer compound is dissolved in an organic solvent so as to have a concentration of 0.05 to 10% by weight is prepared, and a known method such as immersion or spraying is used. After coating on the surface of the substrate, 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 or a mixed solvent thereof is used. Of these, ethanol or methanol is preferable because it does not denature the plastic substrate and is easy to dry.

  By applying the polymer compound of the present invention to a solid phase substrate, it is possible to easily impart a property of suppressing nonspecific adsorption of a physiologically active substance to a base material. From these things, the base material which apply | coated this high molecular compound can be used suitably for the board | substrate for biochips.

Examples of the solid phase substrate used in the present invention include a plastic substrate, a glass substrate, and a substrate having a metal vapor deposition film. Specific examples of the plastic substrate include cycloolefin polymer, polypropylene, polyethylene poly, polysalophone, polyimide, polycarbonate, polymethyl petaacrylate and the like.
The solid phase substrate may have a flow path used as a microfluidic disk.

  The low molecular compounds used in the present invention include synthetic compounds and naturally derived compounds, but are not particularly limited. All low molecular weight compounds are immobilized on a substrate, and low molecular weight compounds that specifically interact with proteins are searched. The low molecular weight compounds that have been confirmed to interact with proteins so far include biotin, rapamycin, digoxin, digitoxin, cyclosporin A, digoxigenin, FK506, leptomycin, lactacystin, trapoxin, fumagillin, radicicol, eponemycin, myriocin, parsenolide And doxorubicin.

  By immobilizing a low molecular weight compound using the biochip substrate of the present invention, a biochip for screening a low molecular weight compound having specific selectivity with a protein is obtained.

  As a method of using the present invention, when there is no active ester, interaction between a specific low molecular weight compound and a protein by reacting a specific protein such as cell extract protein, blood extract protein, avidin, FKBP, mTOR, etc. There is a usage method to detect.

  In case of having an active ester, after immobilizing the aminated low molecular weight compound, the surface is treated with an alkali compound or a compound having a primary amino group to inactivate the active ester other than the portion where the low molecular weight compound is immobilized. Turn into. Thereafter, as described above, there is a method of detecting an interaction between a protein and a specific low molecular weight compound to be reacted with the protein.

  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, aminohexylcellulose, 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 can be preferably used, and aminoethanol and glycine are most preferable.

  The synthesis example of the high molecular compound used for this invention is shown below.

(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 h, the solution was further stirred at room temperature for 2 h. 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 4.5 units of ethylene glycol residue was contained.

(Synthesis Example 1 of polymer compound)
Polyethylene glycol methyl ether methacrylate (also known as methoxypolyethylene glycol methacrylate) (PEGMA average Mn = about 188, manufactured by Aldrich) and 3-methacryloxypropyldimethylethoxysilane (MPDES GELEST, INC.) Were sequentially added to 0.90 mol / L,. A monomer mixed solution was prepared by dissolving in dehydrated ethanol so as to be 1 mol / L. Thereto was further added 0.002 mol / L 2,2-azobisisobutyronitrile (AIBN Wako Pure Chemical Industries, Ltd.) and stirred until uniform. Then, after making it react at 60 degreeC under argon gas atmosphere for 4 hours, the reaction solution was dripped in diethyl ether, and precipitation was collected. The obtained polymer compound was measured by 1H-NMR in a deuterated chloroform solvent, and the peak attributed to the methyl group bonded to Si of MPDES appearing near 0.13 ppm, and the terminal methoxy group of PEGMA appearing near 3.4 ppm. The composition ratio of the polymer compound was calculated from the assigned peaks and the respective integrated values. Table 1 shows the results.

(Synthesis Example 2 of polymer compound)
Polyethylene glycol methyl ether methacrylate (also known as methoxypolyethylene glycol methacrylate) (PEGMA average Mn = about 300 Aldrich) and 3-methacryloxypropyldimethylethoxysilane (MPDES GELEST, INC.) Were sequentially added to 0.90 mol / L,. A monomer mixed solution was prepared by dissolving in dehydrated ethanol so as to be 10 mol / L. Further 0.01 mol / L 2,2-azobisisobutyronitrile (AIBN Wako Pure Chemical Industries, Ltd.) was added thereto and stirred until uniform. Then, after making it react at 60 degreeC by argon gas atmosphere for 2 hours, the reaction solution was dripped in diethyl ether, and precipitation was collected. The obtained polymer compound was measured by 1H-NMR in a deuterated chloroform solvent, and the peak attributed to the methyl group bonded to Si of MPDES appearing at around 0.13 ppm and the terminal methoxy group of PEGMA appearing at around 3.38 ppm. The composition ratio of the polymer compound was calculated from the assigned peaks and the respective integrated values. Table 1 shows the results.

(Synthesis Example 3 of polymer compound)
Polyethylene glycol methyl ether methacrylate (also known as methoxypolyethylene glycol methacrylate) (PEGMA average Mn = about 475 made by Aldrich) and 3-methacryloxypropyldimethylethoxysilane (made by MPDES GELEST, INC.) Were 0.90 mol / L,. A monomer mixed solution was prepared by dissolving in dehydrated ethanol so as to be 1 mol / L. Thereto was further added 0.002 mol / L 2,2-azobisisobutyronitrile (AIBN Wako Pure Chemical Industries, Ltd.) and stirred until uniform. Then, after making it react at 60 degreeC by argon gas atmosphere for 1.5 hours, the reaction solution was dripped in diethyl ether, and precipitation was collected. The obtained polymer compound was measured by 1H-NMR in a heavy ethanol solvent, and the peak attributed to the methyl group bonded to Si of MPDES appearing at around 0.15 ppm and the terminal methoxy group of PEGMA appearing at around 3.35 ppm. The composition ratio of the polymer compound was calculated from the assigned peaks and the respective integrated values. Table 1 shows the results.

(Synthesis Example 4 of polymer compound)
Polyethylene glycol methyl ether methacrylate (also known as methoxy polyethylene glycol methacrylate) (PEGMA average Mn = about 1100 manufactured by Aldrich) and 3-methacryloxypropyldimethylethoxysilane (MPDES GELEST, INC.) Were sequentially added in 0.90 mol / L,. A monomer mixed solution was prepared by dissolving in dehydrated ethanol so as to be 10 mol / L. Thereto was further added 0.002 mol / L 2,2-azobisisobutyronitrile (AIBN Wako Pure Chemical Industries, Ltd.) and stirred until uniform. Then, after making it react at 60 degreeC under argon gas atmosphere for 1 hour, the reaction solution was dripped in diethyl ether, and precipitation was collected. The obtained polymer compound was measured by 1H-NMR in a heavy ethanol solvent, and the peak attributed to the methyl group bonded to Si of MPDES appearing at around 0.16 ppm and the terminal methoxy group of PEGMA appearing at around 3.35 ppm. The composition ratio of the polymer compound was calculated from the assigned peaks and the respective integrated values. Table 1 shows the results.

(Synthesis Example 5 of polymer compound)
Polyethylene glycol methyl ether methacrylate (also known as methoxy polyethylene glycol methacrylate) (PEGMA average Mn = about 188, manufactured by Aldrich), p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate (MEONP), 3-methacryloxypropyldimethylethoxysilane (MPDES GELEST, INC) Were dissolved in dehydrated ethanol so as to be 0.90 mol / L, 0.05 mol / L, and 0.05 mol / L, respectively, to prepare a monomer mixed solution. Thereto was further added 0.002 mol / L 2,2-azobisisobutyronitrile (AIBN Wako Pure Chemical Industries, Ltd.) and stirred until uniform. Then, after making it react at 60 degreeC under argon gas atmosphere for 4 hours, the reaction solution was dripped in diethyl ether, and precipitation was collected. The obtained polymer compound was measured by 1H-NMR in a deuterated chloroform solvent, and the peak attributed to the methyl group bonded to Si of MPDES appearing near 0.13 ppm, and the terminal methoxy group of PEGMA appearing near 3.4 ppm. The composition ratio of the polymer compound was calculated from the peak attributed to it, the peak attributed to the benzene ring of MEONP appearing in the vicinity of 7.4 and 8.29 ppm, and the integrated values of each. Table 1 shows the results.

(Synthesis Example 6 of polymer compound)
Polyethylene glycol methyl ether methacrylate (also known as methoxy polyethylene glycol methacrylate) (PEGMA average Mn = about 300 Aldrich), p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate (MEONP), 3-methacryloxypropyldimethylethoxysilane (MPDES GELEST, INC) Were dissolved in dehydrated ethanol so as to be 0.90 mol / L, 0.05 mol / L, and 0.05 mol / L, respectively, to prepare a monomer mixed solution. Further 0.01 mol / L 2,2-azobisisobutyronitrile (AIBN Wako Pure Chemical Industries, Ltd.) was added thereto and stirred until uniform. Then, after making it react at 60 degreeC by argon gas atmosphere for 2 hours, the reaction solution was dripped in diethyl ether, and precipitation was collected. The obtained polymer compound was measured by 1H-NMR in a deuterated chloroform solvent, and the peak attributed to the methyl group bonded to Si of MPDES appearing near 0.13 ppm and the terminal methoxy group of PEGMA appearing near 3.38 ppm. The composition ratio of this polymer compound was calculated from the peak attributed to it, the peak attributed to the benzene ring of MEONP appearing in the vicinity of 7.4 and 8.3 ppm, and the respective integrated values. Table 1 shows the results.

(Synthesis Example 7 of polymer compound)
Polyethylene glycol methyl ether methacrylate (also known as methoxy polyethylene glycol methacrylate) (PEGMA average Mn = about 475 manufactured by Aldrich), p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate (MEONP), 3-methacryloxypropyldimethylethoxysilane (MPDES GELEST, INC) Were dissolved in dehydrated ethanol so as to be 0.90 mol / L, 0.05 mol / L, and 0.05 mol / L, respectively, to prepare a monomer mixed solution. Thereto was further added 0.002 mol / L 2,2-azobisisobutyronitrile (AIBN Wako Pure Chemical Industries, Ltd.) and stirred until uniform. Then, after making it react at 60 degreeC by argon gas atmosphere for 1.5 hours, the reaction solution was dripped in diethyl ether, and precipitation was collected. The obtained polymer compound was measured by 1H-NMR in a heavy ethanol solvent, and the peak attributed to the methyl group bonded to Si of MPDES appearing at around 0.15 ppm and the terminal methoxy group of PEGMA appearing at around 3.35 ppm. The composition ratio of this polymer compound was calculated from the peak attributed to it, the peak attributed to the benzene ring of MEONP appearing in the vicinity of 7.6 and 8.4 ppm, and the respective integrated values. Table 1 shows the results.

(Synthesis Example 8 of polymer compound)
Polyethylene glycol methyl ether methacrylate (also known as methoxypolyethylene glycol methacrylate) (PEGMA average Mn = about 1100 Aldrich), p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate (MEONP), 3-methacryloxypropyldimethylethoxysilane (MPDES GELEST, INC) Were dissolved in dehydrated ethanol to give 0.9 mol / L, 0.05 mol / L, and 0.05 mol / L, respectively, to prepare a monomer mixed solution. Thereto was further added 0.002 mol / L 2,2-azobisisobutyronitrile (AIBN Wako Pure Chemical Industries, Ltd.) and stirred until uniform. Then, after making it react at 60 degreeC under argon gas atmosphere for 1 hour, the reaction solution was dripped in diethyl ether, and precipitation was collected. The obtained polymer compound was measured by 1H-NMR in a heavy ethanol solvent, and the peak attributed to the methyl group bonded to Si of MPDES appearing at around 0.16 ppm and the terminal methoxy group of PEGMA appearing at around 3.35 ppm. The composition ratio of this polymer compound was calculated from the peak attributed to it, the peak attributed to the benzene ring of MEONP appearing in the vicinity of 7.6 and 8.4 ppm, and the respective integrated values. Table 1 shows the results.

(Substrate 1)
A saturated cyclic polyolefin resin was processed into a slide glass shape (dimensions: 76 mm × 26 mm × 1 mm) to prepare a solid phase substrate, and oxygen plasma treatment was performed.

(Substrate 2)
Glass slide manufactured by Matsunami (Part No .: SD90001)

(Application of polymer compound and production of bichip substrate)
The polymer compound obtained in Synthesis Example 1-8 was dissolved in ethanol to give 0.3% by weight. Substrates 1 and 2 were immersed in a 0.3 wt% ethanol solution of Synthesis Example 1-8 and air-dried to produce bichip substrates 1-16, respectively.
The uncoated substrates 1 and 2 were used as biochip substrates 17 and 18, respectively.

(Example 1-4, Example 9-12, Comparative Example 1)
Biotin (manufactured by Wako Pure Chemicals: 023-08711) was spotted on the biochip substrates 1-4, 9-12, and 17 to obtain biochips.

(Example 5-8, Example 13-16, Comparative Example 2)
EZ-Link (TM) biotin PEO amine (manufactured by Wako Pure Chemicals: 500-45431) was spotted on the biochip substrates 5-8, 13-16, 18 and treated at 65 ° C. for 1 hour, followed by 0.1N water A biochip was obtained by treatment with an aqueous sodium oxide solution at room temperature for 10 minutes and drying.

(Evaluation)
A biotin-streptavidin reaction was performed by applying a solution in which Cy3-labeled streptavidin was mixed with Cy3-labeled 10% serum solution at a rate of 1 ug / ml to the surface of the obtained biochip. The plate was washed with 0.1% sodium dodecyl sulfate in PBS for 5 minutes. The amount of fluorescence was measured for each spot. The results are shown in Table 2.

A microarray scanner “ScanArray” manufactured by Packard BioChip Technologies was used to measure the amount of fluorescence in Examples and Comparative Examples. The measurement conditions were laser output 90%, PMT sensitivity 60%, excitation wavelength 649 nm, measurement wavelength 670 nm, and resolution 50 μm.
In Examples, a biochip with a low background was obtained by specific reaction of immobilized biotin and avidin reacted later. Similarly, in the comparative example, a signal due to a specific interaction between biotin and avidin was confirmed, but the background was high.

Claims (15)

A biochip in which a low molecular weight compound is immobilized on the surface of a solid phase substrate, a monomer having an alkylene glycol residue, a monomer having a crosslinkable functional group, and a single amount having an active ester group on the substrate surface A biochip characterized in that a polymer material obtained by polymerizing a body is formed on a substrate surface . The biochip according to claim 1, wherein the monomer having an alkylene glycol residue is a monomer represented by the following general formula [1].

(Wherein R1 represents a hydrogen atom or a methyl group, R2 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) Represents an integer, and the repeated Xs may be the same or a chain of different alkylene glycol residues. The biochip according to claim 2, wherein the monomer having an alkylene glycol residue is methoxypolyethylene glycol (meth) acrylate. The biochip according to claim 3, wherein the average chain of ethylene glycol in the methoxypolyethylene glycol (meth) acrylate is 3 to 100. The monomer having a crosslinkable functional group is represented by the following general formula [2] (wherein R3 represents a hydrogen atom or a methyl group, and Z represents an alkyl group having 1 to 20 carbon atoms. and it may be .A1, of A2, A3, at least one is a hydrolyzable group, others claim 1, wherein the biochip is represented by an alkyl group.).
The biochip according to claim 5 , wherein, in the general formula [2], at least one of A1, A2, and A3 is an alkoxyl group. The biochip according to any one of claims 1 to 7 , wherein the high molecular weight substance immobilizes a low molecular weight compound via the active ester group . The monomer having an active ester group is represented by the following general formula [3] (wherein R2 represents a hydrogen atom or a methyl group, Y represents an alkyleneoxy group or an alkyl group having 1 to 10 carbon atoms, and W represents an active ester. 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 a chain of different alkyleneoxy groups. The biochip according to claim 7, which is represented by:
The biochip according to claim 8, wherein the monomer having an active ester group is p-nitrophenyloxycarbonyl-4,5-ethylene glycol methacrylate or N-hydroxysuccinimide-4,5-ethylene glycol methacrylate. The biochip according to any one of claims 1 to 9 , wherein the solid phase substrate is made of plastic. The biochip according to claim 10 , wherein the plastic is a saturated cyclic polyolefin. The biochip according to any one of claims 1 to 9 , wherein the solid phase substrate is made of glass. Biochip according to claim 1 to 12, wherein any one, characterized in that the solid phase substrate flow path is provided A method for using a biochip, wherein the biochip according to any one of claims 1 to 11 is reacted with a biological substance. A method for using a biochip comprising reacting a biological material after subjecting the biochip according to any one of claims 7 to 9 to alkali treatment.