JP2003014743A - Biological material chip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of detecting a specific bonded target material using a biological material chip. SOLUTION: In the biological material chip, at least one protein or A which is the residue of a protein-bonded material (excluding nuclei acids) is bonded to a solid-phase carrier having a porous matrix containing pores having diameters between about 2 nm and about 1,000 nm at porosity between about 10% and about 90% and a thickness between about 0.01 μm and about 70 μm by performing covalent coupling through a sulfonyl radical as expressed by the following formula (1): solid-phase carrier-L-SO2 -X-A (wherein, L and X respectively denote a coupling radical and- CR<1> (R<2> )-CR<3> (R<4> )- and R<1> , R<2> , R<3> , and R<4> respectively denote hydrogen atoms, a 1-6C alkyl radical, a 6-20C aryl radical, and a 7-26C aralkyl having a 1-6C alkyl chain, and A denotes the protein or protein-bonded material (excluding nuclei acids)).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a chip in which a protein or protein-binding substance (excluding nucleic acid), which is very useful for proteome analysis / proteomics, is immobilized on a solid phase surface, and detection using the chip. The present invention also relates to a method and a manufacturing method thereof.

[0002]

2. Description of the Related Art Technological development for efficiently analyzing gene functions of various organisms is rapidly progressing.
For the analysis of the nucleotide sequence of A or DNA fragment, DN
A detection tool called a chip, which has a large number of DNA fragments or nucleotide derivatives such as synthetic oligonucleotides immobilized on the surface of a solid substrate, is used. A detection molecule such as DNA or a fragment thereof or a synthetic oligonucleotide, such as a nucleotide derivative bound and immobilized on the surface of a solid substrate, is also called a probe molecule. A typical DNA chip is a microarray in which a large number of probe molecules are aligned and immobilized on a solid phase carrier such as a slide glass. The DNA chip-related technology relating to the production and use of this DNA chip is considered to be applicable to the detection of biomolecules other than DNA. Therefore, it is new to drug discovery research, the diagnosis of diseases and the development of prevention methods. Expected to provide the means.

On the other hand, the genome analysis is almost completed, and "proteome / proteomics" research is underway to provide essential information for finally understanding the meaning of genetic information and simulating the life activity of cells. . Protelome refers to the entire set of proteins translated and produced in specific cells, organs, and organs, as well as higher-level information analysis such as chemical structure, total amount, expression time, post-translational modification, and aggregate formation. This field of research is called "proteomics".

[0004] The proteome research consists of protein profiling, protein identification / precision analysis, interaction network analysis, and construction of a proteome database, which is applied to life science research.

Of these, yeast two? Immunoprecipitation method, BIA-MS method, column switching-mass spectrometry method and the like are performed as methods utilizing the hybrid method, phage display method and affinity capture (proteome analysis method, 163-121, Yodosha, 2000). . None of the above-mentioned interaction analysis methods has reached high throughput analysis.

[0006] Schreiber et al. Reported on a protein microarray for high-throughput protein interaction analysis (Science).
e, 289, 1760-1763, 2000). In this method, an aqueous protein solution is spotted on a slide glass having an aldehyde group, blocked with a BSA solution, reacted with the protein solution, and detected by a fluorescence scanner. In this case, there is a problem that the Schiff base, which is a reaction product of an aldehyde group and an amino group, has low stability (usually, hydrolysis easily occurs).

In addition, as a method for immobilizing a protein on a solid phase, Japanese Patent Publication No. 7-53108 describes a method of immobilizing a solid phase by introducing a hydrophobic polypeptide at the end of the protein. Has been done.

Japanese Patent No. 922040 discloses protein A
Methods for immobilizing antibody proteins with molecular membranes have been described.

Japanese Patent Publication No. 7-43380 (corresponding to US Pat. No. 5,049,962) discloses a detection tool for use in a ligand-receptor assay, which comprises microporous polymer particles having reactive groups on the surface. Analytical devices having receptor molecules bound to the surface are described.

International publication WO 00/61282 describes a porous solid phase carrier. By mainly applying particles of an inorganic substance to the surface, the thickness from the support is 0.0
It is 1 to 70 μm, and the porosity is 10 to 90%. The use of this porous solid phase carrier has an advantage that the amount of biological polymer immobilized can be increased because the surface area becomes large.

[0011]

DISCLOSURE OF THE INVENTION The present invention provides a chip in which at least one protein or protein-binding substance (excluding nucleic acid) is bound and immobilized on a reactive solid phase carrier which can be rapidly and stably bound and immobilized. And a method for detecting a specific binding target substance using the chip. A further object of the present invention is to provide a method for manufacturing the above chip.

[0012]

According to a first aspect of the present invention, the porous region has a pore size of from about 2 nm to about 1000 nm, a porosity of from about 10% to about 90% and from about 0.01 μm.
In a solid-phase carrier having a porous substrate having a thickness of about 70 μm, at least one protein or protein-binding substance (excluding nucleic acid) A has a sulfonyl group as represented by the following formula (I). There is provided a biological material chip characterized by being bound by a covalent bond via. In the solid-phase support _{-L-SO 2 -X-A (} I) [ formula (I), L represents a linking group, X is, -C
Represents R ¹ (R ² ) -CR ³ (R ⁴ )-; R ¹ , R ² , R ³
And R ⁴ are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, or a total carbon atom number of 7 having an alkyl chain having 1 to 6 carbon atoms. To 26 aralkyl groups, A is a protein or protein-binding substance (excluding nucleic acid)
Indicates the residue of. ]

According to a second aspect of the present invention, the above-mentioned chip of the present invention and a target substance which specifically binds to a protein or a protein-binding substance (excluding nucleic acid) carried on the surface of the chip. Contacting with a sample containing:
And a step of detecting the formation of a mutual bond between the protein or protein-binding substance and the target substance.

According to a third aspect of the present invention, the following formula (I
I) A vinylsulfonyl group or a reactive precursor group thereof is formed on a solid phase carrier having a porous substrate having a surface on which a vinylsulfonyl group or a reactive precursor group thereof is reacted to form a covalent bond. At least one protein or protein-binding substance having a reactive group (provided that
There is provided a method for producing a chip of the present invention, which comprises contacting (excluding nucleic acid). —L—SO ₂ —X ′ (II) [In the above formula, L represents a linking group that binds —SO ₂ —X ′ and the solid phase carrier; X ′ represents —CR ¹ ═CR
² (R ³ ) or —CH (R ¹ ) —CR ² (R ³ ) (Y) is represented; R ¹ , R ² and R ³ are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. , 6 to 20 carbon atoms
Or an aralkyl group having a total of 7 to 26 carbon atoms having an alkyl chain having 1 to 6 carbon atoms, Y is a group substituted by a nucleophile, or eliminated as "HY" by a base. Represents the group to

The preferred embodiments of the present invention are described below.
A mode in which the protein or protein-binding substance (excluding nucleic acid) immobilized on the surface is an antibody, an antibody fragment, a ligand, an antigen, a hapten, or a receptor;
Aspects in which the protein or protein-binding substance (excluding nucleic acids) fixed to the surface is avidins; aspects in which avidins are avidin, streptavidin, or modified forms thereof capable of forming a stable complex with biotin A mode in which the protein immobilized on the surface is a nucleic acid recognition protein; a mode in which the nucleic acid recognition protein is a double-stranded DNA recognition protein;

Double-stranded DNA recognition protein is double-stranded DN
A-recognizing antibody; a double-stranded DNA recognizing protein is a DNA transcription factor; a double-stranded DNA recognizing protein is a protein having a Zn finger motif or a ring finger motif; a porous substrate composed of an organic polymer Aspects in which the porous substrate is particles formed of an inorganic substance; Aspects in which the porous substrate contains silicon, alumina or titanium; Solid phase carriers are glass, plastic, electrode surface, sensor chip surface An embodiment; an embodiment in which the target substance is labeled with at least one component capable of generating a detectable signal; an embodiment including a step of blocking the chip with an aqueous solution of amino acid, peptide or protein;

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention has a porous region of about 2 nm.
At least one protein or protein-binding substance (provided that the pore size is about 1000 nm, the porosity is about 10% to about 90%, and the porous substrate has a thickness of about 0.01 μm to about 70 μm). A) which is a residue of (excluding nucleic acid) is bound by a covalent bond via a sulfonyl group as shown in the following formula (I). In the solid-phase support _{-L-SO 2 -X-A (} I) [ the above equation, L represents a linking group, X is, -C
Represents R ¹ (R ² ) -CR ³ (R ⁴ )-; R ¹ , R ² , R ³
And R ⁴ are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, or a total carbon atom number of 7 having an alkyl chain having 1 to 6 carbon atoms. To 26 aralkyl groups, A is a protein or protein-binding substance (excluding nucleic acid)
Indicates the residue of. ]

Representative examples of proteins or protein-binding substances (excluding nucleic acids) to be immobilized include, but are not limited to, those having an antigenic determinant such as antibody or antibody fragment, ligand, antigen and hapten. ,
Examples include receptors, ligands, avidins, and nucleic acid recognition proteins. A typical example of the nucleic acid recognition protein is a double-stranded recognition protein.

Examples of avidins include avidin, streptavidin, and modified forms thereof that can form a stable complex with biotin. The ability to form such a stable complex means that a complex having a dissociation constant close to that of the biotin-avidin complex (10 ⁻¹⁵ M) can be formed. A variant is
It means a modified or fragment of naturally-occurring avidin or streptavidin, or a recombinant thereof.

The nucleic acid recognizing protein is not particularly limited, and examples thereof include double-stranded DNA recognizing substances.
The double-stranded DNA recognizing substance refers to a substance that recognizes double-stranded DNA and specifically binds to it. Specific examples of the double-stranded DNA recognizing substance include a DNA transcription factor, a mismatch repair protein, a double-stranded DNA recognizing antibody, and a peptide nucleic acid. Furthermore, examples of the double-stranded DNA recognizing substance also include those having a Zn finger motif or a ring finger motif.

A DNA transcription factor is a substance that binds to a promoter region on a gene and controls transcription of DNA into mRNA (Tamura Takaaki: Transcription factor (Yodosha 19
1995)). Therefore, the transcription factor is a double-stranded D of a specific sequence.
It is known to specifically bind to NA.

Among many transcription factors, Zinc Fi
nger Protein or Zinc Finger
The transcription factor group having r or Ring Finger motif has a very high appearance rate in eukaryotes, and 1% in the genome seems to encode this. Pabo is Zi
The tertiary structure of the nc Figer motif was analyzed to elucidate the mechanism of binding to DNA (Science, 25.
2, 809 (1991)). Furthermore, Choo et al.
We have succeeded in producing a group of non-natural Zinc Finger Proteins that bind to a specific sequence by a gene recombination method (Nature 372, 64).
2 [1994], PNAS91, 11163 (199
4)). In addition, Scripts Research
The Institute group is Phase Disp
New Zinc Finger Prot by lay
Successful production of ein group (PNAS95,281
2, [1998]: 96, 2758 (1999)). Thus, the group of DNA transcription factors represented by Zinc Finger Protein originally has the property of binding to double-stranded DNA, and according to recent studies, it is also possible to produce recombinants that recognize arbitrary DNA sequences. Is becoming. By immobilizing such a protein, it is possible to efficiently capture the double-stranded DNA on the support.

In addition, helices as nucleic acid binding substances
Examples also include those having loop helix proteins and Ets domains.

As the protein-binding substance to be immobilized (excluding nucleic acid), hapten, biotin (biotin, biocytin, desthiobiotin, oxybiotin, or a derivative thereof capable of forming a stable complex with avidin) is used. The stable dissociation constant of the biotin-avidin complex (1
It means that it is possible to form a complex with a dissociation constant close to 0 ^-15 M). Furthermore, peptides,
Examples include sugars, hormones, medicines, and antibiotics.

The protein or the protein-binding substance to be immobilized on the solid phase carrier having a porous substrate forms a covalent bond via a sulfonyl group by its internal amino group or mercapto group. Furthermore, an amino group, an imino group, a hydrazino group, a carbamoyl group, a hydrazinocarbonyl group, a mercapto group, a carboximide group, or the like may be introduced into a protein to form a covalent bond via a sulfonyl group.

A protein or a protein-binding substance (for example, an antibody or avidin, a solid phase carrier having a porous substrate,
The chip on which the nucleic acid recognizing protein) is immobilized is brought into contact with a target substance (eg, ligand, biotin, nucleic acid) that specifically reacts with the immobilized protein or protein-binding substance in the presence of an aqueous medium, and the target substance is immobilized. Can be fixed. The specific binding target substance to be immobilized (eg, ligand, biotin, nucleic acid) is bound to a detectable label (eg, fluorescent label, enzyme label, etc.) so that the immobilization can be detected from the outside. Is desirable

The solid phase carrier is preferably a substrate having a smooth surface. Examples of the material of the solid phase carrier include non-porous substances such as glass and plastic.

Typical methods of forming porous regions are by adding material (eg, deposition) and removing material (eg, selective etching). In the addition method, a porous region is formed on the surface of the underlying substrate to increase the effective surface area. The porous region can be formed by depositing the following substances, optionally using a solvent and a catalyst. The porous region may be, for example, colloidal silica, tetramethoxysilane (TMO) typically used in sol-gel processes.
It can be formed from organosilicon compounds such as S), metal alkoxides, silsesquioxanes or other silanes or combinations of these materials. For these precursors,
The morphology (pore size, porosity, thickness) of the formed porous region can be adjusted by appropriately selecting parameters such as the composition, concentration, pH, aging time and temperature of the solution. Inorganic materials other than those mentioned above (for example, materials based on aluminum or titanium) can also be used in the same manner as above.

Alternatively, the porous region matrix can be templated. In the templating process, a material such as a polymer is deposited with a matrix and then burned out, leaving behind a porous structure with selected properties.
The templating material may be any of polymers such as polystyrene latex, polymers in solution, or combinations of these materials. The heat burn-off process is typically carried out by heating in air and can be carried out at temperatures from 150 ° C. or higher to the melting temperature (or glass transition temperature) of the material forming the matrix of the porous layer. The matrix material can be fired after the templated material is burned out. By varying the time and temperature of the firing process, varying degrees of densification and pore characteristics can be achieved.

The porous region is formed by spin coating, dip coating, extraction (aerosol), individual spots deposited on the surface,
It can be formed on the surface of the underlying substrate by a variety of processes, including the use of barriers (physical or chemical) to specifically deposit the coating on channels, pads, spots or patterned surfaces. The thickness of the porous region can be adjusted by varying either or both the precursor and the layer forming conditions. For example, the thickness can be adjusted depending on the concentration of the reagent used. Similarly, the layer thickness can be increased by providing multiple depositions. During application of successive reagents, further processing may be performed such as baking the substrate to remove solvent from the film prior to application of the next agent. The thickness of the film can also be changed by adjusting the speed of rotation or deposition,
For example, slower speeds produce thicker films and faster speeds produce thinner films. In addition, varying the draw speed from the dip coating bath affects the layer thickness, with slower speeds producing thinner films and higher speeds producing thicker films. It is also possible to influence the film thickness by controlling the solution conditions. When using, for example, the TMOS technique, the solution can initiate gelation, which increases the viscosity and thus the thickness of the deposited layer.

The coatings, particles or other components can be spun on the surface of the substrate, the substrate can be dipped in a solution containing the agent, the agent can be sprayed or otherwise applied to the surface of the substrate. The substrate can be treated to create high porosity areas on the entire surface of the substrate or only at selected locations, such as by spotting reagents in grids, circular spots, areas, cells or any desired shape.
It is also possible to utilize degradation techniques to increase the porosity of the substrate. For example, the porous regions can be etched into the surface of the substrate (ie, the material of the surface of the underlying substrate material). This surface can be prepared as an etched glass, such as a phase-separated sodium borosilicate glass. In certain embodiments of the degradation procedure to form a porous matrix, the pore size can also be controlled by the annealing time and temperature of the annealing step performed before etching (longer annealing, higher Temperature increases pore size). The depth of the porous region can also be controlled by etching parameters (solution concentration, composition, time, etc.) according to the substrate material.

Examples of the porous substance include porous ceramics, porous activated carbon, woven fabrics, non-woven fabrics, filter papers, membrane films, etc. When the substance itself is porous, it is sprayed onto a solid phase carrier or an adhesive. Can be pasted with. It is also possible to coat the precursor of the porous substance on the solid phase carrier to make it porous on the solid phase surface. The Solgel reaction is a typical example. Colloidal silica is generally used, but not limited to this.

Examples of the organic polymer which constitutes the porous substrate include monosaccharides such as glucose and fructose or their derivatives, polysaccharides such as dextran, amyloses and starch or their derivatives such as carboxymethyl starch. -Chi. Polymeric polymers such as PVA, polyacrylic acid, cellulose, carboxymethyl cellulose, and polyacetal are mentioned.

In the present invention, the protein or protein-binding substance (excluding nucleic acid) A on the chip is a solid phase carrier having a porous substrate by a covalent bond via a sulfonyl group as shown in the following formula (I). Is bound to. Solid phase carrier _{-L-SO 2 -X-A (} I)

In the formula (I), L represents a linking group,
X ^{is, -CR 1 (R 2) -CR} 3 (R 4) - a expressed;
R ¹ , R ² , R ³ and R ⁴ are, independently of each other, a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, and a carbon atom having 6 to 20 carbon atoms.
Or an aralkyl group having a total of 7 to 26 carbon atoms having an alkyl chain having 1 to 6 carbon atoms, and A represents a residue of a protein or a protein-binding substance (excluding nucleic acid).

In the formula (I), L is --SO ₂ --X--
A divalent or higher linking group that binds A to the solid phase carrier is shown. As a specific example of -L-, L is any linking group selected from an aliphatic group, an aromatic group, a heterocycle, and a hydrocarbon chain optionally interrupted with a hetero atom, and a linking group selected from a combination thereof. Groups can be used and L can be a single bond.

In the formula (I), examples of the alkyl group having 1 to 6 carbon atoms include methyl group, ethyl group, n-propyl group, n-butyl group and n-hexyl group, A methyl group is particularly preferable. Specific examples of the aryl group having 6 to 20 carbon atoms include a phenyl group and a naphthyl group. R ¹ and R ²
Both R and R ³ are preferably hydrogen atoms. Examples of the aralkyl group having 7 to 26 carbon atoms and having an alkyl chain having 1 to 6 carbon atoms include the above-mentioned examples of the alkyl group having 1 to 6 carbon atoms and the aryl group having 6 to 20 carbon atoms. A combination of these and examples is given.

The present invention further comprises contacting the above-mentioned chip of the present invention with a sample containing a target substance that specifically binds to the protein or protein-binding substance (excluding nucleic acid) carried on the surface of the chip. And a step of detecting the formation of mutual binding between the protein or protein-binding substance and the target substance.

The type of "specimen" referred to in the present invention is not particularly limited, and examples thereof include blood such as peripheral venous blood, white blood cells, serum, urine, feces, semen, saliva, cultured cells, tissues such as various organ cells. Any sample containing cells and other nucleic acids can be used. A sample such as the tissue cells as described above may be used as it is, but preferably, a sample in which the cells in the sample are destroyed to release nucleic acids, ligands, etc. is used as the sample. Destruction of cells in the specimen sample can be performed by a conventional method, for example, physical action such as shaking or ultrasonic wave can be applied from the outside.
In addition, nucleic acid extraction solutions (eg SDS, Triton-X, Twee
Nucleic acid can also be released from cells using a surfactant such as n-20 or a solution containing saponin, EDTA, protease and the like). When a nucleic acid is extracted using a nucleic acid extraction solution, the reaction can be promoted by incubating at a temperature of 37 ° C or higher.

The present invention further provides a solid phase carrier having a porous substrate having a vinyl sulfonyl group represented by the following formula (II) or a reactive precursor group thereof on the surface, the vinyl sulfonyl group or its reactivity. The present invention relates to a method for producing a chip of the present invention, which comprises contacting at least one protein or protein-binding substance (excluding nucleic acid) having a reactive group which reacts with a precursor group to form a covalent bond. —L—SO ₂ —X ′ (II) [In the formula (II), L represents a linking group that bonds —SO ₂ —X ′ and the solid phase carrier; X ′ represents —CR ¹ ═CR ²
(R ³ ) or —CH (R ¹ ) —CR ² (R ³ ) (Y); R ¹ , R ² and R ³ are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, Represents an aryl group having 6 to 20 carbon atoms, or an aralkyl group having 7 to 26 total carbon atoms having an alkyl chain having 1 to 6 carbon atoms,
Y represents a group that is substituted by a nucleophile or a group that is eliminated as "HY" by a base]

In the formula (II), the number of carbon atoms is 1 to 6
Examples of the alkyl group of are methyl group, ethyl group, n-
Examples thereof include a propyl group, an n-butyl group, and an n-hexyl group, and a methyl group is particularly preferable. Specific examples of the aryl group having 6 to 20 carbon atoms include a phenyl group and a naphthyl group. R ¹ ,
Both R ² and R ³ are preferably hydrogen atoms. 7 total carbon atoms having an alkyl chain of 1 to 6 carbon atoms
Examples of the aralkyl group having 26 to 26 include the above-mentioned examples of the alkyl group having 1 to 6 carbon atoms and 6 to 20 carbon atoms.
In combination with the example of the aryl group of.

In the formula (II), Y is --OH, --O.
“HY” by a group substituted by a nucleophile such as R ⁰ , —SH, NH ₃ , NH ₂ R ⁰ (wherein R ⁰ is a group other than a hydrogen atom, such as an alkyl group) or a base.
Is represented by a halogen atom, —OSO ₂ R ¹¹ , —OCOR ¹² , —OSO ₃ M, or a quaternary pyridinium group (R ¹¹ has 1 to 6 carbon atoms). alkyl group, an aryl group or a total aralkyl group having a carbon number of 7 to 26 carbon atoms containing an alkyl chain having 1 to 6, carbon atoms is 6 to 20; R ¹² has a carbon number of atoms 1 to 6 represents an alkyl group or a halogenated alkyl group having 1 to 6 carbon atoms; M represents a hydrogen atom, an alkali metal atom or an ammonium group).

The alkyl group of R ^11, aralkyl group having an aryl group, and R ¹¹ of R ¹¹ may have a substituent. Examples of such a substituent include a hydroxyl group, an alkoxy group having 1 to 6 carbon atoms, an alkenyl group having 1 to 6 carbon atoms, a carbamoyl group having 2 to 7 carbon atoms, and 1 to 6 carbon atoms. Alkyl group having 7 to 16 carbon atoms, aryl group having 6 to 20 carbon atoms, sulfamoyl group (or its Na salt, K salt, etc.), sulfo group (or its Na salt, K salt, etc.) ), A carboxylic acid group (or its Na salt, K salt, etc.), a halogen atom, an alkenylene group having 1 to 6 carbon atoms, an arylene group having 6 to 20 carbon atoms, a sulfonyl group, and a combination thereof. An atom or group selected from the group can be mentioned.

In the formula (II), L is --SO _2-
A divalent or higher-valent linking group that binds X ′ to the solid phase carrier is shown. As a specific example of -L-, use is made of an arbitrary linking group selected from an aliphatic group, an aromatic group, a heterocycle, and a hydrocarbon chain optionally interrupted by a hetero atom, and a linking group selected from a combination thereof. And again,
L may be a single bond. Preferred specific examples of the above-mentioned "-X '" group are shown below.

[0045]

[Chemical 1]

"-X '" is (X1) in the above specific examples,
(X2), (X3), (X4), (X7), (X8),
(X13) or (X14) is preferable,
More preferably, it is (X1) or (X2). Particularly preferred is a vinyl group represented by (X1).

The covalent bond via a sulfonyl group used in the present invention has a high resistance to hydrolysis, and therefore can be easily and stably stored, and an amino group or a mercapto group can be stored in advance. It can react rapidly with the provided proteins to form stable covalent bonds.

Since the protein has an amino group or a mercapto group, it is usually unnecessary to newly introduce another reactive group. However, since the three-dimensional structure of a protein is largely involved in its function, it is preferable to introduce a reactive group at a specific position unrelated to the activity when the activity of the protein decreases.

When the protein or protein-binding substance (excluding nucleic acid) is spotted, it is carried out by spotting an aqueous liquid on the surface of the reactive solid phase carrier. Specifically, after dissolving or dispersing a protein or a protein-binding substance in an aqueous medium to prepare an aqueous liquid, the aqueous liquid is
It is preferable that the solution is dispensed into a 96-well or 384-well plastic plate, and the dispensed aqueous liquid is dropped onto the surface of the solid phase carrier using a spotter device or the like. As described above, a spotter device can be used for spotting the protein or protein-binding substance, but the activity of the protein or protein-binding substance may be reduced depending on the properties of the pin head. In that case, it may be preferable to use an inkjet device or the like.

In order to prevent the protein or the protein-binding substance from drying and denaturing after the spotting, a substance having a high boiling point may be added. The substance with a high boiling point is a substance that can be dissolved in an aqueous liquid in which the spotted protein or protein-binding substance is dissolved or dispersed, does not interfere with hybridization with the sample to be detected, and has a high viscosity. Is preferably not so large. Such substances include glycerin, ethylene glycol, dimethylsulfoxide and low molecular weight hydrophilic polymers. Examples of hydrophilic polymers include polyacrylamide, polyethylene glycol, and sodium polyacrylate. The molecular weight of this polymer is preferably in the range of 10 ^{3 to} 10 ⁶ . As the substance having a high boiling point, it is more preferable to use glycerin or ethylene glycol, and it is particularly preferable to use glycerin. The concentration of the high-boiling substance can be adjusted by the activity of the protein after spotting.

For the same purpose, it is also preferable that the solid-phase carrier after spotting the protein or the protein-binding substance is placed in an environment having a humidity of 90% or more and a temperature range of 20 to 40 ° C.

The fixed amount (number) of the protein or protein-binding substance is preferably in the range of 1 to 10 ⁵ types / cm ² with respect to the surface of the solid phase carrier. By spotting,
An aqueous liquid of a protein or a protein-binding substance is fixed on the surface of a solid support in the form of dots. The dot shape is
It is almost circular. The distance between the dots, the size, and the volume of the aqueous liquid when spotted vary depending on the application.

FIG. 1 schematically shows the structure of a protein chip which is a typical embodiment of the present invention.

When the protein A having a reactive group (Z) is spotted on the surface of the porous solid support (P1) of FIG.
Reaction between X and protein occurs, but solid phase carrier (P1)
There is also unreacted X on the surface of which the protein is not bound. Such X may cause a non-specific reaction in a reaction with a labeled ligand or the like which is performed later, and may measure non-specific binding. Therefore, X is blocked in advance. It is preferable to process. The blocking treatment is preferably carried out by bringing the surface of the solid phase carrier (P2) into contact with one having an amino group or a mercapto group. In order to prevent nonspecific binding of the reacting ligand, it is preferable to perform a blocking treatment with a protein blocking agent, that is, BSA, casein, gelatin or the like. By doing so, BSA and the like will be present on the surface of the solid phase carrier (P2) other than the spotted portion, and the ligand cannot be bound. When reacting a nucleic acid, the blocking treatment can be carried out by bringing an anionic compound having an amino group or a mercapto group into contact with the protein blocking agent. When the nucleic acid reacts with the protein, the nucleic acid has a negative charge, so that the negative charge is also generated on the surface of the solid phase carrier (P2) to prevent the nucleic acid from reacting with unreacted X. be able to. Such anionic compounds include those that react with X and have a negative charge (COO-, SO3
-, OSO3-, PO3-, or PO2-) can be used, but amino acids are preferable, and glycine or cysteine is particularly preferable. Also, taurine can be preferably used.

The protein chip, which is a typical embodiment of the present invention, is used for protein interaction analysis, protein expression analysis and drug discovery research. Furthermore, when the protein is a nucleic acid binding protein, it can be used for mutation analysis or polymorphism analysis depending on the recognition nucleic acid sequence.

The detection principle is a reaction with a labeled ligand or nucleic acid. RI method and non-RI method (fluorescence method, biotin method, chemiluminescence method, etc.) are known as labeling methods.
It is not particularly limited. For example, in the case of the fluorescence method,
As the fluorescent substance used for the fluorescent label, any substance can be used as long as it can bind to a base portion of a nucleic acid or an amino acid residue of a protein, but a cyanine dye (for example,
Cy3, Cy5, etc. of commercially available Cy DyeTM series),
Rhodamine 6G reagent, N-acetoxy-N2-acetylaminofluorene (AAF) or AAIF (AAF
Iodine derivative) can be used.

In the case of a protein chip on which a nucleic acid recognizing protein is immobilized, it is preferable to directly detect the target nucleic acid in the sample without amplification by the PCR method or the like, but it may be detected after the amplification in advance. The target nucleic acid or its amplified product can be easily detected by labeling it in advance. To label nucleic acids, use the enzyme (Reverse Tr
anscriptase, DNA polymer
se RNA Polymerase, Termina
A method using ldeoxytransferase, etc.) is often used, but a labeling substance may be directly bound by a chemical reaction. For such labeling methods,
It is described in a book as a known technique (Shintaro Nomura, Deisotope Experiment Protocol 1, Shujunsha 1994).
Year, Deisotope experiment protocol 2, Shujunsha 199
8 years, Masaaki Muramatsu DNA microarray and latest PCR
Law-marked substance Shujunsha 2000). The labeling substance is preferably a substance capable of producing a detectable signal. When the labeling substance is a substance capable of amplifying a signal, such as an enzyme or a catalyst, the detection sensitivity of DNA is greatly improved.

However, since the above-mentioned labeling operation is generally complicated, a more preferable detection method is as follows.
A method of measuring the nucleic acid in the sample without labeling it beforehand can be mentioned. For this, for example, a DNA intercalating agent that recognizes double-stranded DNA, a so-called DNA intercalator can be used. The use of the DNA intercalator not only simplifies the detection operation, but also improves the detection sensitivity. For example, when detecting 1000 bp of DNA, the so-called labeling method can introduce only a few labeling substances at most, but when using an intercalator, 100 or more labeling substances can be introduced.

The DNA intercalator may be a substance capable of forming a detectable signal by itself, but may have a signal forming substance bound to its side chain,
It may be bound to the intercalator via a specific binding pair such as biotin-avidin, antigen-antibody, hapten-antibody. In the present invention, the detectable signal is
For example, fluorescence detection, luminescence detection, chemiluminescence detection, bioluminescence detection, electrochemiluminescence detection, radioactivity detection, electrochemical detection,
A signal that can be detected by colorimetric detection is preferable, but the signal is not limited thereto.

When the ligand is a target, a cyanine dye (for example, commercially available Cy Dy
eTM series Cy3, Cy5, etc.), Rhodamine 6G reagent, N-acetoxy-N2-acetylaminofluorene (AAF) or AAIF (iodine derivative of AAF)
The product obtained by reacting the succinimide compound can be used. The present invention will be described in more detail with reference to the following examples, which are intended to facilitate understanding of the present invention and are intended to limit the present invention thereto. is not.

[0061]

[Example 1] Detection of ligand by antibody-immobilized slide (1) Preparation of vinylsulfonyl group-introduced porous solid phase carrier (A) Colloidal silica (SNO-TEX PS-S [Nissan Chemical Industry] ] / 15% suspension with an average particle size of about 10 nm 15
g, methanol 13 g, water 5 g, TMOS 1 g,
Stir for 10 minutes and rest for 1 hour. The solution was filtered at 0.22 micron. A slide glass was immersed in this solution and dried at room temperature for 2 hours. Then at 70 ℃ for 1 hour,
Heated at 100 ° C. for 2 hours. This slide glass is reacted with 200 ml of a 2 mass% solution of Shin-Etsu Silicone KBE 903 (Shin-Etsu Chemical Co., Ltd.) for 3 minutes using a commercially available slide washer. After completion of the reaction, the plate is washed with 200 ml of ultrapure water for 1 minute (using a slide washer). While exchanging the ultrapure water, the above-mentioned washing condition is repeated twice. After washing with water, and after drying for 10 minutes in a dryer at 45 ℃, 1
Put in an oven set at 10 ° C. and heat-treat for 10 minutes. After cooling, the mixture is reacted with 3 wt% 1,2-bis (vinylsulfonylacetamide) ethane / pH 8.0 borate buffer solution for 120 minutes using a slide washer. After completion of the reaction, the product is washed with ultrapure water for 20 seconds x 3 times. Dry 2
It was carried out for 30 minutes with a dryer set at 5 ° C. to obtain a porous solid phase carrier (A).

A film refractive index of about 1.3 was obtained by measuring the deflection of the surface of the porous solid phase carrier (A). Since the refractive index of the porous film is the volume average of the solid phase and the pore space, an estimated value of the porosity can be obtained. From the above, the porosity of the above film was estimated to be about 30 to 40%. The film thickness is about 65.
It was requested to be 00 angstrom. Pore size is calculated by assuming three equal size particles in a triangular layer:
Guessed. In this arrangement, the pore diameter is about 0.15 times the diameter of the particles, so in the case of the above 10 nm particles, the smallest pore is estimated to be on the order of 2 nm. The above measurement was performed in the same manner as the method described in International Publication WO00 / 61282.

(2) Immobilization of antibody Goat Anti-Human IgG (Jacks
on ImmunoResearch) is diluted with PBS (100, 20, 4, 0.8, 0.16 ng / μ)
L, 1 μL), the solid phase carrier (A) prepared in (1) above
Spotted on. Immediately after the solid-phase carrier after spotting was left in a saturated saline solution chamber at 25 ° C. for 3 hours, 1% BSA /
1 in 0.05% Tween20-PBS (PBS-T)
Blocking was carried out by immersing for a period of time to obtain an antibody slide (B).

(3) Reaction with Ligand and Detection The antibody slide (B) prepared in (2) above was hybridized.
Well (GraceBio-Labs) was adhered. Human IgG-Cy5 (Jackson I
mmunoResearch) with 1% BSA / PBS-
Dilute to 2 μg / ml with T and add 100 μL to Hybri
After adding to the well, 2 in the moisture chamber
Incubated at 5 ° C for 1 hour. Then this one,
Wash 3 times with PBS-T, rinse with PBS, 700 rp
It was dried by centrifuging at m for 5 minutes. When the fluorescence intensity on the surface of the slide glass was measured with a fluorescence scanning device, it was 26.3 at the position where the antibody was spotted at 100 ng / μL, which was much higher than the background fluorescence intensity. Therefore, by using an antibody-immobilized solid-phase carrier in which an antibody is bound to the porous solid-phase carrier of the present invention via a sulfonyl group, a ligand having reactivity with the antibody immobilized on the antibody-immobilized solid-phase carrier is used. Can be detected efficiently

[Example 2] Detection of ligand by antibody-immobilized slide (1) Immobilization of antibody Rabbit Anti Streptavidin
Dilute (Polyscience) with PBS (100
ng / μL), 10 μL each was dispensed to a 384 plate. Cartesian Arlayer (PixSys550
0) was applied to the solid phase carrier (A) prepared in Example 1 (1) above. Immediately after applying the solid phase carrier at 25 ° C,
1/4 × after standing in saturated saline solution chamber for 1 hour
Block Ace (Dainippon Pharmaceutical) /0.05% Tween
Blocking was performed by immersing in 20-PBS (PBS-T) for 1 hour to obtain an antibody slide (C).

(2) Labeling of ligand 1 mg of Streptavidin (Wako Pure Chemical Industries)
It was dissolved in 0 μl of 0.1 M NaHCO 3 (pH 8.0). Cy3-monofunctional
(Amersham Pharmacia Biote
ch) tube and incubated at room temperature for 30 minutes. There, 100 μl of 1M Tris · HCl
The reaction was stopped by adding (pH 8.0) and further incubating at room temperature for 30 minutes. This reaction solution was applied to a NAP-5 column (Amersham Pharmac).
IA Biotech), and Cy3 labeled Str
eptavidin was obtained.

(3) Reaction with Ligand and Detection Secur on the antibody slide (C) prepared in (1) above.
eSeal Hybridization Chamber
r (SA500, Grace Bio-Labs) was brought into close contact. Cy3-Strept labeled in (2) above
Avidin was diluted to 1 μg / ml with 1/4 × Block Ace / PBS-T, and 500 μL was added to SA500, followed by incubation in a moisture chamber at 25 ° C. for 1 hour. Then, this was washed with PBS-T three times, rinsed with PBS, and then centrifuged at 700 rpm for 5 minutes to be dried. When the fluorescence intensity on the surface of the slide glass was measured with a fluorescence scanning device, it was 2
The intensity was 1,300, which was larger than the background fluorescence intensity. Therefore, by using an antibody-immobilized solid-phase carrier in which an antibody is bound to the porous solid-phase carrier of the present invention via a sulfonyl group, it is possible to react with an antibody immobilized on the antibody-immobilized solid-phase carrier in a microarray system. It can be seen that a ligand having a property can be efficiently detected.

[0068]

Industrial Applicability According to the present invention, it is possible to provide a biological material chip in which at least one member of a specific binding partner is bound and immobilized on a reactive solid phase carrier which can be rapidly and stably bound and immobilized. became.

[Brief description of drawings]

FIG. 1 is a schematic diagram of the structure of a protein chip that is a typical embodiment of the present invention.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/566 G01N 33/566 (72) Inventor Yukio Sudo 3-11-46 Izumi, Asaka-shi, Saitama Fuji Photo Film Co., Ltd. (72) Inventor Hiroshi Shinoki 3-11-46 Fuji, Asaka-shi, Saitama Prefecture Photo Fuji Photo 3-11-46 Izumi Osamu, Inventor, Asaka-shi, Saitama Fuji Photo F-term within the film Co., Ltd. (reference) 4B029 AA07 AA23 BB15 BB17 CC10 FA01 4H045 AA40 BA62 BA63 DA50 DA76 DA86 EA50 FA81

Claims (30)

[Claims] 1. The porous region is about 2 nm to about 1000 nm.
Pore size of about 10% to about 90% porosity and about 0.01
In a solid-phase carrier having a porous substrate having a thickness of μm to about 70 μm, at least one residue of protein or protein-binding substance (excluding nucleic acid) A is represented by the following formula (I): biological material chip, characterized in that it covalently bonded via a sulfonyl group; in the solid phase support _{-L-SO 2 -X-a (} I) [ formula (I), L a linking group Where X is -C
Represents R ¹ (R ² ) -CR ³ (R ⁴ )-; R ¹ , R ² , R ³
And R ⁴ are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, or a total carbon atom number of 7 having an alkyl chain having 1 to 6 carbon atoms. To 26 aralkyl groups, A is a protein or protein-binding substance (excluding nucleic acid)
Indicates the residue of. ] 2. The chip according to claim 1, wherein the protein or protein-binding substance (excluding nucleic acid) immobilized on the surface is an antibody, an antibody fragment, a ligand, an antigen, a hapten, or a receptor. 3. The chip according to claim 1, wherein the protein or protein-binding substance (excluding nucleic acid) immobilized on the surface is an avidin. 4. The chip according to claim 3, wherein the avidins are avidin, streptavidin, or a modified form thereof capable of forming a stable complex with biotin. 5. The chip according to claim 1, wherein the protein immobilized on the surface is a nucleic acid recognition protein. 6. The chip according to claim 5, wherein the nucleic acid recognition protein is a double-stranded DNA recognition protein. 7. The double-stranded DNA recognition protein is double-stranded D
The chip according to claim 6, which is an NA-recognizing antibody. 8. The chip according to claim 6, wherein the double-stranded DNA recognition protein is a DNA transcription factor. 9. The chip according to claim 6, wherein the double-stranded DNA recognition protein is a protein having a Zn finger motif or a ring finger motif. 10. The chip according to claim 1, wherein the porous substrate is composed of an organic polymer. 11. The chip according to claim 1, wherein the porous substrate is particles formed of an inorganic substance. 12. The chip according to claim 1, wherein the porous substrate contains silicon, alumina or titanium. 13. The solid phase carrier is glass, plastic, an electrode surface, or a sensor chip surface, according to claim 1.
The chip according to any one of 1. 14. A sample comprising the chip according to any one of claims 1 to 13 and a target substance that specifically binds to a protein or a protein-binding substance (excluding nucleic acid) carried on the surface of the chip. And a step of detecting the formation of a mutual bond between the protein or protein-binding substance and the target substance. 15. The method for detecting a target substance according to claim 14, wherein the target substance is labeled with at least one component capable of generating a detectable signal. 16. The method for detecting a target substance according to claim 14 or 15, which comprises a step of blocking the chip with an aqueous solution of amino acids, peptides or proteins. 17. A vinylsulfonyl group represented by the following formula (II) or a reactive precursor group thereof is provided on a solid phase carrier having a porous substrate having on its surface a vinylsulfonyl group or a reactive precursor group thereof. The method for producing a chip according to claim 1, which comprises contacting at least one protein or a protein-binding substance (excluding nucleic acid) having a reactive group that reacts with to form a covalent bond. —L—SO ₂ —X ′ (II) [In the above formula, L represents a linking group that binds —SO ₂ —X ′ and the solid phase carrier; X ′ represents —CR ¹ ═CR
² (R ³ ) or —CH (R ¹ ) —CR ² (R ³ ) (Y) is represented; R ¹ , R ² and R ³ are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. , 6 to 20 carbon atoms
Or an aralkyl group having a total of 7 to 26 carbon atoms having an alkyl chain having 1 to 6 carbon atoms, Y is a group substituted by a nucleophile, or eliminated as "HY" by a base. Represents the group to 18. The method for producing a chip according to claim 17, wherein the protein or protein-binding substance (excluding nucleic acid) immobilized on the surface is an antibody, an antibody fragment, a ligand, an antigen, a hapten, or a receptor. . 19. The method for producing a chip according to claim 17, wherein the protein immobilized on the surface is an avidin. 20. The method for producing a chip according to claim 19, wherein the avidins are avidin, streptavidin, or modified forms thereof that can form a stable complex with biotin. 21. The method for producing a chip according to claim 17, wherein the protein immobilized on the surface is a nucleic acid recognition protein. 22. The method for producing a chip according to claim 21, wherein the nucleic acid recognition protein is a double-stranded DNA recognition protein. 23. The method for producing a chip according to claim 22, wherein the double-stranded DNA recognizing protein is a double-stranded DNA recognizing antibody. 24. The double-stranded DNA recognition protein is DNA
The chip manufacturing method according to claim 22, which is a transcription factor. 25. The method for producing a chip according to claim 22, wherein the double-stranded DNA recognition protein is a protein having a Zn finger motif or a ring finger motif. 26. The method for producing a chip according to claim 17, wherein the porous substrate is composed of an organic polymer. 27. The chip manufacturing method according to claim 17, wherein the porous substrate is particles formed of an inorganic substance. 28. The method for producing a chip according to claim 17, wherein the porous substrate contains silicon, alumina or titanium. 29. The solid phase carrier is glass, plastic, an electrode surface, or a sensor chip surface, according to claim 17 to 2.
9. The method for manufacturing a chip according to any one of 8. 30. After immobilization on a solid phase carrier having a porous substrate by contacting at least one protein or protein-binding substance (excluding nucleic acid),
30. The method for producing a chip according to claim 17, wherein the free vinylsulfonyl group or its reactive precursor group on the surface of the solid support is subjected to a blocking treatment with an amino acid, peptide or protein aqueous solution. .