JP2003021633A - Biological material chip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a chip comprising at least one of proteins or protein binding materials other than nucleic acids and a reactive solid support, wherein proteins or protein binding materials are immobilized rapidly and stably on the support and a method for detecting specific bonding target materials using the chip. SOLUTION: The biological material chip is characterized by bonding A to the solid phase support having unevenness through a covalent bonding via sulfonyl group as shown in formula (I), wherein the A is a residue of at least one proteins or protein binding materials other than nucleic acid; Solid phase support-L-SO2 -X-A (I) In formula (I), L represents a linker group, X represents- CR<1> (R<2> )-CR<3> (R<4> )-; R<1> , R<2> , R<3> and R<4> are each independently hydrogen, alkyl group of 1 to 6 carbon atoms, aryl group of 6 to 20 carbon atoms, or aralkyl group of 7 to 26 total carbon atoms having alkyl chain of 1 to 6 carbon atoms, and A represents a residue of proteins or protein binding materials other than nucleic 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. However, in this case, (1) when detected by fluorescence, luminescence, RI, etc., scattering by excitation light, luminescent substance, etc. increases, and the background becomes high; (2) liquid permeation during spotting is large. Therefore, it is difficult to reduce the spot diameter, and it is difficult to create a high-density array; (3) Since the labeled sample enters the spaces between the particles, it is necessary to sufficiently perform the washing process, If it is enough, the background is high and things are common:

[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 the first aspect of the present invention, A, which is a residue of at least one protein or protein-binding substance (excluding nucleic acid), is added to a solid-phase carrier having irregularities. A biological material chip, which is bound by a covalent bond via a sulfonyl group as represented by the following formula (I), is provided. 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. ]

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
A reaction of reacting with the vinylsulfonyl group or its reactive precursor group to form a covalent bond on a solid-phase carrier having a surface having a vinylsulfonyl group or its reactive precursor group represented by I) There is provided a method for producing a chip of the present invention, which comprises contacting at least one protein or protein-binding substance having a functional group (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-recognition antibody; a double-stranded DNA recognition protein is a DNA transcription factor; a double-stranded DNA recognition protein is a protein having a Zn finger motif or a ring finger motif; unevenness is formed by an inorganic substance A particle having an average particle size of 50 μm or less containing silicon, alumina, or titanium; the solid phase carrier is glass, plastic,
Embodiments that are electrode surfaces and sensor chip surfaces; embodiments in which the target substance is labeled with at least one component capable of producing a detectable signal; embodiments that include a step of blocking the chips with an aqueous solution of amino acids, peptides or proteins;

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention provides a solid-phase carrier having irregularities in which at least one protein or protein-binding substance (excluding nucleic acid) residue A has the following formula (I): The present invention relates to a biological material chip, which is bound by a covalent bond via a sulfonyl group. 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 irregularities forms a covalent bond via a sulfonyl group due to the amino group or mercapto group contained therein. Furthermore, amino groups on proteins,
An imino group, a hydrazino group, a carbamoyl group, a hydrazinocarbonyl group, a mercapto group, or a carboximide group may be introduced to form a covalent bond via a sulfonyl group.

A chip in which a protein or a protein-binding substance (for example, an antibody, avidins, a nucleic acid recognizing protein) is immobilized on a solid-phase carrier having irregularities is specific to the immobilized protein or the protein-binding substance in the presence of an aqueous medium. The target substance can be immobilized by bringing it into contact with a target substance that reacts with (for example, a ligand, biotin, or nucleic acid). 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.

As the solid phase carrier, if it does not adversely affect the bond formation between members of the specific binding partner, its shape may be, for example, flat plate, microwell, bead, stick or the like. You can also It is preferable that the surface of those substrates is a substrate having a particularly hydrophobic or hydrophilic surface and a smooth surface. Alternatively, a substrate having a surface with unevenness and low planarity can be used. As the material of the solid phase carrier, glass, cement,
Ceramics such as ceramics or new ceramics,
Polyethylene terephthalate, cellulose acetate, bisphenol A polycarbonate, polystyrene, polymers such as polymethyl methacrylate, silicon, activated carbon, porous glass, porous ceramics, porous silicon, porous activated carbon, woven / knitted fabric, nonwoven fabric, filter paper, short fiber , Various porous materials such as membrane filters. The size of the pores of the porous material is 2 to 10
It is preferably in the range of 00 nm, preferably 2 to 500 n
The range of m is particularly preferable. The material of the solid phase carrier is particularly preferably glass or silicon. This is because surface treatment is easy and analysis by an electrochemical method is easy. The thickness of the solid phase carrier is 1
It is preferably in the range of 00 to 2000 μm. Further, for convenience of operation, the solid phase carrier may be processed into a magnetic material or an electrode.

As the solid phase carrier having irregularities of the present invention,
Various solid phase carriers that have been conventionally used for the production of DNA chips or proposed for the production of DNA chips are preferable, and they can be used as they are as protein chips. Examples of such solid phase carriers include:
Examples thereof include a glass substrate, a resin substrate, a glass substrate surface-treated with a silane coupling agent, a resin substrate, or a glass substrate or a resin substrate having a coating layer on the surface. The solid phase carrier is preferably a silicate glass substrate, a silicate glass substrate surface-treated with a silane coupling agent, or a silicate glass substrate coated with an organic coating layer. Further, it may be an electrode substrate used in an electrochemical analysis method. Further, various functional substrates such as the above-mentioned substrate for surface plasmon resonance (SPR) biosensor and charge coupled device (CCD) may be used. Further, in addition to these substrates, a particulate solid phase carrier or the like can be used.

As the uneven substance, an inorganic substance is preferable, and examples thereof include colloidal silica, alumina sol, titanium oxide, metal oxides such as ZrO ₂ , plastics having a hydroxyl group, and latex. The particle size is preferably 50 μm or less, and particularly in the case of detecting confocal laser fluorescence, in order to suppress the background, 100
It is preferable to use fine particles of nm or less. As a method for coating the solid phase carrier with the uneven substance, the uneven substance is mixed with the silane coupling agent, and the solid phase carrier is brought into contact with and reacted to easily give unevenness to the solid phase carrier. You can It is particularly effective when the solid phase carrier is glass. It can also be produced by bringing an uneven material into contact, drying and then treating with a silane coupling agent. When the solid phase carrier is coated with a particulate substance to form irregularities, the surface area becomes considerably larger than that on a smooth surface, and there is an advantage that the amount of the bound DNA fragment to be immobilized can be significantly increased. Furthermore, the single layer is more advantageous than the multi-layer form for the following reasons. 1. When detection is performed by fluorescence, luminescence, RI, etc., scattering due to excitation light, luminescent substances, etc. is reduced, and the background is reduced. 2. Since the penetration of the liquid at the spot is small, the spot diameter can be made small and a high-density array can be created. 3. In the post-hybridization operation, the post-treatment washing step for removing unreacted labeled sample becomes easy. In the case of a multilayer, the labeled analyte enters the voids between the particles. For this reason, it is necessary to sufficiently perform the washing process, and if insufficient, the background is often high.

In the present invention, the protein or protein-binding substance (excluding nucleic acid) A on the chip is bound to the solid phase carrier having irregularities by a covalent bond via a sulfonyl group as shown in the following formula (I). Has been done. 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 and 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.

Further, the present invention provides a vinylsulfonyl group represented by the following formula (II) or a reactive precursor group thereof on a solid phase carrier having irregularities on the surface thereof, the vinylsulfonyl group or a reactive precursor thereof. 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 that reacts with a 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.

[0041]

[Chemical 1]

"-X '" means (X1),
(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 not necessary to newly introduce a 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 protein-binding substance from drying and denaturing after spotting, a substance having a high boiling point may be added. The substance having a high boiling point is a substance that can be dissolved in an aqueous liquid obtained by dissolving or dispersing the spotted protein or protein-binding substance, does not interfere with the reaction with the sample to be detected, and has a high viscosity. It is preferably a material that is not very 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 10
It is preferably in the range of ^{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 to place the solid-phase carrier after spotting the protein or the protein-binding substance 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 the reactive group (Z) is spotted on the surface of the uneven solid support (P1) shown in FIG. 1, a reaction between X and the protein occurs.
Unreacted X to which the protein is not bound is also present on the surface of 1). 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. Blocking treatment is a solid phase carrier (P2)
It is preferable to bring the surface of the product into contact with a material 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 an anionic compound is a compound that reacts with X and has a negative charge (COO −,
Any of those having 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 that the target nucleic acid in the sample is directly detected without amplification by PCR method or the like, but it may be detected after being amplified 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 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 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 the target, the 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.

[0057]

[Examples] [Example 1] Detection of ligand by antibody-immobilized slide (1) Preparation of uneven solid phase carrier (A) having vinylsulfonyl group introduced Colloidal silica (Snowtex PS-S [Nissan Chemical Industry]) 200 ml of a 5% by weight suspension having an average particle diameter of about 10 nm) was prepared, and the washed glass slide 2 was placed therein.
Place 0 in a commercial basket and let it soak for 30 seconds. After pulling it up, it is drained and dried at 45 ° C. for 10 minutes. Next, attach this glass slide to Shin-Etsu Silicone K.
200 ml of a 2 wt% solution of BE903 (Shin-Etsu Chemical Co., Ltd.)
Then use a commercially available slide washer for 3 minutes. 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
After drying for 10 minutes with a dryer at 45 ° C., it is placed in an oven set at 110 ° C. and heat-treated 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. Drying was carried out for 30 minutes with a dryer set at 25 ° C. to obtain an uneven solid phase carrier (A). Atomic force microscope (AFM) (A)
When the surface of the sample was observed, it was found that the thickness was about 10 nm, and it was confirmed that it was coated with a single layer.

(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 by a fluorescence scanning device, it was 39.2 at the position where the antibody was spotted at 100 ng / μL, which was much higher than the background fluorescence intensity. Therefore, by using the antibody-immobilized solid-phase carrier in which the antibody is bound via the sulfonyl group on the uneven solid-phase carrier of the present invention, it has reactivity with the antibody immobilized on the antibody-immobilized solid-phase carrier. It turns out that the ligand 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) was used.
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, 1
The intensity was 9,320, which was much higher than the background fluorescence intensity. Therefore, by using an antibody-immobilized solid-phase carrier in which an antibody is bound via a sulfonyl group on the uneven solid-phase carrier of the present invention, an antibody immobilized on the antibody-immobilized solid-phase carrier in a microarray system is also used. It can be seen that a reactive ligand can be efficiently detected.

[Example 3] Detection of ligand by antibody-immobilized slide (1) Solid support having vinylsulfonyl group introduced (D)
Preparation of coated glass slides for DNA microarray (Type
e2 Densified amino group introduction type [Matsunami Glass Industry]) 2
0 sheets were placed in a commercially available basket and 3% by weight of 1,2-bis (vinylsulfonylacetamide) ethane / pH 8.0
Immerse in a borate buffer solution and react for 120 minutes using a slide washer. After the reaction is complete, use ultrapure water for 20
Rinse with water 3 times per second. Drying is performed with a dryer set at 25 ° C. for 30 minutes.

(2) Immobilization of antibody Goat Anti Human IgG (Jacks
on ImmunoResearch) was diluted with PBS (100 ng / μL) and dispensed in 10 μL aliquots on a 384 plate. Cartesian Arlayer (PixSy
s5500) was applied to the solid phase carrier (D) prepared in Example 3 (1) above. Immediately after leaving the solid-phase carrier after spotting at 25 ° C. in a saturated saline solution chamber for 1 hour,
1/4 x Block Ace (Dainippon Pharmaceutical) /0.05% T
The antibody slide (E) was obtained by blocking by immersing it in ween20-PBS (PBS-T) for 1 hour.

(3) Reaction with Ligand and Detection Secur on the antibody slide (E) prepared in (1) above.
eSeal Hybridization Chamber
r (SA500, Grace Bio-Labs) was brought into close contact. Human IgG-Cy5 (Jackso
n ImmunoResearch) with ¼ × Block Ace / PBS-T 5, 2, 1, 0.2, 0.
After diluting to 1 μg / ml and adding 500 μL into SA500, it was incubated at 25 ° C. for 1 hour in a moisture chamber. Then use this with PBS-T 3
It was washed twice, rinsed with PBS, and then centrifuged at 700 rpm for 5 minutes to dry it. When the fluorescence intensity on the surface of the slide glass was measured with a fluorescence scanning device,
2, 1, 0.2, 0.1 μg / ml Human I
33,5 for slides containing gG-Cy5, respectively
25, 23, 629, 15, 001, 8, 667, 6,
053, and almost linearity was established in the range of 0.2 to 2 μg / ml (FIG. 2).

[0066]

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.

FIG. 2 is a graph showing the results of detecting a ligand using the antibody-immobilized slide according to the present invention.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 31/22 121 G01N 37/00 102 37/00 102 C12N 15/00 F (72) Inventor Yukio Sudo Saitama 3-11-46 Izumi, Asaka-shi, Japan (72) Inventor, Hiroshi Shinoki, Hiroshi Shinoki Saitama 3-11-46 Izumi, Asaka-shi (72) Inventor, Osamu Seshimoto Saitama 3-11-46 Izumi, Asaka-shi, Japan F-Term inside Fuji Photo Film Co., Ltd. (reference) 2G042 BD19 CB03 FB05 HA02 4B024 AA11 HA11 4H045 AA11 AA20 AA30 DA00 DA50 DA76 EA50 FA80

Claims (28)

[Claims] 1. A solid phase carrier having irregularities, in which at least one residue of protein or protein-binding substance (excluding nucleic acid), A, is shared via a sulfonyl group as in the following formula (I). biological material chip wherein the linked by bonds; 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. ] 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 irregularities are particles formed of an inorganic substance. 11. The chip according to claim 1, wherein the irregularities are particles containing silicon, alumina or titanium and having an average particle size of 50 μm or less. 12. 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. 13. A sample comprising the chip according to claim 1 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. 14. The method for detecting a target substance according to claim 13, wherein the target substance is labeled with at least one component capable of generating a detectable signal. 15. The method for detecting a target substance according to claim 13 or 14, comprising a step of blocking the chip with an aqueous solution of an amino acid, a peptide or a protein. 16. A reaction with a vinyl sulfonyl group or its reactive precursor group on a solid phase carrier having a surface having a vinyl sulfonyl group or its reactive precursor group represented by the following formula (II) The method for producing a chip according to claim 1, which comprises contacting at least one protein or protein-binding substance (excluding nucleic acid) having a reactive group that forms a covalent bond with each other. —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 17. The method for producing a chip according to claim 16, 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. . 18. The method for producing a chip according to claim 16, wherein the protein immobilized on the surface is an avidin. 19. The method for producing a chip according to claim 18, wherein the avidins are avidin, streptavidin, or modified forms thereof capable of forming a stable complex with biotin. 20. The method for producing a chip according to claim 16, wherein the protein immobilized on the surface is a nucleic acid recognition protein. 21. The method for producing a chip according to claim 20, wherein the nucleic acid recognition protein is a double-stranded DNA recognition protein. 22. The method for producing a chip according to claim 21, wherein the double-stranded DNA recognizing protein is a double-stranded DNA recognizing antibody. 23. The double-stranded DNA recognition protein is DNA
The method for manufacturing a chip according to claim 21, which is a transcription factor. 24. The method for producing a chip according to claim 21, wherein the double-stranded DNA recognition protein is a protein having a Zn finger motif or a ring finger motif. 25. The method for manufacturing a chip according to claim 16, wherein the irregularities are particles formed of an inorganic substance. 26. The method for producing a chip according to claim 16, wherein the irregularities are particles containing silicon, alumina or titanium and having an average particle diameter of 50 μm or less. 27. The solid phase carrier is glass, plastic, an electrode surface, or a sensor chip surface, according to claim 16 to 2.
6. The method for manufacturing a chip according to 6. 28. After immobilizing on a solid-phase carrier having irregularities by contacting at least one protein or protein-binding substance (excluding nucleic acid), a free vinylsulfonyl group or the free vinylsulfonyl group on the surface of the solid-phase carrier. 28. The method for producing a chip according to claim 16, wherein the reactive precursor group is subjected to a blocking treatment with an amino acid, peptide or protein aqueous solution.