JP2001242116A - Protein chip and method for detecting protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a protein chip and a method for detecting protein, for the purpose of performing the analysis of the interaction of protein with other protein as a part of research of protein through an electrochemical technique. SOLUTION: The protein chip is characterized, in that protein is fixed on the surface of a substrate and adapted in order, to detect protein forming a specific bond with respect to the protein on the surface of the substrate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a protein analysis technique, which is particularly useful for studies on protein expression, functional analysis, interaction with other biochemical molecules, identification and modification of proteins, etc. To a protein chip in which is fixed.

[0002]

2. Description of the Related Art Various reactions exhibited by living organisms are caused and controlled by the interaction of in vivo molecules, particularly proteins. Therefore, in order to understand biological reactions at the molecular level, it is essential to elucidate the interactions between molecules in the living body. For that purpose, it is required to clarify protein expression, post-translational modification (phosphorylation, dephosphorylation, etc.), interaction between the protein and other substances, and the like.

[0003] Extensive research into the properties of proteins has been performed for proteome (complete set of proteins expressed by the genome) analysis. The main task is to analyze protein expression levels, post-translational modifications and interactions, and to clarify the phenomena occurring at the protein expression level in health and disease states. On the other hand, 20
The Human Genome Project, which will elucidate the nucleotide sequences of all human genes by 2003, has been progressing at a rapid pace with the advent of a nucleotide sequence analysis system with significantly improved processing capacity. However, the function of the genome is not determined only by the base sequence. That is, not all proteins encoded on the genome gene are simultaneously expressed, and the presence / absence / amount of gene expression varies depending on the organ, tissue, drug, time, aging, and type of disease. In vivo proteins also have inhomogeneities such as post-translational modifications and higher tertiary structures.

Therefore, one of the promising means of proteome analysis is the development of mass spectrometry for analyzing proteins with high sensitivity. In mass spectrometry, spots are cut out from an electrophoresis gel, subjected to reduction or trypsin digestion treatment, and then the molecular weight of the degraded protein is measured, and the protein is identified by searching a database. As an improved method of the above mass spectrometry, MALDI-T
OF MS (matrix assisted ionization-time of flight mass spectrometer: Koster, H. et al., Nature Biotechnol., 14,
1123-1128 (1996) and Griffin, TJ et al., Nature B
iotechnol., 15, 1368-1372 (1997)). M
ALDI uses a matrix as a reagent to promote ionization of a sample, irradiates a laser beam onto the matrix provided with the sample, evaporates the sample with energy from the matrix in an excited state, and performs a gas phase reaction. This is a method of ionizing a sample. TO
F is the time required for the ionized sample to reach the detector installed on the opposite side of the sample plate from low molecular weight molecules after free flight in a high vacuum flight tube. Is measured to determine the molecular weight.

[0005] SEIDI (Surface Enhanced Laser D) combining a TOF mass spectrometer with a protein chip.
esorption / Ionization) Protein chip system
A powerful tool for proteome analysis. The SELDI protein chip is a chip in which the substrate surface is chemically modified (cations, anions, hydrophobicity, hydrophilicity, metal ions, etc.) or biochemically modified (antibodies, receptors, etc.). Even chips are being developed. In this system, protein, DN
A, A solid phase reaction with various low molecular substances is performed, and the molecular weight of the analyte is measured by a TOF mass spectrometer. As a result, it is possible to analyze differences in protein expression between cells, the interaction between a specific protein and another substance, quantify trace substances in a sample, measure post-translational modification, and measure enzyme activity. This method eliminates the need for labeling the sample and allows detection of the sample down to the level of 1 attomole to 1 femtomolar.

[0006] As a protein chip, a sensor chip in which a protein is immobilized on the surface of a gold film whose surface is coated with carboxydextran is also known. In a method for detecting a protein-protein interaction using a sensor chip (mainly, screening of a monoclonal antibody), a sample is added to the chip surface via a cassette-type microchannel system, and a bilayer of the protein and the sample is added. The change in mass on the chip surface due to the binding and dissociation between them is detected as a surface plasmon resonance (SPR) signal. In this method, it is not necessary to label the sample.

[0007] SELDI protein chips and sensor chips are generally effective when the interaction between a protein and another substance is strong (eg, ligand-receptor, antigen-antibody, etc.), and is used for detecting weak interaction. Is not an effective method. Also, with the current technology of SELDI protein chips and sensor chips, it is difficult to analyze many samples simultaneously in a short time.

[0008]

DISCLOSURE OF THE INVENTION The present invention provides a protein chip, which aims to analyze the interaction between a protein and another protein as a part of protein research by utilizing an electrochemical technique. And a method for detecting a protein.

[0009]

Means for Solving the Problems The present invention resides in a protein chip for detecting a protein which forms a specific bond with a protein on the surface, wherein the protein is immobilized on the substrate surface. .

[0010] Preferred embodiments of the protein chip of the present invention are as follows. (1) The substrate is an electrode provided with a terminal for outputting to the outside. (2) There are a plurality of electrodes, and proteins having different amino acid sequence sites are immobilized on each of the plurality of electrode surfaces. (3) The electrodes are gold deposited on the electrically insulating substrate surface at regular intervals, and the protein is bound to the gold surface at one end via a thiol bond.

According to the present invention, a sample protein labeled with an electrochemically active substance is brought into contact with the surface of the above-described protein chip of the present invention in a liquid phase, and the protein fixed on the chip and the sample protein are contacted. Forming a complex with the complex on the substrate; and detecting the amount of label on the complex-fixed chip by electrochemical measurement through the substrate to which the complex is fixed. There is also a method for detecting a sample protein that forms a specific bond with a protein immobilized on a protein chip, which is a feature.

According to the present invention, further, a sample protein labeled with an electrochemically active substance is formed between the protein on the chip and the sample protein on the surface of the protein chip of the present invention in the liquid phase. Contacting while changing the liquid phase factor affecting the binding state to form a complex of the protein immobilized on the chip and the sample protein on the substrate; and immobilizing the complex. Comprising a step of detecting a change in the amount of label on the complex-fixed chip due to a change in the factor by electrochemical measurement through the substrate, wherein the protein is fixed to the protein chip. There is also a method for detecting a sample protein that forms a specific bond.

A preferred embodiment of the method for detecting a sample protein that forms a specific bond with a protein immobilized on a protein chip affects the binding state formed between the protein on the protein chip and the sample protein. The temperature of the liquid phase or the ionic strength of the liquid phase is used as a factor of the liquid phase.

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Protein chip (1) of the present invention
Is an analytical element in which a protein is immobilized on the surface of a substrate, and is an analytical element for detecting a protein that interacts with the protein on the surface. FIG. 1 schematically shows the most preferable protein chip (1) of the present invention and a typical protein detection method of the present invention (particularly, formation of a complex between a protein immobilized on a protein chip and a protein). Show.

The most preferred protein chip (1) of the present invention is an analytical element having a protein (5) fixed on the surface of an electrode (3) having a terminal (4) for outputting to the outside. preferable. The number of electrodes may be one or more. When the number of electrodes is two or more, the protein immobilized on each electrode surface may be the same (but completely the same) protein,
It is preferable that the amino acid sequence sites are different from each other.

Here, the term “complex” means a substance formed by the interaction between a protein immobilized on a protein chip and the protein, and the term “interaction” means
It refers to a general term for specific binding observed between proteins such as van der Waals force, Coulomb force, ionic bond, and electrostatic attraction. The term “immobilized” includes not only a state in which the protein is chemically bonded to the substrate surface but also a state in which the protein is temporarily immobilized by electrostatic bonding or the like. "Protein" also includes oligopeptides in the range of 2 to 10 residues and polypeptides in the range of 11 to 50 residues in terms of the number of amino acid residues, complex proteins include glycoproteins and phosphorylated proteins, , Including fibrous and spherical.

As shown in FIG. 1, the basic principle of the protein detection method of the present invention is as follows: a typical protein chip (1) of the present invention is labeled with an electrochemically active substance (7) in a liquid phase. The contacted protein (8) forms a complex (10) of the protein (5) immobilized on the electrode (3) and the labeled protein (8), and the complex (1)
0) is to convert the interaction observed in 0) into an electric signal. FIG. 1 shows a state in which an interaction is recognized between the protein (5) and the labeled protein (8), and a complex having electrochemical activity is formed on one electrode.

Hereinafter, the protein chip of the present invention will be described in detail.

The protein chip of the present invention has a protein immobilized on one substrate surface or on a substrate surface divided into a plurality of regions. When there are a plurality of regions, the number is preferably in the range of 8 to 500, and particularly preferably in the range of 25 to 400. When an electrode is used as the substrate, it is preferable that the electrodes are regularly arranged so as not to be in contact with each of the surfaces of the substrate having no electric insulation, which are divided into a plurality of regions. Examples of the material of the electrode include carbon electrodes such as graphite, glassy carbon, pyrolytic graphite, carbon paste, and carbon fiber; noble metal electrodes such as platinum, platinum black, gold, palladium, and rhodium; titanium oxide, tin oxide, and manganese oxide. Oxide electrodes such as lead oxide, Si, Ge, ZnO,
Examples thereof include semiconductor electrodes such as CdS, TiO ₂ , and GaAs, and electron conductors such as titanium. It is particularly preferable to use gold or glassy carbon. These electron conductors may be covered with a conductive polymer, or may be covered with a monomolecular film. As a result, a stable protein chip can be produced.

As the electrode having a plurality of electrodes disposed on an electrically insulating substrate, it is preferable to use a substrate obtained by treating the surface of a non-conductive substrate with the above-described electronic conductor.
It is particularly preferable to use one that has been subjected to a vapor deposition treatment with gold. Before the surface treatment with the electron conductor, the substrate may be provided with a layer made of a charged hydrophilic polymer substance or a layer made of a crosslinking agent on the substrate. By providing such a layer, unevenness of the substrate can be reduced. Further, depending on the substrate, a hydrophilic polymer substance having an electric charge can be contained in the substrate, and a substrate subjected to such treatment can be preferably used. A document in which a plurality of electrodes are arranged on an electrically insulating substrate is described in the literature (Sosn
owski, RG et al., Proc. Natl. Acad. Sci. USA, 94, 111.
9-1123 (1997)), a silicon chip on which nucleic acids are not immobilized can also be preferably used. Further, like a printed wiring board, an electrode may be printed on the board.

The substrate other than the electrodes is preferably an electrically insulating hydrophobic carrier or an electrically insulating low hydrophilic carrier. In addition, even if the surface has unevenness and low flatness, it can be preferably used. Materials for the substrate include glass, cement, ceramics such as ceramics or new ceramics, polyethylene terephthalate, cellulose acetate, polycarbonate such as bisphenol A, polystyrene, polymers such as polymethyl methacrylate, silicon, activated carbon, porous glass,
Porous ceramics, porous silicon, porous activated carbon,
Examples thereof include porous materials such as woven and knitted fabrics, nonwoven fabrics, filter paper, short fibers, and membrane filters. The size of the pores of the porous material is preferably in the range of 2 to 1000 nm, particularly preferably in the range of 2 to 500 nm. The material of the substrate is various polymers described above,
Particularly preferred is glass or silicon. This is due to the ease of surface treatment and the ease of analysis by electrochemical methods. The thickness of the substrate is not particularly limited.
m is preferably in the range.

The protein may be immobilized at any position, or may be immobilized at two or more positions, but is particularly preferably immobilized at the C-terminal or N-terminal. Known methods (covalent bond, ionic bond, physical adsorption, etc.) can be used as the method for immobilizing the protein. When the substrate surface has been vapor-deposited with gold, for example, a protein into which a cysteine residue is introduced is prepared, and the protein is transferred to the gold surface through a coordination bond between the mercapto group of the cysteine residue and gold. Can be fixed. The sequence position of the cysteine residue in the protein is not necessarily the terminal of the protein as long as it does not hinder the formation of a higher-order structure, but it is particularly preferable that the protein is fixed at the C-terminal or the N-terminal. For this reason, it is particularly preferable that the protein is at the C-terminal or N-terminal. If the substrate surface is coated with glassy carbon, the glassy carbon layer is oxidized with potassium permanganate to form a carboxylic acid group on the substrate surface (or on the surface of the glassy carbon layer). Is introduced, the protein is immobilized on the substrate by an amide bond.

As the protein, a protein fragment obtained by appropriately cleaving a protein obtained from an arbitrary biological material such as a cell, a polypeptide or an oligopeptide artificially synthesized can be used. In addition, a DNA fragment taken from any biological material is ligated to a DNA molecule of another organism in a test tube to prepare a recombinant DNA molecule, and the recombinant DNA molecule is transferred to a host cell, and then the host cell is cultured. It is also preferred to use proteins produced by (so-called recombinant DNA technology). Recombinant DNA technology is effective in that it can express or propagate a specific type of protein. DNA fragments of any organism can be obtained not only by a method of removing from biological material, but also by a method of artificial synthesis, a method of synthesizing by PCR, or a method of extracting mRNA from biological material and using this as a template to obtain cDNA (reverse transcriptase). Complementary DN
Those prepared by a method for synthesizing A) may be used. As the protein into which a cysteine residue has been introduced, it is preferable to use a protein produced by subjecting a DNA fragment having a cysteine residue to recombinant DNA technology.

For example, when detecting an interaction with a protein contained in a crude product at a specific stage of a translation step in protein synthesis of yeast, an open reading frame (ORF: stop codon) taken out of yeast is detected. It is preferable to use a protein which is expressed by incorporating a relatively long DNA sequence without a) into an expression vector.

The immobilization of the protein is preferably carried out by spotting an aqueous solution containing the protein on the substrate. When the immobilization is performed by a method other than chemical bonding, the surface of the substrate is treated with poly-L-lysine, polyethyleneimine, polyalkylamine, etc. in order to stably immobilize the protein on the substrate surface, and then the substrate after the treatment is treated. It is preferable to apply an aqueous solution of a protein containing a hydrophilic polymer on the surface. Also,
It is also preferable that after the aqueous solution of the protein containing the hydrophilic polymer is spotted, a heat treatment or an ultraviolet treatment is further performed. This is because heat treatment or ultraviolet treatment forms a crosslink between the protein and the substrate surface treated with polylysine or polyethyleneimine, and as a result, the protein is more stably immobilized. As the hydrophilic polymer, a cationic, anionic or zwitterionic polymer can be used, but one that does not interfere with electrochemical detection and one that does not inhibit the formation of a complex with the sample protein Is preferred.

After the aqueous solution of the protein is spotted, the protein is fixed on the surface of the substrate when left at a predetermined temperature (preferably room temperature) for several hours. After spotting, incubation may be performed if necessary. The conditions for spotting are
It depends on the type and size of the substrate to be used, the number of types of spotted proteins, and the like. The spotting can be performed by a manual operation, but it is also preferable to perform the spotting using a DNA spotter apparatus useful in the DNA microarray method. That is, using a DNA spotter device,
It is also preferable to spot an aqueous solution of the protein on the substrate divided into a plurality of regions so as to correspond to each region. After spotting, it is preferable to wash and remove unfixed proteins.

Confirmation of the fixation of the protein to the substrate surface
For example, it can be carried out by labeling a protein to be spotted in advance with an electrochemically active substance to be described later, and measuring the amount of current flowing between the substrate and the electrochemically active substance after spotting.

Hereinafter, the method for detecting a protein of the present invention will be described.

Examples of sample proteins to be detected include proteins, peptides, and composite peptides and proteins modified with sugar, lipid, phosphate, and the like. Extracts, tissues, leukocytes, body fluids (blood,
Serum, urine, semen, saliva, etc.), feces, and other proteins can be used without purification.

In the protein detection method of the present invention, the sample protein is labeled with an electrochemically active substance in order to measure the complex of the protein immobilized on the protein chip and the sample protein by an electrochemical technique. Signs include:
Two or more different types of electrochemically active substances different in peak current value depending on the applied potential may be used. Examples of the electrochemically active substance include the following organometallic compounds and substances having no metal atom but having conductivity.

As the organometallic compound, a π complex of a type in which an electron is donated from the orbit of the central metal atom to the free orbit of the atom of the ligand through a π bond in addition to the electron donation of the central metal atom from the ligand. It is preferable to use Further, as the organometallic compound, a coordination polymer in which repeating units are bonded to each other through a coordination bond can also be preferably used. The π complex has a monodentate coordination with respect to a metal atom,
Any of bidentate, multidentate or cross-linking coordination may be used. Specific examples of the π complex include a metallocene complex represented by the following formula. M is
Represents a metal atom.

[0032]

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The metal atom of the metallocene complex is Fe, N
i, Co, Mo, Zn, Cr, Tl, Ta, Ti, C
u, Mn, W, V, Ru or Os, preferably Fe, Co, Ni, Ru, Os, V or Cr
And more preferably Fe (where the metallocene complex is ferrocene).

As the π complex, a cyclobutadiene complex, a cyclopentadienyl complex, a phenanthroline complex, a bipyridyl complex or a triphenylphosphine complex represented by the following formula can also be preferably used. Examples of the phenanthroline complex include a tris (phenanthroline) zinc complex, a tris (phenanthroline) ruthenium complex,
Mention may be made of tris (phenanthroline) cobalt, di (phenanthroline) zinc, di (phenanthroline) ruthenium and di (phenanthroline) cobalt and phenanthroline platinum complexes. Examples of the bipyridyl complex include a bipyridyl platinum complex, a tris (bipyridyl) zinc complex, a tris (bipyridyl) ruthenium complex, a tris (bipyridyl) cobalt complex, a di (bipyridyl) zinc complex, a di (bipyridyl) ruthenium complex, and a di (bipyridyl) cobalt complex Can be mentioned.

[0035]

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[0036]

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As the bipyridine complex, [Pd (bp
y) ₂ ] ²⁺ , [CoCl ₂ (bpy) ₂ ] ⁺ , [Fe (bp
y) 3] ⁿ or [Cr (bpy) 3] ⁿ . Here, bpy represents 2,2′-bipyridine, and n represents an integer of −1 to 3. Examples of triphenylphosphine complexes include CoCH ₃ (PPh ₃ ) ₃ , C
It is preferably oH (N ₂ ) (PPh ₃ ) ₃ , RuH ₂ (PPh ₃ ) ₄ or RhH (PPh ₃ ) ₄ . Ph is
Indicates a phenyl group.

As conductive compounds, chlorochloroheme, chlorophyllide, chlorophyll, heme, vitamin B
A porphyrin complex such as ₁₂ can also be preferably used.

Examples of the conductive compound containing no metal atom include viologen, 2,2'-bipyridine, 1,10-phenanthroline, and catecholamine represented by the following formula. Provided that R ^A and R ^B independently represent an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, an aryl group having 6 to 12 carbon atoms, N, O And 2 to 12 carbon atoms containing 1 to 4 heteroatoms selected from the group consisting of
Is preferably a group selected from the group consisting of: The above alkyl group, alkoxy group, aryl group and heterocyclic group are all alkyl groups having 1 to 6 carbon atoms, alkoxy groups having 1 to 6 carbon atoms and aryl groups having 6 to 10 carbon atoms. May be substituted with a group selected from the group consisting of R ^A and R ^B are more preferably an alkyl group having 1 to 6 carbon atoms or an aryl group having 6 to 12 carbon atoms,
Particularly preferred is a phenyl group.

[0040]

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[0041]

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A sample protein labeled with an electrochemically active substance (hereinafter referred to as “protein probe”) can be easily obtained by a known method. In FIG.
A method for preparing a typical protein probe will be described. The protein probe (A) is obtained by reacting the active ester form of ferrocene (C) with the sample protein (D). The amino group of the sample protein (D) is N
It may be a terminal amino group or a δ-amino group of a lysine residue constituting (D).

The detection method of the present invention comprises the following steps. A step of bringing a protein probe into contact with the surface of the protein chip of the present invention in a liquid phase to form an electrochemically active complex between the protein and the protein probe immobilized on the chip; and immobilizing the complex This is a step of detecting the amount of label on the chip on which the complex is immobilized by electrochemical measurement via the substrate that has been performed. In the protein detection method of the present invention, “electrochemical measurement” means that the protein chip of the present invention is immersed in an electrolyte solution containing a protein probe, and then, using a counter electrode, in the electrolyte solution. After forming a pair of electrolytic systems with the electrode to which the composite is fixed, a potential is applied between the electrode to which the composite is fixed and the counter electrode, and the composite fixed electrode and the electrochemically active material are applied. To detect the amount of current flowing between the two. The method of immersing a protein chip in an electrolyte solution containing a protein probe also includes a partial immersion method of the protein chip, such as spotting an electrolyte solution containing a protein probe on the protein chip. After contact with the protein probe, washing is preferably performed to remove glycerol, salt, urea and the like.

The method for measuring the amount of current may be any method as long as the method can measure the amount of current flowing between the composite fixed electrode and the electrochemically active substance. It is preferable to use cyclic voltammography (CV), differential pulse voltammography (DPV), linear sweep voltammography, potentiostat, or the like. It is particularly preferable to immerse the counter electrode and the composite fixed electrode in an electrolyte solution to form a pair of electrolytic systems, and measure differential pulse voltammography.

Although not an electrochemical measurement method, the amount of current may be measured using a scanning electrochemical microscope (SECM). Scanning electrochemical microscopes generally use a probe /
Consists of a sample holder, bipotentiostat, high-resolution data processing, and three-dimensional micropositioner. Scans the xy plane of a small substrate using the equipped probe, and chemically changes near the substrate surface. (Redox reaction) is a general term for a system that gives a three-dimensional SECM image diagram. As the probe, an insulating glass, a noble metal or a carbon fiber microelectrode embedded in a polymer is used.

In the method using a scanning electrochemical microscope, a protein chip is immersed in an electrolyte solution containing a protein probe, and then a probe is guided to each of the partitioned regions on the substrate in the electrolyte solution. Thus, the amount of current can be measured. Details of the detection method using a scanning electrochemical microscope are described in Japanese Patent Application No. 11-12 / 1999.
No. 1563.

In the method for detecting a protein of the present invention, the interaction between the protein on the protein chip and the protein probe and the change in the interaction are determined by the specific protein formed between the protein chip-immobilized protein and the protein probe. Detection can also be performed while continuously changing the liquid phase factors that affect the binding state. Factors affecting the binding state include ionic strength, temperature, organic solvents and the like. The method of detecting while changing the above factors is considered to be particularly effective in a system with a weak interaction, unlike the above-described detection at a constant temperature and a constant ionic strength.

The protein chip of the present invention can be used in various ways, but any method can be used as long as it detects a protein that forms a specific bond with a protein on the protein chip. Hereinafter, specific examples will be described.

Specific Example 1: The protein chip of the present invention comprises:
It can be used to identify genes that cause diseases such as cancer and inflammation, or genes that function at each stage of development and cell cycle. For example, a protein chip immobilized with a protein produced by incorporating the ORF of budding yeast whose entire nucleotide sequence has been elucidated into an expression vector is produced, and yeast at a specific time such as a logarithmic growth phase or a stationary phase for the protein is prepared. By detecting the interaction of the protein extracted from the cells, the expression level of the yeast protein at that time can be analyzed. Alternatively, gene-disrupted strains or overexpressed strains can be used as yeast cells, and the expression level in those strains can be analyzed. Furthermore, if the separated and purified expressed protein is used as a sample, the type of the expressed protein can be specified.

Specific Example 2: The protein chip of the present invention can also be used as a biosensor. For example, after contacting a cell lysate containing a PDC subunit with a protein chip on which an antibody against pyruvate dehydrogenase complex (PDC) is immobilized, washing is performed under different conditions, whereby the strength of the interaction between the subunits is increased. Can be detected.

Specific Example 3: The protein chip of the present invention can also be used for protein purification. By sequentially changing the washing conditions, it is possible to purify several types of proteins.

Specific Example 4: The protein chip of the present invention can quantify a trace amount of protein in a sample. For example, a calibration curve can be prepared by contacting phosphotyrosine having different concentrations with a protein chip on which a phosphotyrosine peptide is immobilized.

The protein chip of the present invention is also useful for detecting antigen-antibody reactions and ligand-receptor binding, which are generally known to have strong interactions. Examples of the former include screening for monoclonal antibodies. An example of the latter is the interaction between a native human growth hormone receptor and a mutant hormone in which one amino acid at the binding site of the receptor is substituted with alanine, and then contacted with a protein chip on which the human growth hormone receptor is immobilized. Due to the difference in potency, it is also possible to develop a novel human growth hormone as a pharmaceutical lead compound.

[0054]

According to the protein chip and the method for detecting a protein of the present invention, the expression level of the protein at a specific expression stage is determined, the type of the expressed protein is determined by the cells, and the interaction with the protein fixed on the protein chip is achieved. The detection of a biofunctional substance having the above, the purification of a protein, and the like can be performed by measuring the amount of electrochemical labeling. Although a fluorescence method using a protein labeled with a fluorescent substance instead of the electrochemically active substance is also conceivable, a high-precision result cannot be expected in the fluorescence method due to the fluorescence of the protein itself. In this regard, the detection method of the present invention has high sensitivity and accuracy, and can easily process many samples simultaneously in a short time.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a typical protein chip of the present invention and a method for detecting a typical protein of the present invention.

FIG. 2 shows a representative method for preparing a sample protein labeled with an electrochemically active substance.

[Explanation of symbols]

REFERENCE SIGNS LIST 1 protein chip 2 substrate 3 electrode 4 terminal 5 protein 6 sample protein 7 electrochemically active substance 8 sample protein labeled with electrochemically active substance 9 protein chip after contacting sample protein labeled with electrochemically active substance 10 on electrode Complex formed by

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Shigeori Takenaka 4-23-21 Mai-no-sato, Koga-shi, Fukuoka F-term (reference) 4H045 AA40 BA60 CA40 DA01 EA50 FA80

Claims (7)

[Claims] 1. A protein chip for detecting a protein that forms a specific bond with a protein on the surface, wherein the protein is immobilized on the surface of the substrate. 2. The protein chip according to claim 1, wherein the substrate is an electrode provided with a terminal for outputting to the outside. 3. The method according to claim 2, wherein a plurality of electrodes are provided, and proteins having different amino acid sequence sites are immobilized on each of the plurality of electrode surfaces.
The protein chip according to 1. 4. The method of claim 1, wherein the electrodes are gold deposited at regular intervals on an electrically insulating substrate surface, and that the protein is bound to the gold surface at one end via a thiol bond. The protein chip according to claim 2 or 3, wherein the protein chip is a protein chip. 5. A sample protein labeled with an electrochemically active substance is brought into contact with the surface of the protein chip according to any one of claims 2 to 4 in a liquid phase, and is immobilized on the chip. Forming a complex of the present protein and the sample protein on the substrate; and detecting the amount of label on the complex-fixed chip by electrochemical measurement through the substrate to which the complex is fixed. A method for detecting a sample protein that forms a specific bond with a protein immobilized on a protein chip, comprising the steps of: 6. A protein chip labeled with an electrochemically active substance on the surface of the protein chip according to any one of claims 2 to 4 in a liquid phase, wherein the protein on the chip and the sample protein are used. Contacting while changing the factors of the liquid phase that affect the binding state formed between the sample and the substrate to form a complex of the protein fixed on the chip and the sample protein on the substrate; and Immobilizing on the protein chip, comprising a step of detecting a change in the amount of label on the complex-immobilized chip due to a change in the factor by electrochemical measurement through a substrate on which the complex is immobilized. A method for detecting a sample protein that forms a specific bond with the protein being used. 7. The method according to claim 1, wherein the temperature of the liquid phase or the ionic strength of the liquid phase is used as a factor of the liquid phase which affects the binding state formed between the protein on the protein chip and the sample protein. A method for detecting a sample protein according to claim 6.