JP2006170617A - Biochip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biochip capable of quantifying the reactivity in the material fixed on a chip, especially the phosphorization efficiency of peptide fixed to a substrate by a simple technique. <P>SOLUTION: The biochip is constituted by fixing a mixture, which is composed of at least two kinds of materials, to the surfaces of a plurality of kinds of substrates and characterized in that the mixture ratio of at least two kinds of the materials constituting the fixed mixture is specified. This biochip is especially useful for quantifying the phosphorization reactivity of the substrate fixed on the chip by analysis due to SPR. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel method for quantifying the reaction rate of a substance immobilized on a chip, for example, the rate of phosphorylation. More specifically, the phosphorylation rate in the immobilized substrate is quantified based on the signal by using an analysis technique based on surface plasmon resonance (hereinafter sometimes referred to as SPR).

  Analysis technology using a biochip is widely used as an analysis means / tool in proteomics. For example, analysis of networks related to post-translational modification of proteins, particularly protein phosphorylation, is important in understanding the life phenomena that are carried out in cells. For this purpose, there is a demand for means for comprehensively analyzing in detail including activity regulation and function regulation by phosphorylation modification. In recent years, research on intracellular signal transduction has progressed dramatically, not only how signals are transmitted to the nucleus from cell surface receptors activated by growth factors and cytokines, but also cell cycle, adhesion, The reality of various signaling pathways that control movement, polarity, morphogenesis, differentiation, life and death, etc. has become clear. These signal transduction pathways do not function independently, but function as a system by cross-talking with each other. The causes of various diseases including cancer have been explained as abnormalities in these signal transduction pathways.

  In the signal transduction pathway described above, it is known that various types of protein kinases play an important role in a complex manner. If we can comprehensively analyze the activity of these protein kinases and profile their dynamics in a single cell, we will not only be able to perform basic research in cell biology and pharmacy, but also in fields such as drug development and clinical application. It is expected to contribute greatly. However, at present, a sufficient level of technology capable of simultaneously profiling the dynamics of various protein kinases in a simple and efficient manner has not yet been established. In addition, regarding the quantification of protein phosphorylation by protein kinase, the current situation is that it has hardly been realized.

As a related technique already reported, for example, it has been reported that the activity of cSrc kinase, which is a kind of tyrosine kinase, was evaluated using a peptide array (see, for example, Non-Patent Document 1). In addition, with respect to p60 tyrosine kinase and protein kinase A (hereinafter also referred to as PKA), a phosphorylation detection system using a fluorescently labeled antibody using an array in which each substrate peptide is immobilized on a glass slide. (For example, refer to Non-Patent Documents 2 and 3), or examples for a detection system of kinase reaction on an array using a radioactive substance ([γ ³² P] ATP) (for example, Non-Patent Documents 4, 5 and 6). However, in any of the prior arts, a sufficient technique is not disclosed as a method for efficiently and efficiently profiling the dynamics of various protein kinases. In any of the above methods, it is necessary to use a fluorescent substance or a radioactive substance, and there are major problems in terms of the time required for analysis, the difficulty of handling, the necessity of special techniques and facilities, and the like.

  Further, in the profiling related to phosphorylation as described above, it is necessary to evaluate not only from a qualitative level but also from a quantitative viewpoint according to the purpose. However, it is very difficult to simultaneously quantify how much phosphorylation each individual substrate has when arrayed, and it has been hardly realized in the techniques reported so far. Absent.

Benjamin T. Houseman et al., Nature Biotechnology Volume 20, 270-274 (issued March 2002) Bioorganic & Medical Chemistry Letters Vol. 12, pp. 2085-2088 (issued in 2002) Bioorganic & Medical Chemistry Letters Vol. 12, pp. 2079-2083 (issued in 2002) Current Opinion in Biotechnology Vol. 13, 315-320 (issued in 2002) The Journal of Biological Chemistry Vol. 277, 27839-27849 (issued in 2002) Science 289, 1760-1763 (2000)

  The present invention is intended to provide a method for quantifying the reaction efficiency by performing a simple calculation process from the signal intensity obtained on the array. For example, it provides a useful technique for quantifying the phosphorylation efficiency of proteins and peptides immobilized on an array.

  As a result of intensive studies in view of the above circumstances, the present inventors have calculated a ratio between a signal change amount in an immobilized substrate to be quantified and a signal change amount in a substrate phosphorylated in advance as a standard substrate. Has been found to be extremely useful in quantifying the phosphorylation rate of the immobilized substrate, and the present invention has been achieved.

The present invention has the following configuration.
1. A biochip in which a mixture composed of two or more kinds of substances is immobilized on the surface of a plurality of kinds of substrates, and there are two or more kinds of mixing ratios of substances constituting the immobilized mixture. Biochip as a feature.
2. 1. The biochip according to claim 1, wherein only at least one of the substances constituting the mixture is immobilized on the same substrate surface as the mixture.
3. The biochip according to 1 or 2, wherein the mixture is a mixture of peptides.
4). 3. The biochip according to 3, wherein the mixture contains a peptide having a phosphorylated amino acid residue.
5. The biochip according to any one of 1 to 4, wherein the surface is gold.
6). The biochip according to any one of 1 to 5, wherein the substrate is a transparent substrate.
7). The biochip according to any one of 1 to 6, which is used for surface plasmon resonance (SPR) analysis.
8). The biochip according to any one of 1 to 7, which is used for obtaining a calibration curve for quantifying the reactivity related to any one of the substances constituting the immobilized mixture.
9. 8. The biochip according to 8, which is used for obtaining a calibration curve for quantifying the phosphorylation rate of a substrate.

  The biochip of the present invention does not require a special technique, and particularly when SPR is used, it is not necessary to use a label such as a fluorescent substance or a radioactive substance. This makes it possible to quantify the reactivity and the like of the substance immobilized on the substrate. In particular, it is useful for quantifying the phosphorylation rate of the substrate immobilized on the chip, and is a very useful technique for analyzing various protein kinase dynamics. The present invention is particularly applicable to comprehensive analysis of various types of protein kinase signals, introduction of genes whose functions are unknown, or profiling of intracellular protein kinase dynamics associated with drug administration. This is expected to expand into genomic drug discovery, such as functional analysis from new genes and approaches to drug discovery.

  The present invention provides a biochip in which a mixture of two or more kinds of substances is immobilized on the surface of a plurality of kinds of substrates, and the mixing ratio of substances constituting the immobilized mixture is two or more, preferably five or more. More preferably, there are 10 or more types. More preferably, only at least one substance constituting the mixture is immobilized on the same substrate surface as the mixture as a positive control or a negative control. The substance constituting the mixture is not particularly limited, and can be applied to general biomolecules such as nucleic acids, proteins, peptides, sugars, lipids, etc., but is particularly preferably applied to peptides. In particular, it is applied to a system of a mixture of a peptide (hereinafter also referred to as “phosphorylated peptide”) and a normal peptide (hereinafter also referred to as “mixed peptide”) that has been phosphorylated in advance. It is particularly preferable to use a mixed peptide consisting of a pair of peptides having the same amino acid sequence other than the presence or absence of modification with a phosphate group. In this case, regarding the efficiency of immobilization of both peptides on the substrate depending on the presence or absence of a phosphate group, it is considered that there is basically little difference, which is very convenient for use in quantification.

  The detection method relating to the action on the substrate in the present invention can be performed using a labeling compound such as a radioactive substance, a fluorescent substance, and a chemiluminescent substance as is well known. However, it is more preferable to apply an optical detection method such as surface plasmon resonance (SPR), elliptical polarization (hereinafter referred to as ellipsometry), sum frequency generation (hereinafter referred to as SFG) spectrometry or the like. Among these, SPR is particularly preferable because it is not necessary to obtain a phase difference, and a change in film thickness on the order of nm can be obtained only by obtaining a reflected light intensity. The SPR imaging method is more preferable in that a wide range of observation is possible and substance interaction observation in an array format is possible.

Among them, phosphorylation of a peptide can be performed by applying a test sample capable of having a protein kinase and a nucleoside triphosphate such as ATP (adenosine triphosphate) on the array of the present invention. Optimum phosphorylation reaction conditions vary depending on the type of protein kinase. For example, a test sample that can have protein kinase in a reaction buffer and a nucleoside triphosphate are added, and the temperature is about 10 to 40 ° C. Preferably, the peptide can be phosphorylated by reacting at a temperature of 30 to 40 ° C. for about 10 minutes to 6 hours, preferably about 30 to 1 hour. If necessary, the phosphorylation reaction solution may contain a substance that assists or promotes phosphorylation, such as cAMP, cGMP, Mg ²⁺ , Ca ²⁺ , and phospholipid.

  Examples of protein kinases targeted for kinetic profiling include enzymes that phosphorylate side chains of amino acids such as tyrosine, serine, threonine, and histidine of proteins. For example, cGMP-dependent protein kinase family, cAMP-dependent protein kinase ( PKA) family, myosin light chain kinase family, protein kinase C (PKC) family, protein kinase D (PKD) family, protein kinase B (PKB) family, protein kinase family belonging to MAP kinase (MAPK) cascade, Src tyrosine kinase family And the receptor tyrosine kinase family can be exemplified, but are not particularly limited.

  Here, when a peptide is used as a substrate for a protein kinase, it is ideal and preferable that one type of peptide is phosphorylated only by one type of protein kinase and not by other protein kinases. Peptide sequences that serve as substrates for protein kinases are known or can be appropriately selected based on known sequences. Alternatively, a substrate peptide having an amino acid sequence that is particularly susceptible to phosphorylation may be designed and used by making full use of bioinformatics techniques.

  Ellipsometry can measure the film thickness and refractive index from the change in the deflection state caused by the interference between the light reflected on the surface of the thin film and the light reflected on the back surface of the thin film. That is, it is a means for evaluating the ratio of the absolute value of the reflectance and the ratio of the phase change with respect to p-polarized light and s-polarized light. Among them, the spectroscopic ellipsometry for measuring the ellipsometry while changing the wavelength is preferable because the change in the film thickness of the surface can be detected very sensitively.

  SFG is a kind of second-order nonlinear optical effect, and is a phenomenon in which two types of incident light (frequency ω1 and frequency ω2) having different frequencies are mixed in a medium to generate light of ω1 + ω2 or ω1−ω2. In particular, when visible light is used as ω1 and wavelength-variable infrared light is used as ω2, vibrational spectroscopy similar to infrared spectroscopy can be performed. Since this method has good surface selectivity, vibration spectroscopy of molecules at the monomolecular film level is possible, and it is useful as a very sensitive surface analysis method.

  As mentioned above, in one particularly preferred embodiment, the present invention uses SPR to interact with phosphate groups and any detection probes that exhibit specific effects on phosphate groups in mixed peptides of various phosphorylation ratios. It is particularly preferable to analyze In SPR, an evanescent wave is generated by a polarized light beam applied to a metal and oozes on the surface, excites surface plasmons which are surface waves, consumes light energy, and reduces reflected light intensity. The resonance angle at which the reflected light intensity is significantly reduced varies depending on the thickness of the layer formed on the metal surface. Therefore, the substance or group of substances to be examined is fixed on the surface of the metal, and the interaction with the substance or group of substances in the sample is detected by changing the resonance angle or the reflected light intensity at a certain angle. Is possible. Therefore, the SPR is useful as a quantitative method that does not require labeling with a fluorescent substance, a radioactive substance, etc., and that enables real-time evaluation.

  The type of the light source is not particularly limited, but it is preferable to use light including near infrared light that makes the change in the SPR resonance angle particularly sensitive. Specifically, a white light source capable of irradiating light over a wide range such as a metal halide lamp, a mercury lamp, a xenon lamp, a halogen lamp, a fluorescent lamp, and an incandescent lamp can be used. A halogen lamp that is sufficiently high, has a simple light power supply and is inexpensive is particularly preferable.

  In the case of the chip for SPR used in the present invention, the chip preferably comprises a metal substrate having a metal thin film formed on a transparent substrate, directly or indirectly on the metal thin film, chemically or physically, a substance or A slide on which a collection of substances is immobilized is used. The material of the substrate is not particularly limited, but it is preferable to use a transparent material. Specific examples include glass or plastics such as polyethylene terephthalate (PET), polycarbonate, and acrylic. Of these, glass is particularly preferable.

The thickness of the substrate is preferably about 0.1 to 20 mm, and more preferably about 1 to 2 mm. In order to achieve the object of evaluating the reflection image from the metal thin film, the SPR resonance angle is as small as possible, and there is no fear that the image to be photographed will be fuzzy, and the analysis is easy. Therefore, the refractive index n _D of the transparent substrate or the transparent substrate and the prism in contact with the transparent substrate is preferably 1.5 or more.

  Examples of the metal constituting the metal thin film include gold, silver, copper, aluminum, platinum, and the like. These may be used alone or in combination, but it is particularly preferable to use gold. The method for forming the metal thin film is not particularly limited, and examples of the known method include a vapor deposition method, a sputtering method, and an ion coating method. Of these, the method by vapor deposition is preferred. The thickness of the metal thin film is preferably about 10 to 3000 mm, more preferably about 100 to 600 mm.

  One particularly preferred embodiment of the present invention is first of all the phosphorylated and non-phosphorylated peptides (in this case having the same amino acid sequence except for the presence or absence of modification with a phosphate group) as described above. )), The signal intensity when the detection probe for the phosphate group is allowed to act on the array to which the mixed peptide is immobilized is preferably measured by SPR, and the calibration between the phosphorylation rate and the signal intensity is performed. Create a calibration curve. In this case, calculate the ratio between the signal intensity of the mixed peptide having the compounding ratio of each phosphorylated peptide and the signal intensity of the 100% phosphorylated substrate peptide, and plot this value against the phosphorylation rate. A method of obtaining a calibration curve by the above is preferable. By adopting such a method, it is possible to obtain a calibration curve relating to the phosphorylation rate with excellent reproducibility.

  Thereafter, the phosphorylation rate of the substrate on the chip by the on-chip protein kinase reaction in the actual measurement array can be quantified by using the calibration curve. Using an array in which at least the phosphorylated substrate peptide and the non-phosphorylated substrate peptide constituting the mixed peptide are immobilized, a solution containing a protein kinase such as a cell disruption solution is allowed to act on the array, and a detection probe is further used. In particular, the state of their interaction is detected by SPR. The peptide used here is a peptide having a property of undergoing a phosphorylation reaction by a protein kinase. By calculating the ratio of the signal increase in the non-phosphorylated substrate peptide thus obtained and the signal increase in the phosphorylated substrate peptide, and applying it to the above calibration curve, the phosphorylation rate can be obtained. .

  When a peptide is used, its length is not particularly limited, but generally a peptide having 100 amino acid residues or less is used. Preferably, those consisting of 5 to 60 amino acid residues, more preferably about 10 to 25 amino acid residues are used. The peptide may be a peptide obtained by chemical synthesis based on a known technique, or may be a peptide produced by a genetic engineering technique. In order to facilitate the desorption to the substrate, biotin, a cysteine residue having a thiol group added to one end of the peptide, oligohistidine (His-tag), glutathione-S-transferase It is also useful to use a tag to which a commonly used tag such as (GST) is added.

  The method for immobilizing the peptide on the metal thin film is not particularly limited, but it is more preferable to immobilize the substrate peptide by introducing a functional group that can be easily immobilized on the surface of the metal thin film in advance. preferable. Examples of the functional group include an amino group, a mercapto group, a carboxyl group, and an aldehyde group. In order to introduce these functional groups onto the surface of the metal thin film, it is preferable to use a generally used derivative of alkanethiol.

  At that time, J.H. M.M. See by Brockman et al. Am. Chem. Soc. Based on the method reported in Volume 121, pages 8044-8051 (1999), a method of immobilizing through an alkanethiol layer and modifying the background with PEG (polyethylene glycol) may be used. Good. In addition, in order to further suppress non-specific effects, it is also possible to immobilize a peptide after binding a derivative in which a functional group as described above is introduced to the end of PEG to alkanethiol, which also exerts a spacer effect. It is useful in.

  Specifically, for example, carboxymethyl dextran or a water-soluble polymer such as PEG having a terminal modified with a carboxyl group is bonded to a metal thin film to introduce a carboxyl group on the surface, and EDC (1-ethyl-3, Examples of NHS (N-hydroxysuccinimide) esters using water-soluble carbodiimides such as 4-dimethylaminopropylcarbodiimide) include reacting amino groups of peptides or proteins with activated carboxyl groups. Alternatively, the surface may be modified with maleimide and then immobilized via an amino acid residue containing a thiol group such as cysteine. In this case, the cysteine residue is preferably added to one end of the peptide. In order to reduce the non-specific influence, the latter immobilization method via a thiol group is more preferable, but it is not particularly limited.

  A method of immobilizing a peptide to which a tag such as His-tag or GST described above is added is also very simple and useful. In this case, it is preferable to introduce NTA (nitrilotriacetic acid) and glutathione on the metal thin film after introducing an amino group or a carboxyl group to the metal surface via alkanethiol as described above. In the case of His-tag, the substrate is immobilized after the NTA-introduced array is treated with nickel chloride.

  In the present invention, a detection probe having a binding property to a phosphorylated substrate is used in order to monitor the phosphorylation of the substrate on the array specifically and sensitively. As the detection probe, an antibody that recognizes a phosphorylated amino acid or a phosphorylated peptide can be used. More preferably, a chelate compound is used. The chelate compound generally refers to a complex formed by coordination of a polydentate ligand or a chelating reagent to a metal ion, and particularly a compound having a property of selectively and reversibly binding to phosphoric acid, More preferably, a polyamine zinc complex is used. More preferably, a dinuclear zinc complex having a polyamine compound as a ligand is used. Furthermore, it is more preferable to use a hexaamine dinuclear zinc (II) complex having a dinuclear zinc (II) complex in the basic structure.

  A typical example of such a compound is 1,3-bis [bis (2-pyridylmethyl) amino] -2-hydroxypropanolate (IUPAC name: 1,3-bis [ bis (2-pyrylmethyl) amino] -2-propanolatodiincinc (II) complex) as a ligand is a dinuclear zinc complex (provided that the hydroxyl group of the propanol skeleton is an alcoholate with two zinc divalent ions) However, the present invention is not particularly limited to this compound.

The complex as described above can be synthesized using a general chemical synthesis technique, but can also be synthesized using a commercially available compound as a raw material. For example, the compound (Zn ₂ L) represented by the above formula (I) is obtained by the following method using commercially available 1,3-bis [bis (2-pyridylmethyl) amino] -2-hydroxypropane and zinc acetate as raw materials. Can be synthesized. To a ethanol solution (100 ml) of 4.4 mmol of 1,3-bis [bis (2-pyridylmethyl) amino] -2-hydroxypropane was added 10M aqueous sodium hydroxide solution (0.44 ml), and then zinc acetate dihydrate. (9.7 mmol) is added. A brown oil can be obtained by distilling off the solvent under reduced pressure. To this residue was added 10 ml of water and dissolved, then 1M aqueous sodium perchlorate solution (3 equivalents) was added dropwise while heating to 70 ° C., and the precipitated colorless crystals were collected by filtration and dried by heating. (I) two perchlorate acetate ion adduct _{(Zn 2 L-CH 3 COO} - · 2ClO 4 - · H 2 O) represented by the structural formula can be obtained in high yield. This crystal contains one molecule of crystal water.

  In the present invention, it is preferable to use a polyamine zinc complex modified as described above with biotin. It is preferably modified with biotin via a linear linker structure. Specifically, the structure shown in the formula (II) is exemplified, but not particularly limited.

  The solution concentration of the biotin-modified polyamine zinc complex acting in the present invention is not particularly limited, but is usually in the range of 1 μM to 10 M, preferably 10 μM to 1 M, more preferably 10 μM to 10 mM. The mode of action on the array is not particularly limited, but the amount of liquid necessary for spreading the biotin-modified polyamine zinc complex solution over the entire array surface may be dropped, or the array may be immersed in the solution. Or you may make it act by making a solution contact on an array surface, sending a solution using a pump. The working temperature may be room temperature or may be incubated at about 20 to 40 ° C. The action time is preferably about 10 minutes to 2 hours, more preferably about 30 minutes to 1 hour.

  When a biotin-modified polyamine zinc complex is allowed to act, it may be possible to detect phosphorylation by itself, but by using a modified biotin, in particular, by further avidin or streptavidin action, The effect that the detection sensitivity increases can be expected. It is more preferable to make streptavidin act. The concentration of avidin or streptavidin to be actuated is not particularly limited, but is usually in the range of 1 μM to 10 M, preferably 10 μM to 1 M, more preferably 10 μM to 10 mM. The mode of action on the array is not particularly limited, and is the same as in the case of the biotin-modified polyamine zinc complex.

  In a further preferred embodiment, after avidin or streptavidin is allowed to act, an antibody that recognizes avidin or streptavidin is allowed to act further, so that the detection sensitivity can be further enhanced. The concentration at which the antibody is allowed to act is not particularly limited, but is preferably about 0.01 to 10 μg / ml, more preferably about 0.1 to μg / ml. As the antibody, either a monoclonal antibody or a polyclonal antibody can be applied, but the monoclonal antibody is preferred in terms of specificity. The mode of action on the array is also not particularly limited, and is the same as in the case of biotin-modified polyamine zinc complex, avidin or streptavidin.

  Further, a biotin-modified polyamine zinc complex, avidin or streptavidin may be allowed to act sequentially, but a biotin-modified polyamine zinc complex and a complex of avidin or streptavidin previously formed may be allowed to act directly. In this case, as described above, an antibody that recognizes avidin or streptavidin may be allowed to further act. In forming the complex, it is preferable that the biotin-modified polyamine zinc complex and avidin or streptavidin are reacted in a molar ratio of 1: 1 to 4: 1. The reaction product is preferably purified to remove unreacted product, but the reaction product can be applied as it is.

  The application of such a chelating compound is advantageous in that it can be synthesized at a very low cost by the method described above. In addition, it is stable and easy to use in that it can be stored at room temperature, which is advantageous in terms of distribution. In addition, the method of detecting using an antibody in particular, because it acts regardless of the type of amino acid residue to be phosphorylated, and the reactivity is less likely to depend on the amino acid sequence in the vicinity of the phosphorylated amino acid. It has a great advantage in comparison.

  Examples of the protein kinase include various tyrosine kinases and serine / threonine kinases. The types of these protein kinases are not particularly limited, and can basically be applied to all types of protein kinases.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated more concretely, this invention is not specifically limited to an Example.

[Example 1]
(Peptide immobilization)
4 armPEG (SUNBRIGHT PTE-100SH manufactured by NOF Corporation) whose terminal functional group is a thiol group was dissolved in a mixed solution of 7 ml of ethanol: water = 6: 1 at a concentration of 1 mM. The molecular weight of 4armPEG is 10,000, which is a molecule in which four PEG chains having approximately the same length from the center are present, and is very hydrophilic. In addition, all four ends of PEG are thiol groups, and particularly exhibit metal binding properties to gold. A gold-deposited slide in which 3 nm of chromium was vapor-deposited on an SF15 glass slide of 18 mm square and 2 mm thickness and gold was vapor-deposited at 45 nm was immersed in the 4 armPEG thiol solution for 3 hours to bond 4 armPEG thiol to the entire gold substrate.

  A photomask was placed on the slide and irradiated with a 500 W ultra-high pressure mercury lamp (manufactured by USHIO) for 2 hours to remove 4 armPEG thiol in the UV irradiation part. The photomask has 96 square holes of 500 μm square (consisting of 8 × 12 patterns), and the pitch between the hole centers is designed to be 1 mm. The portion of the photomask with a hole is transparent to UV light and is irradiated onto the slide for patterning. 4 armPEG remains in the part that was not irradiated and functions as a reference part as a background part of the chip.

  It was immersed in a 1 mM ethanol solution of 8-Amino-1-Octanethiol, Hydrochloride (8-AOT, manufactured by Dojindo Laboratories) for 1 hour to form a self-organized surface of 8-AOT on the UV irradiation part. Sulfoccinimidyl 4- [N-maleidomethyl] cyclohexane-1-carboxylate (Pierce; hereinafter referred to as SSMCC), which is a heterobifunctional cross-linker having a succinimide (NHS) group and a maleimide (MAL) group at the terminal, is phosphoric acid. It was dissolved at 0.4 mg / ml in a buffer solution (20 mM phosphate, 150 mM NaCl; pH 7.2), and reacted with 8-AOT on the gold surface for 15 minutes. Since the amino group of 8-AOT and the NHS group of SSMCC reacted and the MAL group remained unreacted, a maleimide group could be introduced on the surface.

With respect to the surface of the protein kinase A (hereinafter referred to as PKA) (J. Biol. Chem. 252, pp. 4888-4894; 1977), phosphorylated substrates and non-phosphorylated on the surface obtained as described above. A mixed peptide consisting of a substrate was immobilized. The mixing ratio of the substrates was prepared in a 10-fold series. Both substrates were dissolved in phosphate buffer (20 mM phosphate, 150 mM NaCl; pH 7.2) at 1 mg / ml and mixed at the desired ratio. The amino acid sequence of the substrate peptide and the arrangement for immobilization thereof are as shown in FIG. 1A. -Indicates a blank spot where the peptide is not immobilized. The prepared substrate solution was spotted 10 nl at a time using a MultiSPRinter ^™ spotter (manufactured by Toyobo). Then, the immobilization reaction was carried out by allowing to stand at room temperature for 16 hours in a wet environment. The maleimide group formed on the surface of the chip reacts with the thiol group possessed by the cysteine residue at the terminal of the substrate peptide, so that the substrate peptide can be covalently immobilized on the surface.

(Blocking of unreacted maleimide groups)
After the surface on which the substrate peptide is immobilized is washed with a phosphate buffer, in order to block unreacted maleimide groups, PEG thiol (SUNBRIGHT MESH-50H manufactured by NOF Corporation) is phosphate buffer so that the concentration becomes 1 mM. Dissolved in (20 mM phosphoric acid, 150 mM NaCl; pH 7.2), 300 μl was poured onto the chip and allowed to react at room temperature for 30 minutes. The molecular weight of PEG thiol used here is 5,000.

(Detection of phosphate groups on the array by SPR)
The array that had been blocked as described above was washed with PBS and water to allow the biotin-modified polyamine zinc complex to act. As the biotin-modified polyamine zinc complex, Phos-tag ^™ BTL-104 (purchased from Nard Laboratory Co., Ltd.) represented by the following formula (II) was used. Phos-tag ^™ BTL-104 had a concentration of 25 μg / ml, and the lysis solution contained 10 mM HEPES-NaOH buffer containing 0.005% Tween 20, 10% (v / v) ethanol, 0.2 M sodium nitrate, 1 mM zinc nitrate. (PH 7.4) was used. The action was carried out for 1 hour at room temperature.

The above-treated array was washed with PBS and water and set in an SPR apparatus (MultiSPRinter ^™ manufactured by Toyobo Co., Ltd.) for analysis. As the running buffer, the same 0.005% Tween 20, 10% (v / v) ethanol, 10 mM HEPES-NaOH buffer (pH 7.4) containing 0.2 M sodium nitrate and 1 mM zinc nitrate was used. Streptavidin (manufactured by Molecular Probes) was dissolved in the same buffer to prepare a 10 μg / ml concentration solution, which was allowed to act on the array surface while being fed using a planner pump (from Model-021 manufactured by Flume). The temperature was set at 30 ° C. When the signal increase in the sensorgram due to the binding of streptavidin was stabilized, the running buffer was fed and washed.

Based on the analysis results described above, SPR imaging was performed. In this SPR analysis, an image is captured every 5 seconds by a CCD camera. From an image captured at the time after the streptavidin (hereinafter also referred to as SA) reaction, an image at the time before the reaction is obtained. Is a result of performing a subtraction process using image calculation processing software Scion Image (manufactured by Scion Corp.). The results are shown in FIG. 1A. A darker spot is confirmed as the mixing ratio of the phosphorylated substrate is higher, and it is confirmed that Phos-tag ^™ BTL-104 is specifically bound in proportion to the ratio of the phosphorylated substrate.

(Create calibration curve)
The ratio of the signal change amount in each mixed substrate and the signal change amount in the phosphorylated substrate obtained in the above SPR analysis was calculated. A graph was prepared by plotting this value against the mixing ratio of the phosphorylated substrate. The result is shown in FIG. 1B. A calibration curve showing a very high correlation with R ² = 0.9686 could be obtained.

[Example 2]
The process up to the step of introducing maleimide groups onto the surface was carried out in the same manner as in Example 1, and the array was washed with PBS and water. Thereafter, six arrays were prepared with the phosphorylated and non-phosphorylated substrates immobilized on the PKA substrate described above. The blocking of unreacted maleimide groups in the prepared array was also performed in the same manner as in Example 1. The array that had been blocked was washed with PBS and water, and phosphorylated with PKA. 400 μl of PKA solution was dropped on the array, and the reaction was carried out at 30 ° C. for 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours. The composition of the PKA solution was 1 μl of PKA catalyst subunit (manufactured by Promega), 375 μl of 50 mM Tris-HCl buffer (pH 7.4), 20 μl of 1M magnesium chloride solution, 4 μl of 10 mM ATP (manufactured by Amersham Bioscience).

The array subjected to the PKA reaction at each time was washed with PBS and water, and Phos-tag ^™ BTL-104 was allowed to act under the same conditions as in Example 1. The array was washed with PBS and water, and set in an SPR device (MultiSPRinter ^™ manufactured by Toyobo Co., Ltd.) for analysis. The same running buffer as in Example 1 was used. In the same manner as in Example 1, the streptavidin solution was allowed to act at a concentration of 10 μg / ml. As a result, the ratio of the signal change amount in the PKA substrate and the signal change amount in the phosphorylated PKA substrate in the sensorgram based on each PKA reaction time obtained was calculated. A graph was prepared by plotting this value against the PKA reaction time. As a result, a time course curve related to phosphorylation by PKA as shown in FIG. 2 could be obtained.

  As shown in FIG. 2, it was confirmed that the phosphorylation reaction of the immobilized PKA substrate almost stabilized in about 2 hours. From the calibration curve in FIG. 1B, the phosphorylation rate received by this substrate was estimated to be about 70%. The result of examining SPR imaging while changing the PKA reaction time is shown in the right column of FIG. The spot pattern of the array is as shown in the lower part of FIG. When the signal in the PKA substrate is compared with time, it is confirmed that the signal is specifically strong.

A similar study was performed by autoradiography using the same array. 400 μl of PKA solution was dropped on the array and reacted at 30 ° C. The composition of the PKA solution was 1 μl of PKA catalyst subunit (manufactured by Promega), 378 μl of 50 mM Tris-HCl buffer (pH 7.4), 20 μl of 1M magnesium chloride solution, and 1 μl of γ ³³ P-ATP (manufactured by Amersham Biosciences). Thereafter, the array was washed three times with PBS and water, and the surface of the array was dried, followed by an exposure treatment for 30 minutes to perform autoradiography measurement. Reading was performed using BAS1800II (manufactured by Fuji Photo Film) using a dedicated IP sheet. The results are shown in the left column of FIG. 3 and show that the RI uptake in the PKA substrate increases with time. From this result, it was verified that the signal obtained in the SPR analysis was due to the phosphorylation reaction.

[Example 3]
In the same manner as in Example 1, with respect to the cSrc substrate (Bioorg. Med. Chem. Lett. 12, pp. 2085-2088; 2002), a mixed peptide comprising a phosphorylated substrate peptide and a non-phosphorylated substrate peptide was mixed at a mixing ratio. Were prepared in 10-fold series and arrayed for calibration. In the same manner as in Example 1, calibration between the phosphorylation rate and the signal ratio was performed. The results are shown in FIG. A curve having a behavior pattern different from that of the PKA shown in FIG. 1B is obtained. For this reason, the range of the phosphorylation rate with quantitative properties is considerably narrow.

  Next, in the same manner as in Example 2, cSrc reaction (30 ° C.) was performed on chip, and the time course was examined. The composition of the cSrc reaction solution (300 μl dropped) was 6 μl of cSrc kinase (manufactured by Upstate; 5 U / μl), 276 μl of 50 mM MES buffer (pH 6.8), 15 μl of 1M magnesium chloride solution, and 3 μl of 10 mM ATP. The result of obtaining the time course curve is shown in FIG. The progress of phosphorylation in this case is not very sharp behavior, but it shows that phosphorylation proceeds with time. The reaction is almost stationary after about 3 hours of reaction. The phosphorylation rate at this time was a very low value of about 10% when applied to the calibration curve of FIG.

  The change with time due to the cSrc reaction was examined by SPR imaging and autoradiography as in Example 2. An array having the same pattern as that shown in FIG. 3 was used. The results are shown in FIG. Also in this case, it can be confirmed in any detection system that the phosphorylation is specifically performed on the cSrc substrate.

The biochip of the present invention does not require a special technique, and particularly when SPR is used, it is not necessary to use a label such as a fluorescent substance or a radioactive substance. This makes it possible to quantify the reactivity and the like of the substance immobilized on the substrate. In particular, it is useful for quantifying the phosphorylation rate of the substrate immobilized on the chip, and is a very useful technique for analyzing various protein kinase dynamics. The present invention is particularly applicable to comprehensive analysis of various types of protein kinase signals, introduction of genes whose functions are unknown, or profiling of intracellular protein kinase dynamics associated with drug administration. As a result, it is expected to expand into genome drug discovery such as functional analysis from new genes and approaches to new drug discovery, and it will greatly contribute to the industry.

It is a figure which shows the calibration curve regarding the result of SPR imaging in Example 1, and the obtained phosphorylated PKA substrate. In Example 2, it is a figure which shows the time course of PKA reaction obtained by SPR analysis. In Example 2, it is a figure which shows the result of having verified the progress of the phosphorylation of the substrate by PKA with time by SPR imaging and autoradiography. It is a figure which shows the calibration curve regarding the phosphorylated cSrc substrate obtained in Example 3. In Example 3, it is a figure which shows the time course of cSrc reaction obtained by SPR analysis. In Example 3, it is a figure which shows the result of having verified the progress of the phosphorylation of the substrate by cSrc with time by SPR imaging and autoradiography.

Claims (9)

  A biochip in which a mixture of two or more kinds of substances is immobilized on the surface of a plurality of kinds of substrates, and there are two or more kinds of mixing ratios of substances constituting the immobilized mixture. Biochip.   The biochip according to claim 1, wherein only at least one of the substances constituting the mixture is immobilized on the surface of the same substrate as the mixture.   The biochip according to claim 1 or 2, wherein the mixture is a mixture of peptides.   The biochip according to claim 3, wherein the mixture contains a peptide having a phosphorylated amino acid residue.   The biochip according to any one of claims 1 to 4, wherein the surface is gold.   The biochip according to any one of claims 1 to 5, wherein the substrate is a transparent substrate.   The biochip according to claim 1, wherein the biochip is used for surface plasmon resonance (SPR) analysis.   The biochip according to any one of claims 1 to 7, wherein the biochip is used for obtaining a calibration curve for quantifying the reactivity relating to any one substance constituting the immobilized mixture.   The biochip according to claim 8, which is used for obtaining a calibration curve for quantifying the phosphorylation rate of a substrate.