JP3839359B2 - Chemically modified solid support and use thereof - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a solid support useful in the field of molecular biology and biochemistry, capable of immobilizing peptides, or useful as a microlaboratory.
[0002]
[Prior art]
With the completion of the Human Genome Project, biological and medical research has taken a new step from genetic decoding to protein analysis, or proteomics. A gene is simply a program code for producing a protein, and almost all biological activities are performed between molecules of a protein having a very complicated structure generated based on the code. It is known that if certain proteins do not function properly, they can interfere with health. Therefore, elucidating the functions of individual proteins can be said to be an indispensable step for further progress in medicine.
[0003]
Conventionally, two-dimensional electrophoresis (2-D PAGE), which separates and analyzes a mixture of extracted proteins based on differences in molecular weight or isoelectric point, is used for analysis of protein properties, expression state, structure, activity and the like. Have been used. However, the two-dimensional electrophoresis method is not suitable for high-throughput analysis, and has problems in terms of detection sensitivity and sample solubilization.
[0004]
On the other hand, many DNA chips have been reported to date. These DNA chips were useful for confirming changes in gene expression in a certain phenotype or physiological state and for creating a database of expression patterns. However, since the expression levels and patterns of genes and proteins do not always accurately correlate, DNA chips cannot be used for quantification of protein expression levels. In addition, since the function of a protein is changed by being subjected to various modifications such as phosphorylation, sugar chain addition, and cleavage after translation, information on the post-translational modification cannot be obtained from DNA analysis.
[0005]
Thus, a protein chip was developed using the DNA array technology as a tool for protein analysis. The principle of a protein chip is the same as that of a DNA chip. Proteins are immobilized on a slide glass or membrane at a high density, and proteins or nucleic acids that interact with them are detected. However, it is not easy to immobilize on a substrate a protein with a very unstable structure that is skillfully entangled with a strong DNA strand, and in many cases, the reactivity of the protein is Since it changes depending on the change in the three-dimensional folding structure, a slight change in the environment surrounding the protein may denature the protein, making it difficult to analyze the target protein while maintaining its function.
[0006]
In addition, conventionally developed protein chips are those in which a polymer such as polylysine is applied to the surface of a slide glass or silicon substrate, and then proteins are immobilized. The polymer such as polylysine is immobilized on the surface of a slide glass or silicon substrate. In the method of immobilizing proteins by applying the protein, the protein immobilization state is unstable, causing problems such as peeling during the washing process, and it is impossible to store the immobilized proteins for a long period of time. It was. In addition, there are problems such as non-specific adsorption of biological substances, protein function inactivation, and detection system UV opacity.
[0007]
[Problems to be solved by the invention]
An object of the present invention is a solid support for stably immobilizing peptides for efficient protein analysis and protein storage, which does not deactivate the function of the protein and is transparent to the detection system UV Furthermore, it is to provide a solid support capable of desorbing the bound peptide.
[0008]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors have found that the above problems can be solved by a solid support in which a substrate having a surface composed of a carbon layer is chemically modified and a metal chelate is further formed. The present invention has been completed.
[0009]
That is, the present invention includes the following inventions.
(1) A solid support in which a part or all of the substrate surface is composed of a carbon layer, and a metal chelate is formed on the layer.
(2) The solid support according to (1), wherein the carbon layer is a diamond-like carbon layer or a diamond layer.
(3) The solid support according to (1) or (2), wherein the carbon layer has a thickness of carbon monomolecule to 100 μm.
(4) A method for analyzing a peptide by immobilizing the peptide on the surface of the solid support according to any one of (1) to (3).
(5) A method for preserving the peptide by immobilizing the peptide on the surface of the solid support according to any one of (1) to (3).
(6) The solid support according to any one of (1) to (3), wherein a peptide is immobilized on a metal chelate formed on the surface thereof.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
The solid support of the present invention is characterized in that a carbon layer is formed on the surface of a substrate and the carbon layer is subjected to specific chemical modification. The chemical modification in the present invention means that a chelate ligand that forms a metal chelate is introduced onto the carbon layer via a spacer to form a metal chelate. By such chemical modification, the peptide can be immobilized on the surface of the solid support.
[0011]
In the present invention, the substrate means a base material for forming a carbon layer, and such a base material is not particularly limited. For example, a metal such as gold, silver, copper, aluminum, tungsten, molybdenum; And glass; silicon; fiber; wood; paper; plastic such as polycarbonate and fluororesin; and mixtures of plastic and the above metals, ceramics, diamond, and the like. In the present invention, glass or silicon is preferably used from the viewpoints of background reduction in fluorescence measurement, measurement by transmitted light, cost, and productivity. The shape and size of the substrate are not particularly limited, but when a flat plate is used, it is usually about 0.1 to 100 mm in width, 0.1 to 100 mm in length, and about 0.01 to 10 mm in thickness.
[0012]
The structure of the spacer is not particularly limited as long as it can introduce a metal chelate ligand without inhibiting the binding of the peptide, but a hydrocarbon chain having 1 to 12, particularly 1 to 6 carbon atoms is preferable. Such hydrocarbon chains may be saturated or unsaturated and may be substituted or unsubstituted. Further, it may be linear, branched or cyclic. For example, an alkylene group having 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms, such as methylene, ethylidene, dimethylmethylene, ethylene, trimethylene, tetramethylene, 1,2-cyclohexylene, 1,4-cyclohexylene; Arylalkylene groups such as methyl) (phenyl) methylene, diphenylmethylene;₂-CH (OH) -CH₂-O-CH₂-CH (OH) -CH₂-An arylene group such as phenylene; and a divalent group having a benzene derivative or cyclohexane derivative structure.
[0013]
In the present invention, the chelate ligand refers to a multidentate ligand that can coordinate with a metal ion to form a chelate compound. Examples of the chelating ligand structure include iminodicarboxylic acid, nitrilotriacetic acid, terpyridine, bipyridine, triethylenetetraamine, biethylenetriamine, ethylenediamine, trimethylenediamine, ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid analog, diethylenetriaminepentaacetic acid , Macrocyclic polyamine dioxo derivatives, sodium pyrophosphate, sodium tripolyphosphate, sodium polyphosphate, tartaric acid, citric acid, gluconic acid, dimethylglyoxime, 2,4-pentanedione, alanine, bipyridine, diaminocyclopentane, diethylenetriamine, dimethyl Glyoxime, ethylenetriaminepentaacetic acid, glycine, hexaethylenehexamine, 2-methyl-1,2-propanediamine, isoquinoline, Acid, O-phenylenediamine, oxalic acid, N, N, N ′, N′-tetrakis (2-aminoethyl) ethylenediamine, 1,10-phenanthroline, γ-picoline-1,2-propanediamine, pyridine, quinoline , Histidine, 3,4-diacetyl-2,5-hexanedione, 2,2 ′, 2 ″ -terpyridine, tetraethylenepentamine, 1,3-propanediamine, tris (2-aminoethyl) amine, triethylene What has structures, such as a tetraamine and urea, is mentioned. In the present invention, those having an iminodicarboxylic acid structure, an iminodicarboxylic acid-like structure, and a triethylenetetraamine structure are preferred.
[0014]
The process for introducing the chelate ligand is not particularly limited. For example, in the state where the substrate surface is chlorinated and aminated, the formed primary amino group is reacted with a halocarboxylic acid to form a chelate coordination. A child can be formed. Amination can be carried out, for example, by irradiation with ultraviolet light in ammonia gas or polyamine gas. Or it can also carry out by making polyamines react in a suitable solvent using a catalyst suitably. Polyamines that can be used in this case are not particularly limited. For example, methylenediamine, ethylenediamine, trimethylenediamine, tetramethylenediamine, pentamethylenediamine, hexamethylenediamine, phenylenediamine, benzidine, diaminostilbene, tolidine, diaminobenzidine. Polyallylamine and the like, and ethylenediamine, trimethylenediamine, tetramethylenediamine, and polyallylamine are preferably used. Examples of the solvent include N-methyl-2-pyrrolidone, N, N-dimethylformamide, N, N-dimethylacetamide, dimethyl sulfoxide, ethyl acetate, tetrahydrofuran, methanol, ethanol, dimethoxyethane, dioxane, chloroform, methylene chloride and the like. As the catalyst or reaction reagent, triethylamine, diazabicycloundecene, pyridine, pyrrolidine, piperidine, morpholine, and the like can be used.
[0015]
Subsequently, a chelate ligand having an iminodiacetic acid-like structure can be formed by using a halocarboxylic acid. Although it does not specifically limit as halocarboxylic acid, For example, chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid etc. are mentioned, For example, chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid Is preferably used.
Alternatively, chelate ligands can be formed by using anhydrous ethylenediaminetetraacetic acid.
[0016]
Alternatively, the substrate on which the carbon layer is formed can be chlorinated and then aminated, and then a carboxyl group can be further introduced to introduce a chelate ligand into the terminal carboxyl group. In this case, chlorination and amination can be carried out by the same method as described above. The carboxyl group can be introduced, for example, by reacting an amino group with an appropriate dicarboxylic acid. A chelating ligand can be introduced into the formed carboxyl group by adding, for example, iminodicarboxylic acid such as iminodiacetic acid, nitrilotriacetic acid, terpyridine, bipyridine, triethylenetetraamine, biethylenetriamine, and salts thereof. . In the present invention, it is preferable to use iminodicarboxylic acid. When the chelate ligand is introduced, it can be carried out in an appropriate solvent using a catalyst or a reaction reagent as appropriate.
[0017]
And a metal chelate can be formed by adding a metal ion with respect to the chelate ligand formed as mentioned above. Examples of metal ions that form chelates include iron ions, nickel ions, cobalt ions, calcium ions, silver ions, copper ions, tin ions, chromium ions, ruthenium ions, magnesium ions, zinc ions, cadmium ions, manganese ions, Examples include barium ions, strontium ions, mercury ions, lead ions, and the like, and nickel ions, cobalt ions, zinc ions, copper ions, and the like are preferable from the viewpoint of affinity for peptides. On the substrate surface on which the chelating ligand is formed, a salt of these metals, for example, nickel sulfate, nickel succinate, nickel hydroxide, cobalt acetate, cobalt amidosulfate, cobalt chloride, cobalt benzoate, cobalt borate, bromide Cobalt, cobalt chromate, cobalt formate, cobalt naphthenate, cobalt nitrate, cobalt oleate, cobalt phosphate, cobalt stearate, cobalt sulfate, cobalt tartrate, zinc chloride, zinc citrate, zinc nitrate, zinc oxalate, zinc sulfate A metal chelate can be formed by adding zinc tartrate, copper chloride, copper fluoride, copper formate, copper gluconate, copper nitrate, copper oxalate, copper phosphate, copper sulfate, and the like. At this time, in order to stably perform complex formation, it is desirable to add citric acid, tartaric acid or the like and adjust the pH to 3 to 11 as necessary. The peptide is coordinated to the metal chelate formed in this manner, thereby being immobilized on the solid support.
[0018]
Metal chelates can also be introduced into the carbon layer by electrostatic, ionic or isotope covalent bonds. It is also possible to introduce a functional group capable of hydrophobic interaction into the carbon layer and introduce a metal chelate therein.
[0019]
In the present invention, peptides include polypeptides, oligopeptides, and proteins, and may be simple proteins or complex proteins, and may be natural or synthetic. The length of the peptide is not particularly limited, but is suitable for immobilizing a peptide having 1 to 10000 amino acids, preferably 1 to 1000 amino acids. The target peptide that can be immobilized on the solid support of the present invention is not particularly limited, and examples thereof include antibodies, enzymes, pathogenic proteins, peptide hormones, receptors, kinases, glycoproteins, metalloproteins, viruses, derived proteins and the like. A protein can be suitably immobilized.
[0020]
The target peptide preferably has an appropriate label so that it specifically acts with the metal ion forming the chelate. Such labels include polyhistidine sequences that specifically interact with metal ions such as zinc, nickel or cobalt ions (eg, hexahistidine sequences), or specifically interact with zinc or copper, respectively. A polylysine sequence comprising at least about 4 lysines or a polyarginine sequence comprising at least 4 arginine residues. Peptides having such a label, for example, link a nucleotide sequence encoding a target peptide to a nucleotide sequence encoding a labeled peptide, which is transcribed and translated in vitro, and then specifically interacts with the label It can be obtained by isolation from the translation reaction using reagents.
[0021]
The carbon layer formed on the substrate in the present invention is not particularly limited, but diamond, diamond-like carbon, amorphous carbon, graphite, hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide. , Tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, vanadium carbide, and the like. The carbon layer is advantageous in that it has excellent chemical stability, can withstand subsequent chemical modification and reaction in peptide binding, and is transparent to the detection system UV because it has no UV absorption. .
[0022]
In the present invention, the carbon layer can be formed by a known method. For example, a microwave plasma CVD (Chemical Vapor Deposition) method, an ECRCVD (Electrical Cyclotron Resonance) method, an IPC (Inductive Coupled Plasma) method, a DC sputtering method, a DC sputtering method, a DC sputtering method, an ECR method. Examples thereof include a vapor deposition method, a laser vapor deposition method, an EB (Electron Beam) vapor deposition method, and a resistance heating vapor deposition method.
[0023]
In the high-frequency plasma CVD method, a source gas (methane) is decomposed by glow discharge generated between electrodes by a high frequency of 13.56 MHz, and a DLC (Diamond Like Carbon, diamond-like carbon) layer is synthesized on the substrate. In the ionized vapor deposition method, the source gas (benzene) is decomposed and ionized using thermoelectrons generated by a tungsten filament, and a carbon layer is formed on the substrate by a bias voltage. The DLC layer may be formed by ionized vapor deposition in a mixed gas consisting of 1 to 99% by volume of hydrogen gas and 99 to 1% by volume of the remaining methane gas.
[0024]
In the arc evaporation method, an arc discharge is generated in a vacuum by applying a DC voltage between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode), and a plasma of carbon atoms is generated from the cathode to generate an evaporation source. Further, by applying a negative bias voltage to the substrate, carbon ions in the plasma can be accelerated toward the substrate to form a carbon layer.
[0025]
In the laser vapor deposition method, for example, a carbon target layer is formed by depositing carbon atoms on a glass substrate by irradiating a graphite target plate with an Nd: YAG (Yttrium aluminum garnet) laser (pulse oscillation) light to melt it. it can.
[0026]
When the peptide is immobilized on the solid support of the present invention and stored, a diamond layer is preferably formed on the substrate surface. This is because diamond has excellent thermal conductivity and can be rapidly cooled.
[0027]
As diamond, any of synthetic diamond, high pressure formed diamond, natural diamond, and the like can be used. Further, the structure may be either a single crystal or a polycrystal. From the viewpoint of productivity, it is preferable to use a diamond layer manufactured by a vapor phase synthesis method such as a microwave plasma CVD method.
[0028]
When the solid support of the present invention is used for storage of peptides, it is desirable that the surface of the solid support is intentionally roughened. This is because such a roughened surface is advantageous for immobilizing a large amount of peptide by increasing the surface area. The shape of the solid support is not particularly limited, such as a flat plate shape, a thread shape, a spherical shape, a polygonal shape, and a powder shape. Further, the solid support may be a composite of diamond and another substance (for example, a substrate having two layers).
[0029]
By immersing the solid support of the present invention in a buffer in which each peptide can exist stably without losing its function, the peptide immobilized by the method of the present invention can be used for a period equivalent to that stored in the buffer. It is stably immobilized on a solid support without losing its function.
[0030]
When the solid support of the present invention is used for peptide analysis, the substrate surface prevents nonspecific adsorption of the peptide, increases the resistance to the reagent, reduces the background in fluorescence measurement, enables measurement by transmitted light, And it is preferable to form with diamond-like carbon from a viewpoint that cost and productivity are also high. As in the case of diamond, the substrate on which the diamond-like carbon layer is formed is not particularly limited, and a glass such as a slide glass, gold, silver, copper, aluminum, tungsten, molybdenum, etc .; a laminate of the above metal and ceramic; glass; Silicon; fiber; wood; paper; plastic such as polycarbonate and fluororesin; and a mixture of plastic and the above metals, ceramics, diamond, and the like can be used. Moreover, when using for analysis of protein etc., it is preferable that the shape of a board | substrate is a shape according to the analyzer, for example, spherical shape or flat plate shape.
[0031]
Using a solid support in which a diamond-like carbon layer is formed on the substrate surface and chemically modified as described above to form a metal chelate, for example, fluorescence detection is performed by immobilizing a fluorescently labeled peptide on the solid support. Results in a low background and a high fluorescence signal, ie a high S / N ratio.
[0032]
The thickness of the carbon layer on the surface of the solid support of the present invention is preferably from a carbon monomolecule to 100 μm, more preferably from a carbon monomolecule to 100 nm, from the viewpoint of visible light transmittance and a decrease in background to fluorescence. More preferably, it is carbon single molecule-10 nm. In addition, although it is slightly inferior in terms of permeability, it can be applied even when the solid support is entirely composed of carbon.
[0033]
The solid support of the present invention is also suitable for analyzing the target peptide bound thereto by mass spectrometry. Mass spectrometer formats that can be used for mass spectrometry in this case include matrix-assisted laser desorption (MALDI), continuous or pulsed electron spray (ESI), and ion spray or thermal spray. Related methods such as, but not limited to, ionization (I) techniques such as Massive-cluster impact ionization (MCI). Such ion sources include linear or non-linear reflection time-of-flight (TOF), single or multiple quadrupoles, single or multiple magnetic sectors, Fourier transform ion cyclotron resonance (FTICR), Fourier transform ion cyclotron resonance (FTICR). Can be adapted to detection modalities including ion capture, and combinations thereof such as ion capture / time of flight. A number of matrix / wavelength combinations (MALDI) or solvent combinations (ESI) can be used for ionization. Proteins at submolar levels are described, for example, by ESI mass spectrometry (Valaskovic et al., Science 273: 1199- 1202 (1996)) and MALDI mass spectrometry (Li et al., J. Am. Chem. Soc. 118: 1662-1663 (1996). ) Can be detected.
[0034]
Electrospray mass spectrometry is described by Fenn et al. (J. Phys. Chem. 88: 4451-59 (1984); WO 90/14148). MALDI-TOF mass spectrometry Hillenkamp et al. ( "Matrix Assisted UV-Laser Desorption / Ionization: A New Approach to Mass Spectrometry of Large Biomolecules, Biological Mass Spectrometry" (Burlingame and McCloskey editing, Elsevier Science Publ.1990), p.49- 60). For ESI, the measurement of the molecular weight of the femtomolar amount of the sample is very accurate because there are numerous ion peaks, all of which can be used for mass calculation.
[0035]
The mass of the target peptide measured by mass spectrometry can be compared to the corresponding known peptide mass. For example, if the target peptide is a mutein, it can be compared with the corresponding normal protein as the corresponding known peptide.
[0036]
The step of analyzing the target peptide using mass spectrometry as described above can be performed by determining the amino acid sequence of the target peptide or a part thereof. Amino acid sequencing can be performed, for example, from the carboxyl terminus using an enzyme such as carboxypeptidase Y, carboxypeptidase P, carboxypeptidase A, carboxypeptidase G or carboxypeptidase B, or using Edman degradation or alanine An enzyme such as aminopeptidase, leucine aminopeptidase, pyroglutamate peptidase, dipeptidyl peptidase, and microcrosomal peptidase can be used from the N-terminus of the target peptide. The released amino acids are then identified by mass spectrometry.
When used for mass spectrometry of such peptides, it is preferable to use a solid support having a diamond-like carbon layer.
[0037]
【Example】
EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.
Example 1
A glass substrate of 25 mm (width) × 75 mm (length) × 1 mm (thickness) was used. On the surface of this glass substrate, a slide glass having a DLC layer formed to a thickness of 10 nm was prepared by ionized vapor deposition using a gas mixed with 95% by volume of methane gas and 5% by volume of hydrogen gas as a raw material.
[0038]
Next, the surface of the slide glass was chemically modified to coordinate nickel ions. That is, after introducing chlorine into a reaction vessel containing a slide glass, the surface of the glass substrate is chlorinated by irradiating light from a high-pressure mercury lamp for 1 minute, and immersed in a mixed solution of 200 ml of dimethylformamide, 12 ml of ethylenediamine, and 38 ml of triethylamine. The mixture was stirred at room temperature for 5 hours, washed three times with 200 ml of water, added to 240 ml of a 1.25 molar aqueous sodium hydroxide solution containing 28.2 g of chloroacetic acid, and stirred overnight at room temperature. This slide glass was immersed in 500 ml of 0.2 molar nickel sulfate aqueous solution containing 15.0 g of tartaric acid, 21.0 g of citric acid, and 30 ml of 28% ammonia water, stirred at room temperature for 3 hours, washed with 200 ml of water three times, When surface analysis was performed by XPS (X-ray photo-electron spectroscopy), a peak derived from nickel was confirmed at 855.0 to 859.0 eV.
[0039]
In the above, the solid support before analysis by XPS was washed 3 times with 200 ml of water, dried and vacuum packed. The vacuum pack is formed by evacuating the vacuum container to 1 Torr or less and then thermally fusing the vacuum bag therein. Even if the sample was left in a vacuum pack at 4 ° C. for 6 months, the analysis result of the nickel peak by XPS was the same as that without aging, and no change was observed in the function of immobilizing as a chelate.
[0040]
Example 2
A solid support in which a metal chelate is formed on a substrate on which a DLC (Diamond like carbon, diamond-like carbon) layer is formed is prepared, and GFP (green fluorescent protein) in which a polyhistidine sequence is fused on the solid support. Spotted by 0.2 μl with a pipettor. And it was made to react for 1 hour in the wet box kept at 37 degreeC. Thereafter, the plate was washed with 1 × PBS for 15 minutes, and fluorescence measurement was performed with LAS1000 (manufactured by Fuji Photo Film Co., Ltd.). The conditions are as follows. CCD (Charge coupled device): 1.3 million pixels (cooled CCD camera), effective pixels: 1384 (H) × 922 (V) pixels, angle of view: 7 cm × 11 cm, exposure time: 10 seconds. Autofluorescence by GFP was measured. The measurement results are shown in FIG. FIG. 1 is a two-dimensional image of the intensity of autofluorescence caused by GFP using a chemiluminescence measuring device for a solid support on which a protein is immobilized. A round spot is a portion where GFP exists. As a result, fluorescence was observed from GFP fused with the polyhistidine sequence. From the above, it was revealed that GFP was immobilized while maintaining the fluorescence function.
[0041]
Further, as described above, a solid support on which GFP (green fluorescent protein) fused with a polyhistidine sequence was immobilized was added to 45 mL of 0.05 M Tris-HCl (pH 7.4) sterilized by autoclaving at 4 ° C. After soaking for 1 week, 2 weeks, and 1 month, autofluorescence by GFP was measured with LAS1000 (manufactured by Fuji Photo Film Co., Ltd.). The measurement conditions are as follows. CCD: 1.3 million pixels (cooled CCD camera), effective pixels: 1384 (H) × 922 (V) pixels, angle of view: 7 cm × 11 cm, exposure time: 10 seconds. The measurement results are shown in FIG. FIG. 3 is a graph showing the intensity ratio of autofluorescence by GFP measured by a chemiluminescence measuring apparatus for a solid support on which a protein is immobilized. At this time, the autofluorescence of GFP was measured at 89 pixels at the spot site, and the average of each pixel was defined as the autofluorescence intensity. As a result, even when stored for 1 month at 4 ° C. with GFP immobilized, the intensity ratio of 0.8 is maintained with respect to the initial autofluorescence intensity ratio of 1, and GFP is stably immobilized. It became clear that the function was retained.
[0042]
Example 3
A solid support in which a metal chelate is formed on a substrate on which a DLC (diamond-like carbon) layer is formed is prepared, and Cy5 fluorescently labeled polyhistidine sequence is fused on this solid support and GFP and Cy3 fluorescently labeled protein A was spotted with a pipettor at 0.2 μl each. And it was made to react for 1 hour in the wet box kept at 37 degreeC. Then, (1) washing with 1 × PBS for 15 minutes, (2) washing with 10 mM imidazole / 1 × PBS for 15 minutes, and (3) washing with 500 mM imidazole / 1 × PBS for 15 minutes. Fluorescence measurement was performed with FLA8000 (Fuji Photo Film Co., Ltd.). The measurement results are shown in FIG. FIG. 2 is a two-dimensional image of the fluorescence intensity of a dye measured by a fluorescence measuring device on a solid support on which a protein is immobilized. A round spot is a fluorescently labeled protein. As a result, in (1), both GFP fused with Cy5 fluorescently labeled polyhistidine sequence and Cy3 fluorescently labeled protein A are immobilized, and in (2), only GFP fused with Cy5 fluorescently labeled polyhistidine sequence. Was immobilized, and in (3), both were not immobilized. From the above, it was revealed that the solid support of the present invention can desorb proteins by changing the concentration of imidazole.
[0043]
Example 4
A glass substrate of 25 mm (width) × 75 mm (length) × 1 mm (thickness) was used. On the surface of this glass substrate, a slide glass having a DLC layer formed to a thickness of 10 nm was prepared by ionized vapor deposition using a gas mixed with 95% by volume of methane gas and 5% by volume of hydrogen gas as a raw material.
[0044]
Next, the surface of the slide glass was chemically modified to coordinate nickel ions. That is, after introducing chlorine into a reaction vessel containing a slide glass, the surface of the glass substrate is chlorinated by irradiating light from a high-pressure mercury lamp for 1 minute, and a 0.8 molar sodium hydroxide aqueous solution containing 19.8 g of iminodiacetic acid The solution was stirred at room temperature overnight in 250 ml. This slide glass was immersed in 500 ml of 0.2 molar nickel sulfate aqueous solution containing 15.0 g of tartaric acid, 21.0 g of citric acid, and 30 ml of 28% ammonia water, stirred at room temperature for 3 hours, washed with 200 ml of water three times, When surface analysis was performed by XPS, a nickel-derived peak was confirmed at 855.0 to 859.0 eV. In the above, the solid support before analysis by XPS was washed with 200 ml of water three times, dried, and then vacuum packed. The vacuum pack is formed by evacuating the vacuum container to 1 Torr or less and then thermally fusing the vacuum bag therein. Even when left in a vacuum pack at 4 ° C. for 6 months, the analysis result of the nickel-derived peak was the same as that without aging, and it became clear that the function as a chelate was maintained.
[0045]
【The invention's effect】
The solid support in which a metal chelate is formed on the carbon layer of the present invention is excellent in optical properties such as UV transparency, and can stably immobilize a peptide over a long period of time without losing its function. Peptide desorption is possible. Therefore, it can be used as a useful means in peptide storage and analysis.
[Brief description of the drawings]
FIG. 1 shows two-dimensional images of the intensity of autofluorescence by GFP using a chemiluminescence measuring device for a solid support on which a protein is immobilized. A round spot is a portion where GFP exists.
FIG. 2 is a two-dimensional image of the fluorescence intensity of a dye supported by a dye on a solid support on which a protein is immobilized. A round spot is a fluorescently labeled protein.
FIG. 3 is a graph showing the results of examining the fluorescence intensity of a solid support on which a protein is immobilized over time.

Claims (5)

A solid support for immobilizing a peptide, wherein a part or all of the substrate surface is composed of a diamond-like carbon layer and a metal chelate is formed on the layer. The solid support according to claim 1, wherein the diamond-like carbon layer has a thickness of carbon monomolecule to 100 μm. A method for analyzing a peptide by immobilizing the peptide on the surface of the solid support according to claim 1. How to store the peptide immobilized peptide on the surface of a solid support according to claim 1 or 2. The solid support according to claim 1 or 2 , wherein a peptide is immobilized on a metal chelate formed on the surface thereof.

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