JP4635532B2 - Biomolecule analysis and recovery method - Google Patents

Description

  The present invention relates to a method for analyzing and recovering biomolecules such as nucleic acids and proteins.

  A method of comprehensively and comprehensively analyzing biomolecules using an array chip such as a DNA chip (DNA microarray) or a protein chip as a tool is often used. These are binding assays that utilize the selective binding specificity inherent to biomolecules, and are multi-item simultaneous analysis techniques that use position information as signal type information (see Patent Document 1, etc.).

Special table hei 3-505157

However, with such a method, the sample once bound to the array chip cannot be collected. For example, after a sample is qualitatively analyzed with an array chip, it may be desired to use it for other analysis (for example, quantitative analysis) or for other applications. If the sample cannot be collected as in the past, these requests cannot be answered. Many important proteins are particularly inconvenient because they are small in quantity.
The present invention has been made in view of the above circumstances, and an object thereof is to enable a sample to be collected after a binding assay using an array chip or the like.

According to the invention according to claim 1 to solve the above-mentioned problem, a target molecule that is an organic molecule to be analyzed with respect to a linker molecule that has a cleavage site that is cleaved under a specific condition and is bound to a carrier surface Binding a probe molecule to which is bound, cleaving the cleavage site in a state in which the target molecule is bound to the probe molecule, and recovering the target molecule bound to the probe molecule by the cleavage And an analysis method characterized by comprising:

Moreover, according to the invention which concerns on Claim 2, the said cleavage is made by light irradiation, The analysis method of Claim 1 characterized by the above-mentioned is provided.

Moreover, according to the invention which concerns on Claim 3, the said irradiated light is an ultraviolet-ray, The analysis method of Claim 2 characterized by the above-mentioned is provided.

Moreover, according to the invention which concerns on Claim 4, it labels with the fluorescent dye with respect to the said target molecule, The analysis method of any one of Claim 1 to 3 characterized by the above-mentioned is provided.

According to the invention of claim 5, the probe molecule to which the target molecule is bound is labeled with a fluorescent dye different from the fluorescent dye that labels the target molecule, and the target to which the probe molecule is bound 5. The analysis method according to claim 4, wherein after the molecule is recovered, the fluorescent dye of the target molecule and the fluorescent dye of the probe molecule are detected.

Moreover, according to the invention which concerns on Claim 6, the fluorescence intensity of the said fluorescent pigment | dye is measured, The analysis method of Claim 4 or 5 characterized by the above-mentioned is provided.

According to the invention of claim 7, the linker molecule bonded to the carrier is a plurality of types of linker molecules having different cleavage conditions, and the linker molecule is selectively changed by changing the cleavage conditions. The analytical method according to any one of claims 1 to 6, wherein the desired target molecule is recovered.

According to the invention of claim 8, the linker molecule is bonded to the surface of the carrier, and then the probe molecule is bonded, and then the target molecule is bonded to the probe molecule, and then the surface of the carrier The analysis method according to any one of claims 1 to 7, wherein the target molecule is recovered by cleaving the linker molecule bound to the protein.
Further, according to the invention according to claim 9, the probe molecule to which the target molecule can bind is prepared in a state of a solution containing the probe molecule, and a solution containing a linker molecule that links the solution to the probe molecule; Is prepared, and the mixed solution is dropped on the surface of the carrier, and the linker molecule to which the probe molecule is bound is bound to the surface of the carrier and bound to the linker molecule immobilized on the surface of the carrier. The target molecule is collected by binding the target molecule to the probe molecule thus formed and cleaving the linker molecule bound to the surface of the carrier. Is provided.

  According to the present invention, a technique for collecting a sample bound by an array chip or the like is provided.

  Hereinafter, an embodiment of the analysis method of the present invention will be described.

  First Embodiment FIG. 1 is a diagram for explaining an analysis method according to a first embodiment, and is an enlarged schematic diagram of a substrate surface of an array chip. As shown in the figure, the analysis method of this embodiment includes the following steps.

(1) A step of immobilizing the linker molecule 200 that binds the substrate 100 and the probe molecule 300 to the surface of the substrate 100 (2) A step of immobilizing the probe molecule 300 such as a probe nucleic acid or an antibody to the linker molecule 200 (3 ) Step of attempting to bind the target molecule (evaluation target molecule) 400 such as labeled DNA or protein to the probe molecule 300 (4) Step of recovering the target molecule 400 by cleaving the linker molecule 200 Will be described in detail.

  <Step (1)> First, the step of immobilizing the linker molecule 200 that binds the substrate 100 and the probe molecule 300 to the surface of the substrate 100 will be described.

Examples of the substrate 100 serving as a carrier include glass, silica (SiO ₂ ), silicon wafer, alumina (Al ₂ O ₃ ), talc, clay, aluminum, iron, mica, asbestos, titanium oxide, iron oxide, and quartz. . Among these, those having a silanol group on the surface of glass, silicon wafer or the like are preferable. The silicon wafer has a silanol group on the surface as long as a natural oxide film is formed, but it can also be formed on the surface by thermal oxidation treatment. A silicon wafer having a silicon nitride film on the surface can also be used.

  Further, the shape of the carrier is not limited to a plate shape, and can be appropriately selected depending on the application. For example, a sheet shape, a honeycomb shape, a fiber shape, a bead shape, a powder shape, and the like may be used.

  The linker molecule 200 only needs to be capable of binding to the substrate 100 and the probe molecule 300 and can be cleaved by light irradiation or the action of a specific compound. That is, the linker molecule 200 may have any molecular structure including a binding portion 210 with the substrate 100, a cleavable cross-linking portion 220, and a binding portion 230 with the probe molecule 300 that is an organic molecule.

  In such a linker molecule 200, examples of the functional group that can be the bonding portion 210 with the substrate 100 include a trimethoxysilyl group and a trichlorosilyl group that react with a silanol group.

  Examples of the functional group that can serve as the bonding part 230 with the probe molecule 300 include an active ester group such as a succinimidyl group highly reactive with an amino group and a maleimide group highly reactive with a thiol group.

  Examples of the functional group of the cleavable portion 220 of the linker molecule 200 include a nitrobenzyl ester group, a nitrobenzyl ether group, a nitrobenzyl thioether group, and a nitrobenzyl carbamoyl group as functional groups that are cleaved by light irradiation.

As other cleavable functional groups,
A disulfide group (-SS-: for example, cleavage in the presence of a reducing agent (10-100 mM) such as 2-mercaptoethanol, dithioleitol, pH 7-9, 25-37 ° C., 5-30 minutes).
Glycol group (-CH (OH) -CH (OH)-: for example, cleavage with 15 mM NaIO ₄ at pH 7.5 at 25 ° C for 4-5 hours),
An azo group (-N = N-: For example, with 0.1M _Na 2 _S 2 _{O 4,} in _{0.1M NaHCO 3 (pH8.0) -0.15M NaCl} , cleaved by holding at room temperature for 25 minutes.) ,
Sulfone group (-SO ₂ -: For example, 0.1 M _Na 2 HPO ₄ - Tris buffer (pH11.6) -6M -0.1% SDS- 2mM in dithiothreitol urea to 2 hours at 37 ° C. Cleavage at).
Cleavage by holding at 25-37 ° C. for 3-5 hours in 50 mM Tris buffer (pH 7.5-8.5) -25 mM CaCl ₂ -1 mM benzamine using ester (—CO ₂ —: 1 M hydroxylamine) ),
Orthoester (cleaved by holding at pH 6.4 for 1 to 3 hours) and the like.

  The immobilization method of the linker molecule 200 differs depending on the type of the substrate 100 and the type of the linker molecule 200 to be immobilized. The linker molecule 200 may be immobilized in one step, or may be immobilized in a plurality of steps.

  For example, when a linker molecule having a succinimidyl group highly reactive with an amino group and having a nitrobenzylthioether group that is cleaved by light irradiation is immobilized on a substrate having a silanol group on the surface, the procedure is as follows.

  First, a thiol group is formed on the substrate surface with a silane coupling agent having a thiol group such as mercaptomethyldimethylethoxylane or 3-mercaptopropyltrimethoxysilane on a substrate having a silanol group on the surface (for example, a glass substrate). To form.

  Next, succinimidyl-4-bromomethyl-3-nitrobenzoate (BNBA-Osu) having a 2-nitrobenzyl bromide group highly reactive with a thiol group is allowed to act to form a succinimidyl group on the surface. Alternatively, after the silane coupling agent and BNBA-Osu are reacted, the product is allowed to act on the substrate surface.

  In addition to BNBA-Osu, succinimidyl-4- (4-bromomethyl-3-nitrobenzyl) aminobutyrate (BNBAC3-Osu), sulfosuccinimidyl-4- (4-bromomethyl-3-nitrobenzyl) Aminobutyrate sodium salt (BNBAC3sulfo-Osu), succinimidyl-6- (4-bromomethyl-3-nitrobenzyl) aminohexanoate (BNBAC5-Osu), sulfosuccinimidyl-6- (4-bromomethyl-3) -Nitrobenzyl) Aminohexanoate sodium salt (BNBAC5sulfo-Osu) and the like can also be used.

  A plurality of types of linker molecules having different cleavage conditions may be immobilized. By doing so, it is possible to selectively recover the target molecule by selectively cleaving the linker molecule by changing the cleavage condition of the linker molecule at the time of recovery.

<Step (2)> Next, a step of immobilizing a probe molecule 300 such as a probe nucleic acid or a protein to the linker molecule 200 will be described.
The probe molecule (ligand) used varies depending on the target molecule (molecule to be evaluated).

  That is, in the case of a DNA array, an oligonucleotide or cDNA (DNA having a base sequence complementary to mRNA) whose sequence is known can be used as a probe molecule.

  In the case of a protein-detection protein array, an antibody can be used as a probe molecule. In addition, as probe molecules other than antibodies, aptamers selected so as to bind to specific molecules by randomly changing a partial sequence of a nucleic acid or protein can be used. Aptamers are easier to select and more efficient than antibodies.

  Also, in the case of a protein-function array for protein functional analysis, if you want to evaluate protein-protein, protein-small molecule, protein-drug, protein-nucleic acid interaction, or enzyme activity, One substance of the combination may be a probe molecule and the other may be a target molecule.

  Moreover, if it is a peptide array for the search of the substrate of an enzyme, or the search of an inhibitor, a substrate and an inhibitor can be used as a probe molecule.

  The probe molecule is immobilized by coating, spotting, or the like on the surface of the substrate on which the linker molecule is immobilized. When the solution is spotted on the substrate, it is kept for 1 hour to 1 day. Then, by drying, a substrate on which the probe molecules are immobilized can be obtained.

  In addition, by washing with an aqueous solution or an organic solvent after spotting, excess probe molecules that do not contribute to the immobilization can be removed, and the immobilization amount can be made uniform.

  Further, different types of probe molecules may be immobilized at specific positions on the substrate by patterning or the like. In this way, comprehensive analysis and combinatorial analysis over other items can be performed simultaneously.

  In the above description, the probe molecule is immobilized after the linker molecule is immobilized on the substrate. However, the present invention is not limited to this, and the linker molecule and the probe molecule may be immobilized simultaneously.

  <Step (3)> Next, the step of attempting to bind the target molecule 400 to the probe molecule 300 will be described. As described above, the combination of the probe molecule 300 and the target molecule 400 differs depending on the purpose of analysis.

  For example, in the case of a DNA array, the target molecule is DNA or RNA. In the case of a protein detection protein array, the target molecule is a protein. In the case of a protein array for analyzing the function of a protein, a target molecule is one of a combination of protein-protein, protein-small molecule, protein-drug, protein-nucleic acid and the like for which interaction and enzyme activity are to be evaluated. In the case of a peptide array, if the probe molecule is a substrate or an inhibitor, the target molecule is an enzyme.

  The target molecule is spotted on the substrate surface together with the solvent, thereby attempting to bind to the probe molecule. After spotting, it is allowed to stand for a predetermined time and then washed with a solvent. Depending on whether the target molecule is bound to the probe molecule, DNA sequence information, protein detection, interaction with an antibody, enzyme activity, and the like can be known.

  If the target molecule is fluorescently labeled, it can be easily determined which probe molecule is bound to the array. Further, the amount can be measured by the fluorescence intensity.

  <Step (4)> Next, the step of recovering the immobilized target molecule 400 will be described. As described above, the linker molecule 200 has the site 220 that is cleaved by some action. Therefore, by giving a cleavage condition, the cleavage portion 220 of the linker molecule 200 is cleaved, and the target molecule 400 is detached from the substrate 100 together with the probe molecule 300 and collected.

  For example, when the linker molecule is cleaved by light irradiation, light irradiation is performed. At this time, light with a wavelength suitable for cleavage may be irradiated.

  In addition, when giving the conditions of cutting | disconnection of bridge | crosslinking, it is preferable to carry out on the conditions by which the structure of a target molecule or a probe molecule is not broken.

  The detached target molecule can be recovered by washing the substrate with an appropriate solvent.

  In the case of a substrate on which a plurality of types of linker molecules having different cleavage conditions are immobilized, the linker molecules can be selectively cleaved and target molecules can be recovered by changing the cleavage conditions.

  For example, in the case of a substrate on which a linker molecule that is cleaved by light irradiation and a linker molecule that is cleaved by a reducing agent such as 2-mercaptoethanol is immobilized, first, a part of a specific target molecule is probed by light irradiation. It can be collected together. Then, other target molecules can be recovered together with the probe molecules by the reducing agent.

  The steps until the target molecule is recovered have been described above.

  The recovered target molecule can be used for other analyzes and applications. For example, a sample for electrophoresis analysis or mass spectrometry (TOF-MS or the like) can be used. It can also be used for applications such as introducing a specific gene into a cell and expressing it.

  In addition, although the fragment | piece of a linker molecule, a probe molecule, etc. are added to the collect | recovered target molecule | numerator, since what was added is known, what is necessary is just to subtract the part and to evaluate analysis.

  Second Embodiment Next, a second embodiment will be described. The second embodiment relates to an analysis system that executes the analysis method of the first embodiment.

  FIG. 2 is a configuration diagram showing an outline of the analysis system according to the second embodiment. As shown in the figure, the analysis system 1 of this embodiment includes a substrate receiving unit 11, a linker molecule immobilization device 12, a probe molecule immobilization device 13, a target molecule binding test device 14, and a recovery device 15. .

  The substrate receiving portion 11 is a portion that receives the substrate 100 before the linker molecule 200 is immobilized, and includes a jig that supports the substrate 100. The shape is not limited to the substrate 100, and the shape is not limited.

  The linker molecule immobilization apparatus 12 is an apparatus that immobilizes a substance that becomes the linker molecule 200 by coating, spotting, or the like on the surface of the substrate 100. It has a function of performing a series of steps of immobilizing the linker molecule 200 to the substrate 100 described in the first embodiment.

  The probe molecule immobilization apparatus 13 is an apparatus that immobilizes the probe molecule 300 to the linker molecule 200. It has a function to perform a series of steps of the immobilization step of the probe molecule 300 described in the first embodiment.

  The target molecule binding test apparatus 14 is an apparatus that attempts to bind the target molecule (substance to be evaluated) 400 and the probe molecule 300. It has a function of performing a series of steps of attempting to bind the target molecule 400 described in the first embodiment.

  The recovery device 15 is a device that provides conditions for cleaving the linker molecule 200 and recovers the target molecule 400. It has a function to perform a series of steps of the recovery step of the target molecule 400 described in the first embodiment.

  The analysis system 1 may be provided with an observation device such as a microscope for observing the binding state (position, color, etc.), or may be provided with a device that automatically observes and analyzes and outputs the observation result. Good.

  The analysis system 1 configured as described above collects target molecules in a form that can be used for other analyzes and applications after a binding assay using a DNA array, protein array, peptide array, or the like.

  Note that the analysis system 1 does not have to include all the above-described apparatuses, and may include a part of the apparatuses. For example, the substrate receiving unit 11 may receive the substrate 100 on which the linker molecule 200 is immobilized in advance, or the substrate 100 on which the linker molecule 200 and the probe molecule 300 are immobilized.

  Further, the analysis system 1 may include other analysis devices such as an electrophoresis analysis device and a mass analysis device. Then, the sample collected by the collection device 15 may be subjected to necessary processing, and then further analysis may be performed.

  As mentioned above, although several embodiment of this invention was described, various deformation | transformation are possible for this invention within the range of the summary.

  For example, in the above embodiment, the linker molecule is cleaved under specific conditions to recover the target molecule. However, the present invention is not limited to this, and the target molecule may be recovered by cleaving the probe molecule using a probe molecule that cleaves under a specific condition.

  For example, when the probe molecule has the following structure, it can be cleaved by light irradiation.

  EXAMPLES Next, although an Example demonstrates this invention in more detail, this invention is not limited only to the following Example.

<Example 1>
1. Immobilization of linker molecule 1-1. Synthesis of 4- (4-acetyl-2-methoxy-5-nitrophenoxy) butanoic acid 1-1-1. Synthesis of methyl 4- (4-acetyl-2-methoxyphenoxy) butanoate (synthesized with reference to CPHolmes, JOC, 62, 2370 (1997))

  4'-hydroxy-3'-methoxyacetophenone (Wako Pure Chemical Industries, 41.00g, 246.7 mmol), methyl 4-bromobutyrate (Wako Pure Chemical Industries, 49.63 g, 274.1 mmol) and potassium carbonate (Japanese Mitsui Pure Chemicals, 51.1 g, 370 mmol) was placed in 200 ml of dimethylformamide and stirred overnight at room temperature. The obtained reaction solution was poured into water to stop the reaction, and then extracted with 500 ml of ethyl acetate. The organic phase was washed with water and then with a saturated aqueous sodium chloride solution, and dried over anhydrous magnesium sulfate. After the organic phase was filtered off, the solvent was distilled off under reduced pressure to obtain methyl 4- (4-acetyl-2-methoxyphenoxy) butanoate in a yield of 95%. The structure of this compound was confirmed by proton nuclear magnetic resonance spectrum (1H-NMR).

1H-NMR (300 MHz, CDCl ₃ , δ ppm); 2.19 (quadruple line, 2H), 2.56 (triple line, 2H), 2.60 (single line, 3H), 3.70 (single line, 3H), 3.91 ( Single wire, 3H), 4.15 (triple wire, 2H), 6.89 (double wire, 1H), 7.51-7.78 (multiple wire, 2H)

1-1-2. Synthesis of methyl 4- (4-acetyl-2-methoxy-5-nitrophenoxy) butanoate

  Methyl 4- (4-acetyl-2-methoxyphenoxy) butanoate (5.0 g, 18.8 mmol) was dissolved in 30 ml of acetic anhydride. While cooling with ice, 70% nitric acid (100 ml) was gradually added. After stirring for 3 hours at room temperature, the reaction was poured into ice water. The precipitate was collected by filtration and recrystallized from methanol-water to obtain methyl 4- (4-acetyl-2-methoxy-5-nitrophenoxy) butanoate in 66% yield. The structure of this compound was confirmed by proton nuclear magnetic resonance spectrum (1H-NMR).

1 H-NMR (300 MHz, CDCl ₃ , δ ppm); 2.20 (quadruple line, 2H), 2.49 (triple line, 2H), 2.56 (single line, 3H), 3.70 (single line, 3H), 3.95 ( Single wire, 3H), 4.15 (triple wire, 2H), 6.75 (double wire, 1H), 7.67 (single wire, 1H).
1-1-3. Synthesis of 4- (4-acetyl-2-methoxy-5-nitrophenoxy) butanoic acid

  Methyl 4- (4-acetyl-2-methoxy-5-nitrophenoxy) butanoate (2.44 g, 7.84 mmol) was dissolved in 100 ml of a 1N aqueous sodium hydroxide-methanol (1: 1) mixture. The mixture was stirred for 1 day at room temperature, hydrochloric acid was added to adjust the pH to 2, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated sodium chloride in this order, and dried over anhydrous magnesium sulfate. Thereafter, the organic solvent was distilled off under reduced pressure, and the residue was isolated and purified by silica gel column chromatography to give 4- (4-acetyl-2-methoxy-5-nitrophenoxy) butanoic acid in a yield of 97%. Obtained. The structure of this compound was confirmed by proton nuclear magnetic resonance spectrum (1H-NMR).

1 H-NMR (300 MHz, CDCl ₃ , δ ppm); 2.20 (quadruple line, 2H), 2.49 (triple line, 2H), 2.56 (single line, 3H), 3.70 (single line, 3H), 3.95 ( Single wire, 3H), 4.15 (triple wire, 2H), 6.75 (double wire, 1H), 7.67 (single wire, 1H).

1-2. Introduction of amino groups on the glass substrate surface

  A glass flat plate (Matsunami Glass Industry Co., Ltd., NEO cover glass 24 × 32) is washed thoroughly in the order of organic solvent (methanol, etc.), pure water, acidic aqueous solution (sulfuric acid-hydrogen peroxide mixture, etc.), and then 1N It was immersed in an aqueous sodium hydroxide solution for 1 minute. And it wash | cleaned fully in order of the pure water, the 1N hydrochloric acid, the pure water, and methanol. The glass plate was immersed in a 5% ethanol solution of trimethoxy-aminopropylsilane (manufactured by Wako Pure Chemical Industries, Ltd.) for 2 hours at room temperature. Then, after sufficiently washing with a water-ethanol (one to one) mixed solution, the mixture was kept at 150 ° C. for 2 hours in a nitrogen atmosphere.

1-3. Immobilization of 4- (4-acetyl-2-methoxy-5-nitrophenoxy) butanoic acid

  A dimethylformamide solution of 5% 4- (4-acetyl-2-methoxy-5-nitrophenoxy) butanoic acid was prepared. Prepare 5% 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Dojindo Laboratories) and 5% triethylamine dimethylformaside solution, mix the same amount of the three solutions, and prepare the reaction solution. In this, a glass flat plate subjected to the amino group formation treatment was immersed and allowed to stand at room temperature for a whole day and night. After that, it was washed in order of methanol, pure water and methanol, and dried.

1-4. Reduction reaction

  The previously treated glass plate was immersed in a solution of sodium borohydride (Wako Pure Chemical Industries, 166 mg) dissolved in 40 ml of tetrahydrofuran and left at room temperature for one day. Then, it wash | cleaned and dried in order of methanol, the pure water, and methanol.

1-5. Synthesis of chloroformate

  Under a nitrogen gas atmosphere, triphosgene (Wako Pure Chemical Industries, 2.1 g, 12.56 mmol) and potassium carbonate (Wako Pure Chemical Industries, 1.5 g 5 mmol) were dissolved in 100 ml of toluene. The previously treated glass plate was immersed in this solution and allowed to stand at room temperature for 2 days. Then, it was thoroughly washed with ether and dried.

2. Immobilization of probe molecule (fluorescent dye-labeled amino group-introduced nucleic acid) Nucleic acid chain consisting of 9 bases modified with fluorescent dye (Cy3) at the 5 'end and amino group at the 3' end via carbon chain (C6) ( Nippon Gene Co., Ltd.) was dissolved in 100 mM phosphate buffer (containing 20% dimethylformamide) at pH 7.0 to prepare a 3 mM nucleic acid solution. The nucleic acid solution was spotted onto the glass plate prepared above using a metal needle. After leaving it overnight, it was thoroughly washed with water.
Immobilization was confirmed by observing fluorescence from a fluorescent dye (Cy3) under a fluorescence microscope.

3. Nucleic acid hybridization reaction A nucleic acid chain (manufactured by Espec Oligo), which has a complementary sequence to the previously immobilized nucleic acid chain and is modified with a fluorescent dye (Cy5) at the 5 ′ end, is produced by 5 × SSC ( A probe solution was prepared by dissolving in a sodium citrate-saline buffer (manufactured by Dojindo Laboratories) -0.5% SDS (sodium dodecyl sulfate, manufactured by Wako Pure Chemical Industries).

  The probe solution was placed on the glass plate on which the nucleic acid had been previously spotted, and incubated at 65 ° C. for 20 hours to carry out a cross-reaction. After the reaction, it was washed twice with 2 × SSC-0.1% SDS solution and four times with 0.2 × SSC-0.2% SDS solution. The fluorescence from Cy5 was observed with a fluorescence microscope to confirm that the hybridization reaction had progressed.

4). Collection by light irradiation To the part on which the sample on the glass plate after the hybridization reaction is placed, use an ultraviolet (356 nanometer light) irradiation device (USHIO, 250 kW high-pressure mercury lamp-Optiplex device). Was irradiated for 20 minutes.
Using a phosphate buffer (containing 20% dimethylformamide), a nucleic acid sample detached from the substrate by a linker photocleavage reaction by light irradiation was collected. After the collected sample solution was concentrated, it was confirmed that the desired recovery was achieved by detecting two types of fluorescent dyes (Cy3 and Cy5) in a capillary electrophoresis apparatus.

<Example 2>
1. Synthesis of 4-bromomethyl-3-nitrobenzoic acid succinimide ester

  4- (Bromomethyl) benzoic acid (Tokyo Kasei Co., Ltd., 5 g) was gradually added to 70% nitric acid (100 ml) with ice cooling. After stirring at room temperature for 2 hours, the reaction mixture was poured into ice water, and the resulting precipitate was filtered off and recrystallized from methylene chloride-heptane to give 4-bromomethyl-3-nitrobenzoic acid. Obtained. This was dried, dissolved in 20 ml of acetonitrile, N-hydroxysuccinide (Wako Pure Chemical Industries, 1.05 g) and dicyclohexylcarbodiimide (Wako Pure Chemical Industries, 2.15 g) were added, and the mixture was stirred overnight at room temperature. . After removing the urea derivative by filtration, the solvent was distilled off under reduced pressure, and the resulting residue was recrystallized from methylene chloride-heptane to obtain 4-bromomethyl-3-nitrobenzoic acid succinimide ester. The structure of this compound was confirmed by proton nuclear magnetic resonance spectrum (1H-NMR).

1H-NMR (300 MHz, CDCl ₃ , δ ppm); 2.94 (single line, 4H), 4.87 (single line, 2H), 7.78 (double line, 1H), 8.34 (double line-double line, 1H ), 8.78 (double wire, 1H).

2. Nucleic acid immobilization on glass plate via photocleavable linker Nucleic acid chain consisting of 9 bases modified with fluorescent dye (Cy3) at 5 'end and thiol group at 3' end via carbon chain (C6) ( Nippon Gene Co., Ltd.) was dissolved in 100 mM phosphate buffer (containing 20% dimethylformamide) at pH 7.0 to prepare a 3 mM nucleic acid solution. An equal volume mixture of this solution and a 10 mM solution of 4-bromomethyl-3-nitrobenzoic acid succinimide ester, pH 8.6, 100 mM phosphate buffer was prepared. Then, on the glass plate (Matsunami Glass Industry Co., Ltd./NEO cover glass 24 × 32) in which amino groups are introduced into the surface using trimethoxyaminopropylsilane, the previously prepared mixed solution is used with a metal needle. I spotted it. This was left overnight and washed thoroughly with water.
Immobilization was confirmed by observing fluorescence from a fluorescent dye (Cy3) under a fluorescence microscope.

3. Nucleic acid hybridization reaction A nucleic acid chain (manufactured by Espec Oligo), which has a complementary sequence to the previously immobilized nucleic acid chain and is modified with a fluorescent dye (Cy5) at the 5 ′ end, is produced by 5 × SSC ( A probe solution was prepared by dissolving in a sodium citrate-saline buffer solution (produced by Dojindo Laboratories) -0.5% SDS (sodium dodecyl sulfate, manufactured by Wako Pure Chemical Industries).
The probe solution was placed on the glass plate on which the nucleic acid had been previously spotted, and incubated at 65 ° C. for 20 hours to carry out a cross-reaction. After the reaction, after washing twice with 2x SSC-0.1% SDS solution and four times with 0.2x SSC-0.2% SDS solution, the fluorescence reaction from Cy5 is observed with a fluorescence microscope, and the hybridization reaction proceeds. I confirmed.

4). Collection by light irradiation To the part on which the sample on the glass plate after the hybridization reaction is placed, use an ultraviolet (356 nanometer light) irradiation device (USHIO, 250 kW high-pressure mercury lamp-Optiplex device). Was irradiated for 20 minutes. Using a phosphate buffer (containing 20% dimethylformamide), a nucleic acid sample detached from the substrate by a linker photocleavage reaction by light irradiation was collected. After the collected sample solution was concentrated, it was confirmed that the desired recovery was achieved by detecting two kinds of fluorescent dyes (Cy3 and Cy5) in a capillary electrophoresis apparatus.

FIG. 1 is an enlarged schematic view of the substrate surface. FIG. 2 is a configuration diagram of the analysis system.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 ... Substrate, 200 ... Linker molecule, 300 ... Probe molecule, 400 ... Target molecule, 1 ... System, 11 ... Substrate receiving part, 12 ... Linker molecule fixing device, 13 ... Probe molecule fixing device, 14 ... Target molecule binding test device , 15 ... Recovery device

Claims (9)

Binding a probe molecule to which a target molecule, which is an organic molecule to be analyzed, is bound to a linker molecule that has a cleavage site that is cleaved under a specific condition and is bound to the surface of the carrier;
Cleaving the cleavage site with the target molecule bound to the probe molecule;
Recovering a target molecule bound to the probe molecule by the cleavage;
The analysis method characterized by having.   The analysis method according to claim 1, wherein the cleavage is performed by light irradiation.   The analysis method according to claim 2, wherein the irradiated light is ultraviolet light.   The analysis method according to claim 1, wherein the target molecule is labeled with a fluorescent dye. The probe molecule to which the target molecule is bound is labeled with a fluorescent dye different from the fluorescent dye labeled with the target molecule,
5. The analysis method according to claim 4, wherein after the target molecule to which the probe molecule is bound is recovered, the fluorescent dye of the target molecule and the fluorescent dye of the probe molecule are detected.   6. The analysis method according to claim 4, wherein the fluorescence intensity of the fluorescent dye is measured.   The linker molecule bound to the carrier is a plurality of types of linker molecules having different cleavage conditions. By selectively changing the cleavage conditions, the linker molecule is selectively cleaved and a desired target molecule is recovered. The analysis method according to any one of claims 1 to 6, wherein:   The linker molecule is bonded to the surface of the carrier, and then the probe molecule is bonded, then the target molecule is bonded to the probe molecule, and then the linker molecule bonded to the surface of the carrier is cleaved. The analysis method according to claim 1, wherein the target molecule is recovered.   The probe molecule to which the target molecule can bind is prepared in the state of a solution containing the probe molecule, and a mixed solution in which this solution and a solution containing a linker molecule for linking the probe molecule are mixed is prepared, and the mixing is performed. The solution is dropped on the surface of the carrier, the linker molecule to which the probe molecule is bound is bound to the surface of the carrier, and the target molecule is bound to the probe molecule bound to the linker molecule immobilized on the surface of the carrier. The analysis method according to any one of claims 1 to 7, wherein the target molecule is recovered by cleaving the linker molecule bound to the surface of the carrier.

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