KR20030020232A - A method of preparing a hydrogel biochip by using satar-like PEG derivatives - Google Patents

Abstract

PURPOSE: Provided is a process for producing a hydrogel biochip having 3-dimensional structure and improved sensitivity by using an epoxy-contained radial polyethylene glycol derivative. CONSTITUTION: The process comprises the steps of: reacting the radial polyethylene glycol having an epoxy group at the end thereof and a low molecular weight hydrophilic polymer to prepare a matrix, wherein the hydrophilic polymer has an average molecular weight of 60-100,000 and is a polyethylene glycol having hydroxy, amino, or thiol; covalently bonding a probe to the matrix to prepare a matrix-probe conjugation body, wherein the probe is nucleic acid(DNA, RNA, or PNA), protein, or oligopeptide; fixing the matrix-probe conjugation body to a solid substrate such as glass, quartz, silicone, plastic, polypropylene, polycarbonate, or activated acrylamide.

Description

A method of preparing a hydrogel biochip by using satar-like PEG derivatives}

The present invention relates to a method for manufacturing a biochip, and more particularly, to a method for manufacturing a biochip using a gel matrix.

With the phenomenal progress of the human genome project, there has been a great demand for a way to rapidly provide vast amounts of genetic information in the diagnosis, treatment and prevention of genetic diseases. Sanger's method, which has been used as a sequencing method, has been steadily developed due to the development and automation of a polymerase chain reaction (PCR) method that replicates DNA, and the process is cumbersome and requires a lot of time and effort. Because of the cost and high level of proficiency required, large amounts of genes could not be analyzed, and new sequencing systems were constantly being sought. As a result, many advances have been made in the last few years related to the production and use of DNA chips.

DNA chips are oligonucleotides of several, many hundreds of bases in size, with known base sequences on solid surfaces such as silicon, surface-modified glass, polypropylene, and activated polyacrylamides of less than one square inch. Microarrays are commonly referred to as probes attached to hundreds to hundreds of thousands of fixed locations. When the target DNA fragment to be analyzed is bound to the DNA chip, different hybridization states are achieved according to the complementary degree of the nucleotide sequences on the target DNA fragment and the probes attached to the DNA chip. The nucleotide sequence of the target DNA can be analyzed by observing and interpreting it through an optical method or radiochemical method.

By using a DNA chip, DNA analysis systems can be miniaturized, enabling analysis of genes with only a small amount of samples, and simultaneously identifying multiple sequences on target DNA. Can provide. In addition, DNA chips can simultaneously analyze vast amounts of genetic information in a short time, as well as identify correlations between genes.In the future, diagnosis of genetic diseases and cancer, detection of mutations, detection of pathogens, and gene expression analysis It is expected that a wide range of applications such as drug development and drug development will be made. In addition, by detecting genes for detoxification materials and applying genetic recombination technology by using them as detectors for microorganisms or environmental pollution, they can be applied to mass production of detoxification materials, medicinal crops, and low-fat meat. Could revolutionize the biotech industry.

DNA chips are divided into oligo chip and cDNA chip according to the type of probe used, and according to the fabricated method, DNA can be separated using photolithography chip, pin type spotting chip, inkjet type spotting chip, and electricity. It may also be classified as an electronic addressing DNA chip.

First-generation DNA chips are oligonucleotides attached directly onto a substrate in a single layer (2D type chip), causing significant errors in the relative comparison of hybridization bonds due to spot to spot variation between attached probes. However, the low sensitivity has the disadvantage of requiring an expensive confocal fluorescence microscope to detect the DNA hybridized on the chip, or during the surface treatment of solid substrates, In order to proceed, there was a need to use a mixture of an organic solvent and an aqueous solution.

US Pat. No. 5,744,305, which is an example of a first-generation DNA chip, uses a photolabile protecting groups and photolithography to arrange various types of peptides, oligonucleotides, and other polymers on a substrate. The contents are disclosed.

In order to improve the shortcomings of the first generation DNA chip, the following second generation DNA chip has been developed. U.S. Patent Nos. 5,736,257 and 5,847,019 form a molecular layer of vinyl groups on a substrate by reacting -OH groups with silanes on the surface of the substrate, and then polymerizing the acrylamide on the molecular layer. The present invention discloses a method of manufacturing a biochip, in which a network layer is formed, and the network layer is activated by light to be patterned to bond biomaterials. This method has the effect of reducing the quantitative fluctuation between the attached probes and improving the chip sensitivity by using the 3D gel of the polyacrylamide network formed on the surface of the molecular layer of the vinyl group, but it has the disadvantage of requiring high cost and long time. have.

US Pat. Nos. 5,552,270, 5,741,700 and 5,770,721 also describe methods for analyzing DNA sequences using second generation DNA chips comprising a matrix with an oligonucleotide array and a solid substrate. The matrix is attached to the solid substrate by gel layers in the form of numerous square "dots" spaced at regular intervals. The chip forms a polyacrylamide gel between two glass slides 30 μm apart, then removes one slide (100 × 100 × 30 μm ³ ), and dries the remaining slides coated with gel to form a gel of dots (100 × 100 × 30 μm ³ ). A portion of the gel is prepared by mechanical or laser removal. The amide groups in the gel layer are chemically modified to form reactive hydrazides, and DNA probes with N-methyluridine at the 5 'end are modified with dialdehyde and then reacted with each other. The oligonucleotide probe is immobilized in three dimensions on the substrate.

The DNA chip is manufactured by a multi-step process requiring a long time of 1 day to 2 days, requires a thorough washing process after each step, it is not easy to obtain a uniform result according to the reaction environment, the gel surface and the target DNA Irregular adsorption of the liver occurs, which has the disadvantage of increasing background noise.

WO 00/65097 and WO 00/2899 disclose three-dimensional DNA chips using hydrogels having isocyanate (NCO) groups. Hydrogel refers to a hydrophilic network polymer that swells by forming a gel in the presence of glassy or water in the dehydrated state. According to this document, isocyanate groups are involved in the polymerization of hydrogels as well as covalent bonds of hydrogels and probes. A hydrogel having an isocyanate group (for example, a polyethylene oxide having an isocyanate group or a copolymer of a polyethylene oxide having a isocyanate group and a polypropylene oxide) and a probe having an amine group at the 5 'end are mixed together in an aqueous solution to gel the probe. The probe is attached to the substrate by immobilization in three dimensions, followed by reaction with an amine group on the glass surface.

However, since isocyanate groups are hydrolyzed so rapidly that they are difficult to control, DNA chip preparation should be performed at low temperature to prevent them, and as the hydrolysis of the isocyanate groups not consumed in the covalent bond and polymerization of the probe proceeds, the polymerization is performed. Due to the increase in viscosity of the physical properties, solidification may proceed in the spotting pin, and the generation of carbon dioxide gas makes it difficult to control the spotting size.

It is a main object of the present invention to provide a hydrogel biochip with improved sensitivity and ease of manufacture.

Another object of the present invention is to provide a method for manufacturing a biochip with improved sensitivity.

1 is a photograph showing the results of hybridization using the full length of a gene that is HNF-1α normal as a target nucleic acid.

Figure 2 is a photograph showing the result of hybridization using a heterozygote in which nucleotide A was inserted immediately after the 5613 nucleotide of the HNF-1α gene exon 9 section as a target nucleic acid.

3 is a photograph showing a result of scanning fluorescence after hybridizing a biochip prepared according to Example 6 of the present invention with a target nucleic acid.

The present invention provides a method for producing a biochip comprising forming a hydrogel comprising a probe and a matrix containing a radial polyethylene glycol derivative having an epoxy group at an end thereof and a hydrophilic polymer crosslinking agent on a substrate.

The method for preparing a hydrogel biochip of the present invention may include forming a matrix of a radial polyethylene glycol derivative having an epoxy group at an end thereof and a hydrophilic polymer crosslinking agent, and preparing a matrix-probe conjugate by reacting the probe with the matrix. have. This will be described in more detail as follows.

First, the radial polyethylene glycol is reacted with an excess of epichlorohydrin and sodium hydroxide to attach an epoxy group at its end. Subsequently, a low molecular hydrophilic polymer, preferably having a hydrophilic group of a hydroxy group, an amino group or a thiol group, having a mean molecular weight of 60 to 100,000 polymers as a crosslinking agent to form a matrix, and then sharing a probe with the nucleophilic group at the terminal By binding, matrix-probe conjugates are prepared. The matrix forming process and the matrix-probe conjugate manufacturing process may be performed sequentially or simultaneously. As used herein, the term probe refers to any substance that can specifically bind to a target material, thereby enabling the prediction of the properties of the target material by analyzing the binding therebetween. Preferred are nucleic acids, proteins or oligopeptides, including DNA, RNA or PNA. The low molecular hydrophilic polymer used in the present invention may be, for example, polyethylene glycol, polyethyleneimine, polypropylene oxide, polyol and the like having an average molecular weight of 60 to 100,000. In addition, in this specification, "radial polyethylene glycol" means the polyethylene glycol derivative which has the branch branched in three or more directions, and exhibits a radial shape. Accordingly, radial polyethylene glycol derivatives having an epoxy group of the present invention can form a three-dimensional polymer matrix when crosslinked by a crosslinking agent.

In the present invention, a hydrogel biochip may be prepared by microspotting the matrix-probe conjugate on a solid substrate, preferably a solid substrate surface-treated with an amine group, to fix the probe to the substrate. The material of the solid substrate may include glass, quartz, silicon, plastic, polypropylene, polycarbonate, or activated acrylamide.

In addition, the method of manufacturing a hydrogel biochip may include forming a mixed solution comprising a radial polyethylene glycol derivative having an epoxy group at the end, a hydrophilic polymer crosslinking agent and a probe, and immobilizing the mixed solution on the substrate. .

The immobilization can be carried out, for example, at a temperature of 40 ° C. to 70 ° C. and a humidity of 60% to 30%. The immobilization conditions are preferably those which proceed at temperature and humidity conditions of 40 ° C. and 60%, 60 ° C. and 40%, 70 ° C. and 30%. The properties of the spot on the substrate formed as a result of this immobilization step may vary depending on the type of crosslinking agent, the mixing ratio, the reaction conditions such as temperature and humidity. Other details of the polyethylene derivative, the hydrophilic polymer crosslinking agent, the probe, and the substrate are as described above.

The present invention also provides a biochip manufactured by the method of manufacturing the hydrogel biochip. The biochip of the present invention includes a solid substrate, a matrix covalently bonded to the substrate, a matrix which is a reaction product of a radial polyethylene glycol having an epoxy group at its end, and a hydrophilic polymer crosslinking agent, and a probe covalently bonded to the matrix.

In addition, the present invention is to apply a sample containing a target material that binds to the probe to the hydrogel biochip prepared according to the method of manufacturing the hydrogel biochip and detecting the target material specifically bound to the probe It provides a method for analyzing the binding between the probe and the target material comprising. In order to facilitate detection, the target material is preferably a probe to which a signal inducing substance such as a fluorescent dye is attached. Detection of the binding between the probe and the target material may vary depending on the type of signaling material attached to the target material. Fluorescence detection, electrochemical detection, detection using mass change, detection using charge change, or optical properties are widely used. It may be performed by various methods such as a detection method using.

Hereinafter, the present invention will be described in more detail with reference to Examples.

These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1 Synthesis of Radial Polyethylene Glycol Derivative Having Epoxy Group

Epichlorohydrin (7.5 mL) and tetrabutylammonium bromide (0.32 g) were added to the aqueous NaOH solution (50 wt%, 2 mL), followed by stirring, followed by pentaerythritol ethoxylate (1 g). Was added slowly and stirred at room temperature for 18 hours. The reaction was confirmed to be terminated by TLC (thin layer chromatography). When the reaction was not terminated, the mixture was further stirred at 60 ° C. for 1 hour. Then, water (30 mL) was added and diluted, followed by extraction three times with methylene chloride (40 mL). The organic solution layer was washed again with saturated NaHCO ₃ (40 mL) three times and separated, anhydrous MgSO ₄ was added, dried and the solvent was removed at low pressure. The residue of epichlorohydrin was then removed by drying in vacuo for two days. The radial polyethylene glycol derivative having the epoxy group thus synthesized was confirmed by titration of an epoxy group using H NMR and 0.1 N HBr / acetic acid (glacial).

Example 2: Synthesis of Diamine Crosslinker

Penta (ethylene glycol) di-tosylate (5 g, 9.2 mmol) was dissolved in DMF (40 mL) solution, NaN ₃ (4.2 g, 64.1 mmol) and pyridine (0.5 mL) were added sequentially, and then at 140 ° C. Stir for 18 hours. The solvent was removed at low pressure, stirred with addition of water (200 mL), followed by extraction with methylene chloride (100 mL) and the organic solvent layer washed three times with brine (100 mL). Anhydrous MgSO ₄ was added to dryness, the solvent was removed at low pressure, and column chromatography (flash column chromatography, EA: nHex = 1: 2) yielded a di-azide intermediate. The intermediate was dissolved in methanol (30 mL), 10% Pd-C (0.1 equivalent) was added, and then the reduction reaction was performed for 18 hours using hydrogen gas. The catalyst was removed using a Celite pad and the pad was washed with ethanol. The filtrate and the ethanol washing solution were mixed and the solvent was removed at low pressure to obtain a diamine crosslinking agent.

Example 3: Preparation of Gel Matrix Solution Using Crosslinking Agent

(1) Preparation of Gel Matrix Solution Using Diamine Crosslinking Agent

Radial polyethylene glycol derivative (100 mg) having an epoxy group synthesized in Example 1 was added to water (4 mL) and stirred, followed by addition of the diamine crosslinking agent (5.8 mg) synthesized in Example 2 and stirred at room temperature for 18 hours. The solution was stored at 4 ° C.

(2) Preparation of Gel Matrix Solution Using PEG as Crosslinking Agent

PEG with molecular weights of 200, 400, 600, 1500 were put in an aqueous solution with 2N NaOH, respectively, and stirred in an ice bath to prepare a PEG crosslinker solution.

Then, after dissolving the radial polyethylene glycol derivative having an epoxy group synthesized in Example 1 in carbonate buffer solution (0.1 M, pH 9.1), the PEG crosslinker solution prepared above was slowly injected and stirred at room temperature for 3 hours. . After confirming that the reaction was terminated, extracted with MC (methylene chloride) and the solvent was removed at low pressure and stored at 4 ℃.

Meanwhile, when the crosslinking agent solution was prepared using PEG having a molecular weight of 100,000, and then the gel matrix solution was prepared by the above-described method, a solid product was obtained. This product was verified by gel permeation chromatography (GPC). It was confirmed that the matrix was not formed but a dimer form product having 5 to 6 epoxy groups. Therefore, it can be seen that PEG which can be used as a crosslinking agent in the present invention is PEG having a molecular weight of less than 100,000.

Example 4 Preparation of Biochips by Spotting Gel Matrix-DNA Conjugate Solutions on Chips

The oligonucleotide probe was added to the gel matrix solution prepared in Example 3 so that the equivalent ratio of the radial polyethylene glycol having a epoxy group, the crosslinking agent, and the oligonucleotide was 4: 1: 4, and stirred and left at 37 ° C. for 14 hours to form a matrix. A DNA conjugate was prepared and then used as a spotting solution.

The spotting solution was spotted onto a glass surface surfaced with amine groups and then left in a wet chamber at 37 ° C. for 4 hours. The reaction was then carried out and stored in a drier so that the glass surface amine groups in unspotted positions were negatively charged to prevent the target nucleic acid from adhering to the glass surface, i.

Example 5: Chip Test

In chip testing, the target nucleic acid is the full length of a hepatocyte nuclear factor-1 (HNF-1α) wild type gene; and the exon of the HNF-1α gene using the multiplex PCR method. ) 9 sections were obtained, and a heterozygote type mutation was made by inserting nucleotide A immediately after the 5613 nucleotide of the section (toward 3 '), and used as a target nucleic acid hybridizing with the probe. By adding Cy3-dUTP in the PCR process of the target nucleic acid, a fluorescent dye-labeled target nucleic acid was prepared.

In addition, the following oligonucleotide (SEQ ID NO: 1 to SEQ ID NO: 34) was used as a probe.

Hybridization conditions of the probe nucleic acid with the target nucleic acid were obtained by using a chip in which a target nucleic acid and a matrix-DNA conjugate were immobilized at a concentration of 20 nM dissolved in 0.1% 6SSPET (Saline Sodium phosphate EDTA buffer containing 0.1% Triton X-100) solvent at 37 ° C. After reacting for 16 hours, the chips were washed with 0.05% 6SSPET and 0.05% 3SSPET at room temperature for 5 minutes, and then air dried at room temperature for 5 minutes. In this case, scanning was performed using Exxon's GenePix 4000B model.

SEQ ID NO: tgaacttTggacttc (a probe complementary to the promoter region of the HNF-1α gene, with the center T of the probe paired with the normal 432th base of the probe, the probe bases on either side of which are located on both sides of the 432th base of the promoter Makes perfect base pair with base sequence.)

SEQ ID NO: tgaacttGggacttc (a probe complementary to a target nucleic acid caused a point mutation (A → C) at the 432th base of the normal promoter mentioned in SEQ ID NO: 1)

SEQ ID NO: 3: gttttggGggggcag (a probe complementary to the promoter region of the HNF-1α gene, with the center G of the probe paired with the normal promoter 592th base, and the probe bases on either side thereof are on both sides of the promoter 592th base) Makes perfect base pair with base sequence.)

SEQ ID NO: gttttggCggggcag (a probe complementary to the target nucleic acid resulting in a point mutation (C → G) at the 592th base of the normal promoter mentioned in SEQ ID NO: 3)

SEQ ID NO: tgcagctGgctcagt (a probe complementary to the exon 1 interval of the HNF-1α gene, the center G of which is paired with the 733th base of exon 1 that is normal, and the probe bases on both sides thereof are the 733th of exon 1) Complete base pairs with base sequences on both sides of the base.)

SEQ ID NO: tgcagctAgctcagt (a probe complementary to the target nucleic acid in which a point mutation (C → T) was induced at base 733 of normal exon 1 mentioned in SEQ ID NO: 5)

SEQ ID NO: 7 gcactggGtgagccg (a probe complementary to the exon 1 interval of the HNF-1α gene, with the center G of the probe paired with the 806th base of exon 1 that is normal, and the probe bases on both sides thereof are the 806th of exon 1) Complete base pairs with base sequences on both sides of the base.)

SEQ ID NO: gcactggAtgagccg (a probe complementary to a target nucleic acid in which a point mutation (C → T) was induced at the 806th base of normal exon 1 mentioned in SEQ ID NO: 7)

SEQ ID NO: caaggggGagtcctg (a probe complementary to the exon 1 interval of the HNF-1α gene, with the center G of the probe paired with the 856th base of normal exon 1 and the probe bases on both sides of the probe 856th Complete base pairs with base sequences on both sides of the base.)

SEQ ID NO: 10: caaggggAagtcctg (a probe complementary to a target nucleic acid with a point mutation (C → T) induced at the 856th base of normal exon 1 mentioned in SEQ ID NO: 9)

SEQ ID NO: 11 cggtcgaGgggagct (a probe complementary to the exon 1 region of the HNF-1α gene, the center G of which is paired with the 875th base of exon 1 that is normal, and the probe bases on both sides thereof are the 875th base of exon 1) Complete base pairs with base sequences on both sides of the base.)

SEQ ID NO: 12 ggcggtcGagctggc (a probe complementary to a mutated target nucleic acid resulting in deletion of 5 bases from the 875th base to the 879th base of normal exon 1 mentioned in SEQ ID NO: 11)

SEQ ID NO: 13: accatctTcgccaca (a probe complementary to the exon 2 region of the HNF-1α gene, the center T of the probe paired with the 1778th base of normal exon 2 and the probe bases on both sides of the 1778th exon 2 Complete base pairs with base sequences on both sides of the base.)

SEQ ID NO: 14 accatctCcgccaca (complementary with a target nucleic acid resulting in a point mutation (A → G) at the 1778th base of normal exon 2 referred to in SEQ ID NO: 13)

SEQ ID NO: 15 cacaacaTcccacag (a probe complementary to the exon 2 interval of the HNF-1α gene, the center T of the probe paired with the 1812th base of normal exon 2 and its probe bases on either side of the 1812th exon 2 Complete base pairs with base sequences on both sides of the base.)

SEQ ID NO: 16: cacaacaAcccacag (a probe complementary to a target nucleic acid resulting in a point mutation (A → T) at the 1812th base of normal exon 2 referred to in SEQ ID NO: 15)

SEQ ID NO: 17 gcgggccGccctgta (a probe complementary to the exon 2 region of the HNF-1α gene, the center G of which is paired with the 1910 base of exon 2 that is normal, and the probe bases on both sides thereof are 1910 th of exon 2 Complete base pairs with base sequences on both sides of the base.)

SEQ ID NO: 18 gcgggccAccctgta (a probe complementary to a target nucleic acid resulting in a point mutation (C → T) at the 1910 base of normal exon 2 referred to in SEQ ID NO: 17)

SEQ ID NO: 19 acctctcGctgcttg (a probe complementary to the exon 2 interval of the HNF-1α gene, the center G of which is paired with the 1940th base of exon 2 that is normal, and the probe bases on both sides thereof are 1940th of exon 2 Complete base pairs with base sequences on both sides of the base.)

SEQ ID NO: 20 acctctcActgcttg (a probe complementary to a target nucleic acid with a point mutation (C → T) induced at the 1940th base of normal exon 2 mentioned in SEQ ID NO: 19)

SEQ ID NO: 21 aaggggcGgaggaac (a probe complementary to the exon 3 region of the HNF-1α gene, the center G of which is paired with the 2659th base of exon 3 that is normal, and the probe bases on both sides thereof are 2659th of exon 3 Complete base pairs with base sequences on both sides of the base.)

SEQ ID NO: 22 aaggggcAgaggaac (complementary probe with target nucleic acid induced point mutation (C → T) at base 2659 of normal exon 3 referred to in SEQ ID NO: 21)

SEQ ID NO: 23 gaggaacCgtttcaa (a probe complementary to the exon 3 region of the HNF-1α gene, the center C of the probe paired with the 2667 th base of normal exon 3 and the probe bases on both sides thereof were 2667 th Complete base pairs with base sequences on both sides of the base.)

SEQ ID NO: 24 gaggaacTgtttcaa (a probe complementary to a target nucleic acid with a point mutation (G → A) induced at the 2667th base of normal exon 3 referred to in SEQ ID NO: 23)

SEQ ID NO: 25 cacctccGtgacgag (a probe complementary to the exon 4 region of the HNF-1α gene, the center G of which is paired with the 3297th base of normal exon 4 and the probe base on both sides thereof is the 3297th exon 4 Complete base pairs with base sequences on both sides of the base.)

SEQ ID NO: 26: cacctccAtgacgag (a probe complementary to a target nucleic acid resulting in a point mutation (C → T) at the 3297th base of normal exon 4 mentioned in SEQ ID NO: 25)

SEQ ID NO: 27 tagacacGcacctcc (a probe complementary to the exon 4 region of the HNF-1α gene, the center G of which is paired with the 3305th base of normal exon 4 and the probe bases on both sides thereof are 3305th of exon 4 Complete base pairs with base sequences on both sides of the base.)

SEQ ID NO: 28 tagacacAcacctcc (a probe complementary to a target nucleic acid with a point mutation (C → T) induced at the 3305th base of normal exon 4 mentioned in SEQ ID NO: 27)

SEQ ID NO: 29: caaccggCgcaaaga (a probe complementary to the exon 4 interval of the HNF-1α gene, the center C of the probe paired with the 3332th base of normal exon 4 and the probe bases on both sides of the 3332th of exon 4 Complete base pairs with base sequences on both sides of the base.)

SEQ ID NO: 30: caaccggTgcaaaga (a probe complementary to a target nucleic acid with a point mutation (G → A) induced at the 3332rd base of normal exon 4 mentioned in SEQ ID NO: 29)

SEQ ID NO: 31 cctgagaTgccggcg (a probe complementary to the exon 9 section of the HNF-1α gene, the center T of the probe paired with the 5613 base of normal exon 9 and the probe bases on both sides thereof are 5613 second of exon 9 Complete base pairs with base sequences on both sides of the base.)

SEQ ID NO: 32 cctgagaTtgccggc (a probe complementary to a target nucleic acid with a mutation in which the A base is inserted immediately after the 5613 base of normal exon 9 mentioned in SEQ ID NO: 31)

SEQ ID NO: 33 tggcgctGagccggt (a probe complementary to the exon 9 segment of the HNF-1α gene, with the center G of the probe paired with the 5688th base of normal exon 9 and the probe sequence on both sides thereof is 5688th exon 9 Complete base pairs with base sequences on both sides of the base.)

SEQ ID NO: 34: tggcgctGagagccg (a probe complementary to a target nucleic acid with a mutation in which a TC base is inserted immediately after the 5688th base of normal exon 9 mentioned in SEQ ID NO: 33)

Scanning results after the hybridization reaction between the probe and the target nucleic acid are shown in FIGS. 1 and 2. FIG. 1 illustrates the case where the full length of a gene that is HNF-1α is used as a target nucleic acid, and FIG. 2 illustrates a case where a heterozygote having nucleotide A inserted immediately after the 5613 nucleotide of the HNF-1α gene exon 9 section is used as a target nucleic acid.

1 and 2, the same probe was spotted by 6 spots, and the probes of SEQ ID NOs 1, 2, 3, and 4 were immobilized spots from the upper left to the right, and the following lines 5, 6, 7, 8 In this way, the probe of SEQ ID NO: 34 is spotted on the chip in this manner. Spot size was 170 ± 5 μm, and the spacing between spots was adjusted to 375 μm.

The spot-to-spot variation of each spot group was 8% for the same probe group, and the coefficient of variation (CV) was 8%, and the chip-to-chip variation was 10.6. It was%. This proves that the three-dimensional immobilization technique can reduce the error between probes when compared with the 25-30% spot variance value observed in the existing 2D type chips. In the above, the variance represents the standard deviation / percent of the mean value.

As shown in FIG. 1, probes without mutation (ie, probes of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33) ), The mutagenized probes (probe of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34) Stronger fluorescence could be observed in the immobilized spot. Therefore, it was found that the target nucleic acid of the full-length gene, which is normal of HNF-1α, forms a perfect base pair with the probe without mutation.

As shown in FIG. 2, probes without mutation (ie, probes of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33) The same result as in FIG. 1 is shown in the spot where immobilized), whereas in the spot where the probe of SEQ ID NO: 32 is immobilized among the mutagenized probes, it was found that fluorescence is strongly different from the result of FIG. Therefore, a mutation in which the nucleotide A is inserted immediately after the 5613 nucleotide of the HNF-1α gene exon 9 segment can be easily detected.

Example 6 Biochip Preparation by Direct Production of Matrix on Substrate

The matrix of the present invention can be prepared in a container separate from the substrate, as well as directly on the substrate.

Oligonucleotide probe (5′-tgttctcttgtcttg-3 ′: SEQ ID NO: 35) in a carbonate buffer (0.25 mM, pH 9.5), a radial polyethylene glycol derivative having a terminal epoxy group synthesized in Example 1, a diamond prepared in Example 2 The min crosslinker was mixed at a ratio of 1: 8: 8. At this time, the concentration of the oligonucleotide was not to exceed 400μM. Next, the same volume of DMSO was mixed with the mixed solution, and then spotted on the glass surface surface-treated with an amine group. After spotting, the chips were placed in an oven controlled at a temperature of 60 ° C. and 40% humidity, incubated for 1 hour and then post-treated with succinic anhydride. This post-treatment prevents non-specific reactions of the amine groups on the surface of the glass substrate that do not participate in the reaction, the epoxy groups of the radial polyethylene derivatives having expo groups, and the amine groups of the probes.

The scanning result of hybridization of the probe and the nucleic acid on the biochip obtained above is shown in FIG. 3. The target nucleic acid used at this time was a synthetic oligonucleotide complementary to the probe. In FIG. 3, C is a result of the biochip manufactured according to the present embodiment, B is a result of the biochip manufactured by the method of Example 4, and A is a biochip prepared by spotting only a DNA solution on a glass substrate. Results are shown. As can be seen in Figure 3, the fluorescence intensity of C increased by 5 to 7 times than that of B, the fluorescence intensity of A increased by 2-3 times than that of B.

As described above, according to the present invention, it is possible to produce a biochip having a three-dimensional structure by a shape protruding spatially from the surface, which not only increases the sensitivity of the chip, but also provides a low cost and simple and efficient A chip can be manufactured in aqueous solution by a process.

Claims (11)

A method of manufacturing a biochip comprising forming a hydrogel comprising a probe and a matrix containing a radial polyethylene glycol derivative having an epoxy group at an end thereof and a hydrophilic polymer crosslinking agent on a substrate. The hydrogel biochip of claim 1, further comprising forming a matrix of the radial polyethylene glycol derivative having an epoxy group at the terminal and the hydrophilic polymer crosslinker and reacting the probe with the matrix to prepare a matrix-probe conjugate. Manufacturing method. The preparation of a hydrogel biochip according to claim 1, comprising forming a mixed solution comprising a radial polyethylene glycol derivative having an epoxy group at its end, a hydrophilic polymer crosslinking agent and a probe, and immobilizing the mixed solution on the substrate. Way. The method of claim 3, wherein the immobilization temperature is 40 ° C. to 70 ° C., and the humidity is 60% to 30%. The method according to any one of claims 1 to 4, wherein the hydrophilic polymer crosslinking agent is polyethylene glycol having an average molecular weight of 60 to 100,00 and having a hydroxy group, an amino group or a thiol group. 5. The method of claim 1, wherein the probe is a nucleic acid (DNA, RNA or PNA), protein or oligopeptide. 6. 5. The method of claim 1, wherein the substrate comprises a surface layer having an amine group. 6. The method of claim 1, wherein the substrate is made of glass, quartz, silicon, plastic, polypropylene, polycarbonate, or activated acrylamide. A hydrogel biochip manufactured by the method of claim 1. A method comprising applying a sample comprising a target substance to bind a probe to a hydrogel biochip prepared according to any one of claims 1 to 4 and detecting the target substance specifically bound to the probe. Analyzing the binding between the probe and the target material. The method of claim 10, wherein the target material is a biomaterial to which a fluorescent material is attached.