KR101035957B1 - Preparation method of single-step biochip that inhibits blurring phenomenon - Google Patents

Abstract

PURPOSE: A method for manufacturing a single step bio-chip is provided to prevent non-specific adsorption. CONSTITUTION: A method for manufacturing a bio-chip comprises: a step of preparing an active surface on which a chemically functional group is exposed to a solid substrate; a step of preparing an adsorption suppression layer by covalent bond of a high molecular or low molecular material on the surface of the solid substrate; a step of performing a micro/nanospotting of a composition containing a target biomolecule and a compound which is able to cleave the covalent bond; and a step of fixing the target biomolecule to the exposed actively functaionly group.

Description

Preparation method of single-step biochip that inhibits blurring phenomenon

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for manufacturing protein chips by micro / nanospotting, and to a method for preventing the spread of bioarrays generated during the manufacturing and cleaning of biochips.

The types of biochips can be classified into DNA chips, protein chips, cell chips, and sugar chain chips according to the biomaterials on the biochips. Currently, DNA chips are the most commercialized, followed by protein chips. . In addition, the classification according to the manufacturing method or the signal detection method of the biochip is possible, but the classification according to the biomaterial is the most used.

The DNA microchip is a large-scale gene expression analysis system that can examine gene activity differences in a single experiment for all genes accumulated on a glass plate (currently capable of accumulating about 40,000). Compared to the existing methods of dot blot, DD-PCR and Northern blot, which can be used to examine a small number of changes in gene activity in a single experiment, there is a big difference in quantity and analysis speed. Various terms, DNA microarrays, biochips, DNA chips, gene-array genechips, and the like are used to refer to DNA microarray technology.

DNA microchips can be divided into two types, cDNA microarrays and Oligo-DNA chips, depending on the characteristics of genes that are densely arranged on glass solid plates. The cDNA microarray is a probe that can represent each gene on a glass plate, and a DNA fragment (PCR product) is integrated and immobilized. This is a typical DNA microarray. The Oligo-DNA chip is called a gene chip in which oligonucleotides (20 to 25 mer) are directly synthesized on a glass plate, and the gene probes are densely integrated.

DNA microchips are fabricated as follows. DNA fragments representing all genes obtained as a result of structural genomics are prepared and finally dissolved in an aqueous solution to prepare each gene in a well plate. Using an automated manufactur- er with multiple pins, the optical microscope integrates high-density DNA into the slide glass. At this time, the glass plate is coated so that the DNA adheres well. The tip of the pin is cracked like a nib, which causes capillary action to contain the aqueous DNA. The pins then approach the glass plate by an automated system and plant DNA in an orderly fashion. The DNA microarray is then completed through a series of processing steps.

The types of glass slide glass used for the production of DNA microchips are as follows.

1) Amine-coated slide glass is a form in which primary amine groups (primary NH3) are attached to the slide surface. The amine group maintains a positive charge at neutral pH (7.0). The phosphate group in the backbone of the DNA retains the negative charge (-PO ₄ ) in the same situation, so when the DNA is initially spotted on the slide, the DNA temporarily attaches to the slide through ionic bonds. UV treatment during the secondary processing leads to covalent bonds between the slide amine group and the DNA. DNA attached to the slide through the covalent bond remains stable on the slide even during processing including heat treatment or washing after the array experiment. It is one of the most used slides in the current cDNA chip fabrication.

2) Aldehyde-coated slide glass is a form in which primary aldehyde (CHO) groups are attached to the glass surface. When the aldehyde slide is used, the molecule to be attached has a primary amine group exposed to the outside or in the case of DNA, it is often used to artificially attach the primary amine group to the DNA. The bonding principle consists of a form in which free electrons of an amine group attack a carbon of an aldehyde group and form a covalent bond. Since the amine group can be easily pasted to the end of the DNA during oligonucleotide synthesis, the slide tends to be used for the production of diagnostic chips using oligonucleotides.

Protein microchips use a spotting or patterning technique to immobilize and analyze each biomolecule (capture molecule) that has a binding capacity on a pretreated surface so that the molecule can be immobilized stably. After adding a sample solution containing a target molecule, it is a device that can analyze the change of the signal that appears when combined with the biomolecule with high-throughput (high-throughput).

The development of technology related to protein chip development started in earnest from DNA hybridization patterns or DNA chips that can be used for high-throughput analysis of mRNA levels. The development of protein chips that can analyze proteins using this technology has begun. The reason for the study of protein chip along with DNA chip is that the genetic information of the gene is expressed in the protein as it is, but in many cases, the protein is synthesized through post-translational modification after the protein is synthesized. Because of the structural changes in, it is finally necessary to compare at the protein level.

Surface chemistry is a technique for stably immobilizing a protein on a solid substrate while maintaining maximum activity. The surface chemistry techniques developed so far can be classified into three categories: Absorption Methods, Covalent Bonding, and Self-Assembling Linker Molecules. There are a variety of protein chip base plates developed using these surface chemistry techniques. First, plates made of glass materials (aldehyde or amine slides, slide glass coated with poly-L-lysine, poly-L-lysine, poly-acrylamide, BC-hydroxysuccinimide, cellulose, collagen, dextranal S AM molecule) varies depending on the coating process. It can be divided into three types according to shape. It was developed as a plane-glass chip, a three-diesel pad chip, and a microwell chip. Nitrocellulose biomembrane filters, nylon or polyvinylidene difluoride polystyrene films have been developed as solid substrates for protein chips.

Biomolecules (Capture Molecules) refers to the biomaterial to be fixed on the surface-treated solid substrate. Antibodies, cell receptors, enzymes and the like can be immobilized on a solid substrate. Oligonucleotide sequence used for affibody (small protein consisting of three helix bundles), aptamer (molecular recognition) for effective immobilization of protein studies of protein immobilization using oligonucleotide sequences etc. The first thing to consider when analyzing a specific protein using protein chips is the selection of the target molecule. If you want to measure the activity of enzymes, or if you want to observe the changes in the function of the protein itself through the protein chip, the protein should be separated and purified with high purity, but eukaryotic-derived proteins may not be expressed in E. coli. Even if it doesn't work well, it doesn't always have the right structure. This can be overcome by using yeast, in vitro translation, etc. When analyzing the expression profiling of proteins, there are tens of thousands of proteins in the biological sample. Since each protein has a very large variation in protein concentration, it is important to secure biomolecules that specifically bind to target molecules.

The chip surface for immobilizing the capture molecule can be divided into two-dimensional structure (2D) and three-dimensional structure (3D) according to the structure. In general, 2D is a monomolecular membrane formed of a reactor such as an amine group, an epoxy group, an aldehyde group, an aldehyde group, and an aldehyde group together with a linker to covalently bond to an amino group of an amino acid in a protein. Alternatively, poly-L-lysine is made into a monomolecular film to bind with proteins through electrical bonding. This method generally has the advantage of immobilizing high concentrations of biomolecules on the surface, but has the disadvantage that proteins can affect the three-dimensional structure due to the rapid evaporation of water. 3D, on the other hand, has the advantage of providing sufficient space for biomolecules to bind to target molecules while maintaining a stable two-dimensional structure by attaching a reactor-bonded acrylamide gel, agarose gel or nitro cellulose membrane to the surface. Have. However, the use of such surfaces also has the disadvantage that the analytical results can be large.

Detection System refers to an efficient analysis system for protein interaction. Currently, fluorescence scanners using fluorescence are commonly used, but in the future, protein interaction studies are being conducted in the original state, and various methods for analyzing label-free proteins have been sought. For example, Surface Plasmon Resonance (SPR), Quartz Crystal Microbalance (QCM), and Atomic Force Microscopy (AFM) have been actively studied. The SPR method is well known as a Biocore product and is the most advanced method already commercialized. QCM can be applied to various fields of biotechnology as protein analysis method when multiplex analysis method is solved in the future. The AFM method is currently undergoing basic research as a protein interaction and bio-analysis system at the nano level, and is expected to be developed to promote commercialization in the future.

Application fields of protein microarray chips are as follows. 1) Can be used for Biomarker Assay. It can be applied to a disease diagnostic protein chip capable of performing multiplex analysis of biomarkers. 2) Can be used for New Drug Screening. New methods for screening new drug candidates using highly integrated protein chips can be developed. It is possible to develop a protein chip based new drug candidate screening system using protein interaction. New drug using protein-protein and protein-ligand binding tests using natural product libraries, phage display peptide libraries, chemical libraries, etc. Candidates can be derived. 3) Can be used for Differential Protein Expression Profile Study. It can be applied as a technology based on the study of protein expression patterns. By analyzing the profile of protein bodies expressed in cells or tissues using antibody chips, it will be effective in discovering new protein markers. It is expected to co-exist with the 2D gel-mass method in the future.

These various technologies work in combination to determine the performance of protein chips. At present, various methods have been tried in each field to examine the optimal conditions and the applicability of the manufactured protein chips. In particular, many studies are being conducted to find biomarkers that can be used for diagnosis of disease or to search for drug candidates, and to develop methods for analyzing protein expression profiling.

In the manufacturing process of a conventional protein chip, various polymers (PEG or dextran) or small molecule materials are used in order to prevent non-specific binding of the chip surface outside the spotted region during the washing process after the micro / nano spotting. By using this process, the active solid surface (slide glass, gold) is inactivated. In the process of inactivation of the peripheral region, a phenomenon in which an excessive amount of the target protein included in the spotted region inevitably spreads to the peripheral active region is observed. This unintentionally diffused target protein also affects the signal analysis of the target molecule to be detected, resulting in an uneven spot shape and an experimental result of signal strength. In addition, such a non-uniformity not only adds difficulty in improving the sensitivity of the biochip by forming background noise in analyzing the results, but also requires a lot of time and effort to reproduce the experiment.

Therefore, in order to solve these problems, the present inventors have made efforts to improve the existing nonspecific binding prevention method and to develop a fast result analysis method in order to perform protein chip experiments more stably. By fixing and deactivating the adsorption inhibitor layer using chemical bonds that can be easily broken by additives when micro / nanospotting the surface, the adsorption inhibitor layer is broken during micro / nanospotting to expose the active surface, A single step biochip fabrication method has been developed that forms covalent bonds between target molecular layers and substrates. The method completed the present invention by confirming that the bleeding phenomenon can be almost completely prevented compared to the existing method.

An object of the present invention is to fix the adsorption inhibitor layer that prevents non-specific adsorption of various biomolecules on the solid surface on the active surface, and then to fix the adsorption inhibitor when micro / nanospotting the target molecular layer. It provides a single step biochip manufacturing method that leaves a layer and simultaneously fixes a target molecular layer to an active site on an exposed solid surface.

In order to achieve the above object, the present invention

1) preparing an active surface exposed to a solid substrate with a chemical functional group to fix the target biomolecule;

2) preparing an adsorption inhibiting layer by covalently bonding a polymer or a low molecular weight material which inactivates the chemical functional group and suppresses nonspecific adsorption to a surface of a solid substrate;

3) Micro / Nanospotting a composition comprising a target biomolecule to be immobilized on a solid substrate surface and a compound cleaving the covalent bond; And

4) It provides a method for producing a biochip, comprising the step of decomposing the adsorption inhibitor layer by the micro / nano-spotted composition, and fixing the target biomolecule to the newly exposed active functional groups.

Compared to the conventional protein chip manufacturing method, the present invention can stably and simply obtain a result, thereby solving the problem of bleeding of the bioarray on the chip when performing the experiment using the protein chip, and shortening the time by a simple method. It can be usefully used for protein chip experiments.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for producing a biochip by a micro / nanospotting method, wherein the polymer prepares an active surface which immobilizes a biomolecule on a solid substrate and prevents nonspecific adsorption by using a chemical bond that is easily decomposed by a specific compound. By immobilizing the low molecular layer on the surface of the solid substrate first, followed by micro / nanospotting of the target protein with a chemical additive that separates the chemical bonds, thereby simultaneously deactivating the previously fixed nonspecific adsorption inhibitor layer and newly exposed active sites. It provides a method for producing a biochip, characterized in that the biomolecule is fixed to.

The manufacturing method is preferably carried out in the following steps, but is not limited thereto.

1) preparing an active surface having a chemical functional group immobilizing a target biomolecule exposed on a solid substrate;

2) preparing an adsorption inhibitor layer by covalently attaching a polymer or a low molecular material that inactivates the chemical functional group and inhibits nonspecific adsorption to a surface of a solid substrate;

3) Micro / Nanospotting a composition comprising a target biomolecule to be immobilized on a solid substrate surface and a compound cleaving the covalent bond; And

4) The adsorption inhibition layer is decomposed by the micro / nanospotted composition and the target biomolecule is immobilized on the newly exposed active functional group.

In the above method, the chemical functional group of step 1) is preferably any one selected from the group consisting of thiol, amine, alcohol, and carboxyl, and more preferably, thiol. Preferred but not limited to this.

In the method, the active surface of step 1) is amino acid, cholesterol, lipid molecule, oligosugars, oligonucleotide, glutathione, peptide, It is preferable to prepare a molecular layer having a molecular weight of 2 million or less that selectively interacts with any one molecule selected from the group consisting of protein and nitrilotricarboxylic acid, but is not limited thereto.

In the method, the polymer or low molecular weight material of step 2) is glycerol, polyethylene glycol, dextran, hyaluronic acid, chitosan, albumin protein and oligonucleic acid. It is preferably any one selected from the group consisting of, more preferably polyethylene glycol, but is not limited thereto.

In the above method, the covalent bond of step 2) is preferably any one selected from the group consisting of a thioester bond, an amide bond, an ester bond, and an ether bond. More preferably, it is a bond.

In the above method, when the covalent bond of step 2) is an ether bond, vinyl ether, aryl ether, and allyl ether, in consideration of the cleavage of the covalent bond under moderate reaction conditions It is preferable to form an (allyl ether) bond, but is not limited thereto.

In the above method, the micro / nanospotting of step 3) is performed by a pin-type microarray, a piezoelectric driving microarray, a contact or non-contact microarray using an elastomer and a patterning by a template. It is preferably performed by any one selected from the group consisting of, but is not limited thereto.

In the above method, the micro / nanospotting of step 3) is preferably performed at 10 to 50 ° C., but is not limited thereto. In addition, it is preferable that the size of the micro / nanospot is performed to have a size distribution of 10 nm to 10 mm, but is not limited thereto.

In the above method, in the case of the compound which cleaves the covalent bond of step 3), when the covalent bond is a cheester, ammonia, diaminoethan, diaminopropane having an amine functional group ) Basic compounds such as 1,2-bisdiaminoethoxyethane (1,2-bis (diaminoethoxy) ethane), hydroxylamine, etc., or acidic compounds such as hydrochloric acid are possible, but the characteristics of pH-sensitive biomolecules In view of the above, hydroxylamine is more preferred, but is not limited thereto.

In the above method, in the case of the compound which cleaves the covalent bond of step 3), when the covalent bond is an amide bond, the acid compound such as hydrochloric acid, nitric acid, sulfuric acid or acetic acid is hydrolyzed because the amide bond is hydrolyzed in an acidic or basic solution. Basic compounds such as sodium hydroxide and potassium hydroxide are possible, but biological catalysts such as proteases that selectively hydrolyze amide bonds or inorganic catalysts containing metal ions, given the properties of pH and temperature sensitive biomolecules. Preferred but not limited to this.

In the above method, when the target biomolecule of step 3) is a specific protein, and at the same time when the covalent bond of step 3) is an amide bond, the compound which cleaves the covalent bond of step 3) does not damage the protein. It is preferable to form through an α-unsaturated bond which is easily hydrolyzed in a weakly acidic aqueous solution without being limited thereto.

In the above method, when the covalent bond of step 3) is α-unsaturated amide, in the case of the compound cleaving the covalent bond of step 3), the α-unsaturated amide bond is easily hydrolyzed by a weakly acidic (pH 5.5) solution. Therefore, acidic compounds such as hydrochloric acid, nitric acid, sulfuric acid, acetic acid, etc., which can make pH 5.5, are preferred, but are not limited thereto.

In the above method, in the case of the compound which cleaves the covalent bond of step 3), when the covalent bond is an ester bond, hydrochloric acid which can make pH 5.5 because the ester bond is easily hydrolyzed in a weakly acidic or weakly basic solution, Acidic compounds such as nitric acid, sulfuric acid, acetic acid, sodium hydroxide and potassium hydroxide are preferred, but not limited thereto.

In the above method, in the case of the compound which cleaves the covalent bond of step 3), when the covalent bond is a vinyl ether or allyl ether bond, the inorganic bond or acetic acid such as hydrochloric acid, nitric acid or sulfuric acid is decomposed because the ether bond is decomposed in weak acidity. And organic acids such as formic acid, and more preferably metal catalysts such as palladium (Pd) and ruthenium (Ru) or metal oxide catalysts such as osmium oxide (OsO 4).

In the above method, the decomposition of the adsorption inhibiting layer of step 4) is characterized in that when the covalent bond of step 3) is a thioester bond, the compound having an amine functional group such as hydroxylamine is carbonyl. It is preferable that the thiol group is exposed by regeneration of the thiol group through a substitution reaction (SN2) attacking or breaking the thioester bond through hydrolysis in an acidic solution such as hydrochloric acid, but not limited thereto.

In the above method, the decomposition of the adsorption inhibiting layer of step 4) may be performed by hydrolysis in an acidic or basic solution in the case of the amide bond, or by using a biological enzyme or an inorganic catalyst that degrades the amide bond. It is preferable to decompose to regenerate the active functional group containing the amine, but is not limited thereto.

In the above method, in the decomposition of the adsorption inhibiting layer of step 4), when the covalent bond of step 3) is an ester bond, decomposition through hydrolysis in a weakly acidic or weakly basic solution is preferred, but not limited thereto.

In the above method, the decomposition of the adsorption inhibiting layer of step 4) may be performed by using hydrolysis in a weakly acidic solution when the covalent bond of step 3) is vinyl ether, allyl ether, or aryl ether bond, or palladium (Pd) Decomposition by a metal catalyst such as ruthenium (Ru) or a metal oxide catalyst such as osmium oxide (OsO 4) is preferred, but is not limited thereto.

The manufacturing method of the biochip according to the present invention is preferably performed as follows, but is not limited thereto.

In the case of active surface preparation, it is desirable to first treat a hot pyranha solution in which sulfuric acid and hydrogen peroxide are mixed to remove contaminants on the solid surface. The piranha solution effectively removes organic contaminants by oxidizing and dissolving organic matter with hydrogen peroxide and hot reaction with organic matter with sulfuric acid. Then, by dropping the MPTMS in a Pyrex container and heat treatment, the MPTMS is bonded to the solid surface, it is possible to form a structure in which the teeth are exposed on the surface. At this time, the process is carried out once in the case of the slide glass, but in the case of the gold surface it is preferable to repeat the experiment twice MPTMS.

In the case of preparation of the adsorption inhibiting layer, polyethylene glycol (PEG), dicyclohexyl-carbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) are added to the solid surface. It is preferable to prepare a solution mixed in dimethylformamide (DMF) and soak it in a Pyrex container.

In the case of micro / nano spotting, it is possible to fix and fix a capture molecule onto a surface through a microarrayer on a solid surface blocked with the PEG solution, and then react at room temperature. desirable.

At this time, the biomolecule (capture molecule) is to introduce a maleimidepropionic acid (MPA) that selectively reacts with BSA and a fluorescent material (for example, 5-carboxyfluorescein, 5-CF) and surface thiols. It is preferable to make NHS (N-hydroxysuccimide ester) which reacts with amine present on the surface of BSA (using DCC / NHS), and to prepare general amide bond by reacting BSA molecules with buffer solution. . At this time, the fluorescent molecules are preferably combined to have a concentration of about 1 to 5% compared to the BSA, MPA-NHS ester is also preferably combined at a concentration of about 1 to 5%. As a result, it is desirable to prepare 1 to 5 dyes and MPA molecules per molecule of BSA. The BAS solution thus prepared is preferably microspotted at a concentration of about 1 mg / ml.

By using the solid surface chip of the present invention, less bleeding results can be obtained than the protein chip made by the conventional method, and the procedure and time can be shortened compared to the existing experiments.

In one embodiment, the present invention provides a method for preparing an active surface, comprising the steps of: 1) preparing an active surface to expose a molten group to a solid substrate surface; 2) inactivating the entire active layer by forming a polyethylene ester modified at both ends with carboxylic acid to form a ester group, thereby preparing the active surface as an inactivation and adsorption inhibiting layer; And 3) fixing the albumin protein as a probe protein through micro / nanospotting on the totally inactivated surface (see FIG. 1).

In step 1), in order to prepare the active surface exposed to the cheol groups on the glass substrate, MPTMS (mercaptopropyl trimethoxy silane) is heated in an oven (130 ° C., 12 hours) to obtain a thiol (thiol, -SH) on the glass substrate. Fixed.

As the step 2), the cheol groups on the substrate were inactivated by two methods, and both layers were inactivated and an adsorption inhibitor layer was prepared by using carboxylated polyethylene glycol (PEG-DA). The first method is dicyclohexylcarbodiimide (DCC, 1.1 equivalents) in polyethylene glycol (PEG diacid, 1 equivalent) and 4-dimethylaminopyridine (DMAP, 0.1 equivalent), which acts as an organic catalyst. ) The sample was mixed with a DMF solvent to inactivate the thiol group through a reaction on a solid surface (reaction at 25 ° C. for 12 hours). In the second inactivation method, PEG-DA is first converted into acyl chloride at both ends of carboxylic acid using thionyl chloride (PEG-AC), and then PEG is exposed to the surface exposed to thiol. -AC (10 mM) was applied to the DMF solution to allow reaction on the surface. At this time, 0.1 equivalent of triethyl amine (TEA) was added to the surface reaction smoothly, and the reaction was performed for about 12 hours. The polyethylene glycol immobilized on the surface also prevents nonspecific adsorption of impurities such as proteins on the solid surface.

As a step 3), the albumin protein (bovine serum albumine, BSA) modified with maleimide and fluorescent dye on a glass substrate was dissolved in a buffer solution and microspotted, followed by reaction on the surface for 3 hours. Then, the results were analyzed through a fluorescence scanner.

In the present invention, by changing the surface blocking process after microspotting, which is a conventional method used to make protein chips, to a pre-spotting process, a cleaner image and a shorter time can be obtained than the conventional method (FIG. 3). Reference).

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

These examples are only for illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example 1 Synthesis of Dicarboxy Polyethylene Glycol Acyl Chloride (PEG-AC)

1 g (0.33 mmol, 1 equivalent) of dicarboxy-polyethylene glycol (PEG-DA) was dissolved in 30 ml of chloroform solution, 0.1 ml of DMF was added as a catalyst, and then stirred for 10 minutes using a stirrer. 2 ml of 1 M thionyl chloride (SOCl ₂ ) was added thereto using a pipette, followed by stirring at room temperature for 24 hours. After the reaction, the residue was evaporated using a rotary evaporator to weigh a synthetic product such as the remaining oil and react with the exposed surface of the thiol.

Example 2 Synthesis of Biomolecules for Spotting When Using a Microarrayer

10 ml (0.5 mg / ml, 10 uM) of PBS buffer solution containing a commercial BSA was prepared. 5-carboxyfluorescein (5-FC) NHS ester and 3-maleimidopropionic acid (MPA) NHS ester were prepared under a chloroform solution using the DCC / NHS method. 5 mg (5 equivalents) of 5-FC NHS ester and 10 mg (5 equivalents) of MPA NHS ester were dissolved in DMF solution and immediately added to BSA buffer solution, followed by stirring for 3 hours. The solution was used for microspotting without further separation. MPA bound to BSA is immobilized via thioether bonds in response to thiol groups exposed to micro / nanospotted regions.

Example 3 Preparation of Biochip Coated with Adsorption Inhibition Layer

<3-1> Teeth Formation on Slide Glass Surface

A piranha solution was prepared using a slide glass (76 × 26 mm / 3 × 1 inch) surface having a ratio of sulfuric acid and hydrogen peroxide of 3: 1. The slide glass was immersed in the mixed solution and treated at high temperature for 30 minutes to remove organic contaminants. Then, washed with water and completely removed with N 2 gas. The slide glass was placed in a Pyrex container and 100 µl of 95% MPTMS [(3-mercaptopropyl) trimethoxysilane] was put together in a Pyrex container and treated in a 120 ° C. oven for 16 hours.

<3-2> Blocking of polyethylene glycol (PEG-AC) on slide glass with thiol group

After putting the polyethylene glycol (PEG) solution (16.7 mM) synthesized in Example 1 into a Pyrex container, the slide glass on which thiol was formed was immersed and stirred for 12 hours.

<3-3> Biomolecule Fixation on the Slide Glass Surface

After adding hydroxylamine to a concentration of 0.05M in the albumin (bovine serum albumin (BSA)) solution synthesized in <Example 2>, the micro glasses were prepared in the slide glass prepared in Example <3-2>. The chip was fabricated using a layer (microarrayer). The immobilization reaction was allowed to react at room temperature for about 3 hours, and then washed with 1 × PBS, pH 7.4 buffer, and then ultrasonically cleaned for 10 minutes. Nitrogen (N ₂ ) gas was blown for the next step to remove condensation on the slide glass and refrigerated. The PEG molecules linked through the thiester bond through the immobilization reaction for 3 hours exposed the thiol group to the surface through the substitution reaction with hydroxylamine, and the PEG molecules were released from the surface forming an amide bond with the hydroxylamine. In this case, the higher the concentration of hydroxylamine, the faster the cleavage of the thioester bond, and the thiol groups on the exposed slide glass are partially modified with maleimide functional groups and thioethers in the BSA contained in the spotting solution. Bonding led to irreversible binding of protein molecules.

<Example 4> Smearing phenomenon of micro spotted bioarray

Comparison between the chip prepared in <Example 3> and the chip not treated with PEG solution as a control was measured with a fluorescence scanner.

As a result, as shown in Figure 2, when the PEG solution is not used, when measuring the result with a fluorescent image syringe after washing, the biomolecules spread to the surroundings outside the spot was not seen, the accurate results, the chip of the present invention In the case of the spot after washing, the shape of the spot is almost undisturbed and less blurring around it was possible to confirm more accurate results (Fig. 2).

The present invention can easily produce a biochip that prevents non-specific adsorption while fixing biomolecules through a single micro / nanospotting.

1 is a step-by-step illustration of the biochip manufacturing process of the present invention:

First step: preparing a solid surface exposed to the chemical functional group (A) for fixing a specific biomolecule;

The second step is to form a molecular layer (C) on the A layer that inhibits nonspecific adsorption of the biomolecules using a polymer or a low molecule, wherein the probe biomolecule to be fixed through micro / nano spotting And chemical bond (B) to be broken by a specific chemical compound at the same time as the chemical bond of the active functional group (A);

Step 3: micro / nanospotting a solution containing a compound for removing a molecular layer (C) for inhibiting non-specific binding with a target biomolecule to be immobilized on a biochip surface;

Step 4: A reaction occurs in which the molecular layer for inhibiting nonspecific adsorption in the micro / nanospotted region is decomposed by a compound included in the micro / nanospot, and simultaneously between the exposed active functional group (A) and the target biomolecule. Coupling is made; And,

Step 5: When the micro / nanospot is washed with the washing solution, the target biomolecule is finally fixed to the solid surface, and the remaining regions have a molecular layer that inhibits the nonspecific reaction.

2 is a view comparing the process of the conventional biochip manufacturing method and the biochip manufacturing method of the present invention.

3 is a mixture of dicarboxyl polyethylene glycol (PEG-DA, molecular weight 600), dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine (DMAP) used in the present invention, and then treated with a slide glass to inactivate it. This is the result of a photogram showing the surface of the PEG after the spotting / washing process using the albumin (MPA-BSA-F) solution containing maleimide that binds to the fluorescent dye (5-FC) and the thiol group. . On the right is a photographic result of a biochip treated with MPTMS without PEG solution as a control.

Claims (9)

1) preparing an active surface exposed to a solid substrate with a chemical functional group to fix the target biomolecule; 2) preparing an adsorption inhibitor layer by covalently attaching a polymer or a low molecular material that inactivates the chemical functional group and inhibits nonspecific adsorption to a surface of a solid substrate; 3) Micro / Nanospotting a composition comprising a target biomolecule to be immobilized on a solid substrate surface and a compound cleaving the covalent bond; And 4) A biochip manufacturing method comprising the step of decomposing the adsorption inhibiting layer by the micro / nano-spotted composition, and fixing the target biomolecule to the newly exposed active functional groups. [Claim 2] The method of claim 1, wherein the microchip / nanospotting is performed only once to prevent the non-specific adsorption reaction without externally activating the solid surface electrically or optically. The method of claim 1, wherein the chemical functional group of step 1) is a biochip, characterized in that any one selected from the group consisting of thiol, amine, alcohol, alcohol and carboxyl group (carboxyl) Way. The method of claim 1, wherein the active surface of step 1) is amino acid, cholesterol, lipid molecules, oligosugars, oligonucleotides, glutathione, peptides. A method for producing a biochip, characterized in that to produce a molecular layer of less than 2 million molecular weight selectively interacting with any one molecule selected from the group consisting of a protein (protein) and nitrilotricarboxylic acid (nitrilotricarboxylic acid). The method of claim 1, wherein the polymer or low molecular weight material of step 2) is glycerol, polyethylene glycol, dextran, hyaluronic acid, chitosan, albumin protein and oligonucleic acid. Biochip manufacturing method, characterized in that any one selected from the group consisting of. The method of claim 1, wherein the covalent bond of step 2) is any one selected from the group consisting of a thioester bond, an amide bond, an ester bond, and an ether bond. Biochip manufacturing method. The method according to claim 1, wherein the micro / nanospotting in step 3) is performed by patterning by pin-type microarrays, piezoelectric driving microarrays, contact or non-contact microarrays using an elastomer and a template. Biochip manufacturing method characterized in that carried out by any one selected from the group consisting of. The method of claim 1, wherein the micro / nanospotting of step 3) is performed at 10 to 50 ° C. 6. The method of claim 1, wherein the micro / nanospotting of step 3) has a size distribution of 10 nm to 10 mm in size of the micro / nanospot.

Images