KR20080114396A - Method for immobilzing biomolecules on substrate using gold nanoparticles - Google Patents

Abstract

A method for fixating a biomass on a substrate using gold nanoparticle is provided to increase an immobilization capacity of the biomass compared with an existing method by uniting gold nanoparticles with many biomasses by a three dimensional structure and be uniformly immobilized and steadily formed compared with the existing covalent bond due to bond by the adsorption with the gold nanoparticles and the substrate. A method for fixating a biomass on a substrate using gold nanoparticles includes steps of: (a) manufacturing gold nanoparticles-biomass composite by mixing the gold nanoparticles and the biomass; and (b) fixing the substrate of the gold nanoparticles-biomass composite through adsorption of the substrate and the gold nanoparticles by spotting the composite on the substrate.

Description

Method for Immobilizing Biomolecules on Substrate Using Gold Nanoparticles}

1 is a schematic diagram showing a method of preparing a complex of gold nanoparticles and amine DNA according to the present invention and immobilizing the complex on a glass substrate.

2 shows a state in which a complex of gold nanoparticles according to the present invention and DNA having both ends substituted with Cy5 and amine, respectively, is immobilized on an aldehyde glass substrate and an amine glass substrate.

FIG. 3 shows photographs of scanning results of hybridizing target DNA 1 and target DNA 2 on gold nanoparticle-DNA chips prepared in Example 3. FIG.

Figure 4 shows a photograph of the immobilized state through the amplification of silver nanoparticles for the defective spots and good spots of the gold nanoparticles-DNA chip prepared in Example 3.

5 is a graph comparing immobilization ratio (a) and hybridization ratio (b) when immobilizing biomaterials without using gold nanoparticles and when immobilizing biomaterials using gold nanoparticles.

Field of invention

The present invention relates to a method of immobilizing a biomaterial on a substrate using gold nanoparticles, and more specifically, (a) preparing a gold nanoparticle-bio material complex by mixing gold nanoparticles and a biomaterial; (b) spotting the complex on a substrate, thereby immobilizing the gold nanoparticle-bio material complex on the substrate through adsorption of the gold nanoparticles with the substrate. A method of immobilizing a substance on a substrate.

Background of the Invention

Biochips collectively refer to micro-integration by attaching biomaterials such as DNA and proteins at high density to a predetermined position on various types of solid surfaces including polymer substrates such as surface modified glass, silicon, and polypropylene. Such a biochip can be applied to various fields because it can analyze many samples simultaneously in a short time.

Biochip's performance, which is rapidly expanding its applicability in recent years, can be classified into three specificities, sensitivity, and reproducibility. Specifically, first, the biochip must be able to accurately analyze the target material to be tested. In other words, it must have a high specificity that can detect only the target substance to distinguish it from many other substances present in the sample. Second, since the target sample of the biochip is a human gene or protein extraction solution, it should be sensitive enough to detect even a very low concentration of the target substance. Third, biochip products must have reproducibility that always shows the same and consistent performance regardless of the time of manufacture or the manufacturer.

One of the major influences on the performance of biochips including these specificities, sensitivity and reproducibility is the immobilization method. In the case of DNA chips, various immobilization techniques have been developed. Representative techniques such as dendrimer and pseudo-3D structures have been developed to increase immobilization density and hybridization efficiency. Immobilization techniques that have been developed in the past are mostly based on chemical synthesis methods, and therefore require special synthesis techniques, complicated procedures, and usually require a lot of time.

On the other hand, another big issue for DNA chips is the development of a technology that can easily check the immobilization state on the chip. The existing test methods can be largely divided into the following two methods. First, there is a method of randomly sampling a part of a DNA chip, reacting with an actual target gene, and analyzing the reaction result by fluorescence to infer and evaluate the immobilization state. Another method infers the immobilization state of the capture DNA in the immobilization solution by mixing the immobilization solution with a fluorescent dye irrelevant to the DNA reaction and performing immobilization and examining the immobilization state of the fluorescent dye. How to evaluate. However, the method of immobilized inspection by sampling has a disadvantage in that it is expensive and takes a long time to perform a hybridization reaction because it uses a target sample labeled with actual fluorescence. There is a disadvantage in that mold and dye costs are required and additional work such as fluorescent scanning is required.

Recently, a method of immobilizing DNA whose ends are modified with SH with gold nanoparticles has been disclosed (C. Minard-Basquin et al. , IEE Proc.-Nanobiotechnol , 97: 103, 2005). However, this method takes a relatively long time of 3 to 4 days to modify the DNA to SH, and has the disadvantage that the DNA can be immobilized only on a limited substrate such as a silicon substrate.

Therefore, in the art, in order to solve the above problems, a method of easily fixing a biomaterial on a substrate, a biochip manufactured by using the method, and a biochip that can easily perform the immobilization state of the biomaterial in a short time. There is an urgent need for a method for measuring the immobilization status of biomaterials.

Thus, the present inventors have made efforts to improve the problems of the prior art, as a result of fixing the biomaterial on the substrate by adsorption using gold nanoparticles, the biomaterial can be easily immobilized on the substrate, manufactured through the immobilization The present invention was completed by confirming that the degree of immobilization of biomaterials in the prepared biochip can be measured simply and in a short time using amplification of silver nanoparticles.

An object of the present invention is to provide a method for immobilizing a biomaterial on a substrate using gold nanoparticles, a biochip manufactured by the method and a method for measuring a biomaterial immobilization state of the biochip.

In order to achieve the above object, the present invention comprises the steps of (a) preparing a gold nanoparticle-bio material composite by mixing gold nanoparticles and biomaterials; And (b) spotting the composite onto the substrate to secure the gold nanoparticle-bio material composite to the substrate through adsorption of the gold nanoparticles with the substrate. A method of immobilizing a biomaterial on a substrate is provided.

In the present invention, the bio-material may be selected from the group consisting of DNA, RNA, PNA and protein, the DNA or RNA may be characterized in that the amine group is introduced.

In the present invention, the substrate may be characterized by being modified with an aldehyde group or an amine group, the bio-material may be further fixed by a covalent bond with the substrate modified with the aldehyde group, the amine group The biomaterial may be further secured by crosslinking with the modified substrate.

The present invention provides a biochip manufactured by the above method, wherein the gold nanoparticle-bio material complex is fixed to the substrate by adsorption of the gold nanoparticles and the substrate.

The present invention is also prepared by the above method, the gold nanoparticles and the biomaterial complex is fixed to the substrate by adsorption of the gold nanoparticles and the substrate, and at the same time characterized in that the biomaterial is covalently bonded to the substrate modified with aldehyde group To provide a biochip.

The present invention is also prepared by the above method, the gold nanoparticles-biomaterial complex is fixed to the substrate by adsorption of the gold nanoparticles and the substrate, at the same time characterized in that the biomaterial is cross-linked to the substrate modified with an amine group To provide a biochip.

The present invention comprises the steps of (a) mixing a solution containing silver ions and a solution for reducing silver ions to prepare a mixed solution for amplifying silver nanoparticles, (b) reacting the biochip and the mixed solution of step (a) Amplifying the silver nanoparticles, and (c) determining the immobilization state of the biomaterial by measuring the nanoparticle amplification result.

In the present invention, the solution containing the silver ions may be a silver acetate solution, the solution for reducing the silver ions may be characterized in that the hydroquinone solution (hydroquinone) solution.

The present invention provides a method for detecting a hybridization reaction, wherein a gold nanoparticle-amine group-introduced DNA complex uses a DNA chip in which gold nanoparticles are immobilized on a substrate by using a linker.

In the present invention, the DNA chip may be characterized in that the amine group introduced DNA is further covalently bonded to the substrate modified with aldehyde group, the DNA chip is further added to the amine group introduced DNA on the substrate modified with the amine group Characterized by being crosslinked

Hereinafter, the present invention will be described in detail.

The present invention is characterized in using gold nanoparticles when immobilizing a biomaterial onto a substrate.

The gold nanoparticles of the present invention can provide a large surface area to which biomaterials can bind due to their three-dimensional structure, thereby allowing the biomaterials to be immobilized on gold nanoparticles at high density. In addition, since the gold nanoparticles are easily bonded onto the substrate by adsorption, the biomaterial is easily immobilized on the substrate (FIG. 1), and the immobilization state of the biochip according to the present invention can be measured through amplification of silver nanoparticles. .

In the present invention, in order to immobilize a biomaterial on a substrate using gold nanoparticles, first, a complex of gold nanoparticles and a biomaterial (hereinafter, referred to as a gold nanoparticle-biomaterial composite) is provided. The gold nanoparticle-biomaterial composite is simply generated by simply mixing gold nanoparticles with a biomaterial and reacting at room temperature. This is basically because the gold nanoparticles have a negative charge on the surface thereof and are positively charged by electrostatic force. This is because it has a characteristic of easily binding with biomaterials having a part of.

In one embodiment according to the present invention, the biomaterial may be DNA, the substrate may be a glass substrate.

In one embodiment of the present invention, the DNA nanoparticles bound to the gold nanoparticles are easily immobilized on the glass substrate by using the properties of the gold nanoparticles adsorbed onto the aldehyde-modified glass substrate and the amine-modified glass substrate modified with amine. You can.

In the present invention, the gold nanoparticles are adsorbed on the surface of the glass substrate, and at the same time, a part of the DNA bound to the gold nanoparticles forms covalent bonds and crosslinks on the aldehyde glass substrate and the amine glass substrate, respectively. The whole particle-DNA complex may be immobilized on the aldehyde glass substrate and the amine glass substrate.

In general, when using a DNA that does not contain an amine group at the end, the base portion of the DNA can bind to the surface of the gold nanoparticles by electrostatic force, but the base portion of the immobilized DNA hybridizes with the target DNA When the immobilized DNA is detached from the surface of the gold nanoparticles. In order to prevent such desorption of DNA, an amine group was introduced at the end of the DNA chain to bind with the gold nanoparticles. In this case, since the amine introduced at the end of the DNA chain is combined with the gold nanoparticles, even if the base portion of the DNA immobilized on the glass substrate is involved in the hybridization reaction, the immobilized DNA may remain intact on the glass substrate (Li, H. et al. , Proc Natl Acad Sci US A. , 14036: 14039, 2005).

Therefore, when the immobilization method using the covalent bond is used, some DNA may bond with the gold nanoparticles through the base portion, and at the same time, an amine group introduced at the end of the DNA chain may form a covalent bond with the aldehyde on the glass surface.

On the other hand, DNA and RNA that can be used as a biomaterial in the present invention is a polymer having genetic information as a nucleic acid, and has a common point that is composed of a number of units called nucleotides. However, DNA and RNA have a different kind of sugar contained in nucleotides, and DNA has a difference in that it is a double-stranded structure composed of two strands, unlike RNA, but the RNA is applied to a process of immobilizing DNA as a biomaterial of the present invention. It will be apparent to those skilled in the art.

In another embodiment according to the present invention, the biomaterial may be a protein and PNA, the substrate may be a glass substrate.

Since proteins and PNAs have amine groups by themselves, a complex of gold nanoparticles and a protein can be immobilized on an aldehyde glass substrate and an amine glass substrate without the need to introduce an amine group as described above. have.

On the other hand, in the biochip manufactured using the immobilization method according to the present invention, it is possible to provide a method for measuring a biomaterial immobilization state of a biochip capable of inspecting a state in which the biomaterial is immobilized on a substrate in the biochip.

According to the method for measuring the biomaterial immobilization state of the biochip of the present invention, when immobilization of the biomaterial using the gold nanoparticle-biomaterial complex, by performing amplification of silver nanoparticles around the immobilized gold nanoparticles, The immobilization state of the biomaterial can be easily checked. In addition, it is possible to examine the immobilization state of biomaterials within about 20 minutes, which is a significantly shorter time compared to the conventional method for measuring biomaterial immobilization state of biochips through a target DNA hybridization reaction which takes about 4-6 hours. . In addition, compared with conventional fluorescence analysis methods that require expensive fluorescence scanners, the amplified silver nanoparticles can be inspected through an optical microscope or the naked eye. would.

Meanwhile, in the conventional biochip immobilization method, a buffer containing a salt is used to improve the orientation of the immobilized biomaterial. In this case, a salt spot is crystallized, and thus a bad spot is likely to occur. On the other hand, immobilizing the biomaterial using gold nanoparticles according to the present invention, not only does not use a buffer but also has the effect of immobilizing the biomaterial uniformly and stably by using nanoscale particles. Can be represented.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

In the following examples, DNA was used as a biomaterial, but various biomaterials such as RNA, PNA, and protein may be used without limitation.

In addition, in the following examples, although a glass substrate is used as the substrate, various kinds of substrates for manufacturing a biochip may be used without limitation.

In addition, in the following examples, a spot method using a Microarray Spotter (Versar ChipWriterTM Compact system, Bio-Rad) was used as the spot method, but various methods for spotting a biomaterial onto a substrate may also be used. It will be included in the scope of.

Example 1 Preparation of Gold Nanoparticles

Gold nanoparticles were prepared by reducing HAuCl ₄ using sodium citrate (Mucic, RC et al. , J. Am. Chem. Soc. , 12674: 12675, 1998). As a result of measuring the size and concentration of the prepared gold nanoparticles using TEM and UV values, it was confirmed that the gold nanoparticles had a size of 13 ± 2 nm and had a concentration of 6.3 nM.

Example 2 Preparation of Gold Nanoparticle-DNA Chips

Step 1 Preparation of Gold Nanoparticle-DNA Complex

The gold nanoparticles prepared in Example 1 and DNA 1 and DNA 2 (Table 1) labeled at both ends of SEQ ID NOs: 1 and 2, respectively, were labeled with Cy5 and amine (NH ₂ ), respectively, and then 12 hours. And the gold nanoparticle-DNA 1 complex and the gold nanoparticle-DNA 2 complex were prepared.

DNA probe Nucleotide sequence of the probe (5'-3 ') DNA 1 Cy5-TCCAGCTACCAGAAAAAAAAAA-NH ₂ (SEQ ID NO: 1) DNA 2 Cy5-GCTACCAGTCCAAAAAAAAAAA-NH ₂ (SEQ ID NO: 2)

<Step 2> Biochip production by immobilization on aldehyde-modified substrate

The gold nanoparticle-DNA 1 complex and the gold nanoparticle-DNA 2 complex prepared in step 1 were mounted on a microarray spotter (Microarray Spotter: VersArray ChipWriterTM Compact system, Bio-Rad), on the surface of the aldehyde-modified glass substrate. After spotting (FIG. 2 (a)), the gold nanoparticle-DNA 1 complex and the gold nanoparticle-DNA 2 complex were immobilized on a glass substrate by reacting for 12 hours in a place where light was blocked. The glass substrates on which the gold nanoparticle-DNA 1 complex and the gold nanoparticle-DNA 2 complex are fixed are sequentially washed with a sodium dodecyl sulfate (SDS) solution and distilled water, respectively, followed by NaBH ₄ (Sodium borohydride). Covalent bonds between the nanoparticle-DNA 1 complex and the gold nanoparticle-DNA 2 complex with the glass substrate were induced. Then washed with SDS and distilled water sequentially and dried to confirm the gold nanoparticles-DNA chip with a scanner (Fig. 2 (b)).

<Step 3> Biochip production using immobilization on amine-modified substrate

The gold nanoparticle-DNA 1 complex and the gold nanoparticle-DNA 2 complex prepared in step 1 were mounted on a microarray spotter (Microarray Spotter: VersArray ChipWriterTM Compact system, Bio-Rad), and the amine-modified glass substrate was used. After spotting on the surface ((c) of FIG. 2), the gold nanoparticle-DNA 1 complex and the gold nanoparticle-DNA 2 complex were immobilized on a glass substrate by reacting for 12 hours in a place where light was blocked. The glass substrates on which the gold nanoparticle-DNA 1 complex and the gold nanoparticle-DNA 2 complex were fixed were crosslinked using a UV cross linker, washed sequentially with SDS solution and distilled water, and then dried to dry the gold nanoparticle- DNA chip was confirmed by a scanner (Fig. 2 (d)).

Example 3 Specific Hybridization Reaction of Gold Nanoparticle-DNA Chips

<Step 1> Preparation of gold nanoparticle-DNA chip for measuring specificity of hybridization reaction

DNA probe name Nucleotide sequence of the probe (5'-3 ') DNA 1 ' NH2-AAAAAAAAAAGACCATCGACCT (SEQ ID NO: 3) DNA 2 ' NH2-AAAAAAAAAAACCTGACCATCG (SEQ ID NO: 4) Destination DNA 1 Cy3-AGGTCGATGGTC (SEQ ID NO: 5) Destination DNA 2 Cy3-CGATGGTCAGGT (SEQ ID NO: 6)

To measure the specificity of the hybridization reaction, DNA 1 'and DNA 2' (Table 2) having amine groups introduced in SEQ ID NOs: 3 and 4, respectively, were reacted with gold nanoparticles for about 12 hours, and then gold nanoparticle-DNA 1 'Complex and gold nanoparticle-DNA 2' complexes were prepared. Thereafter, the prepared gold nanoparticle-DNA 1 'complex and gold nanoparticle-DNA 2' complex were mounted on a microarray spotter (Microarray Spotter: VersArray ChipWriterTM Compact system, Bio-Rad), and the aldehyde-modified glass substrate was used. After spotting on the surface (Fig. 3 (a)), and reacted for 12 hours in a place where the light is blocked by the gold nanoparticle-DNA 1 'complex and gold nanoparticle-DNA 2' complex on the aldehyde glass substrate Immobilized. The glass substrates on which the gold nanoparticle-DNA 1 'complex and the gold nanoparticle-DNA 2' complex were immobilized were sequentially washed with SDS solution and distilled water, and then covalently bonded with NaBH ₄ . Then, each was sequentially washed with SDS and distilled water, followed by drying to prepare a gold nanoparticle-DNA chip.

Step 2 Measurement of Specific Hybridization Reaction

Dilute Target DNA 1 and Target DNA 2 (Table 2) labeled Cy3 in SEQ ID NOs: 5 and 6, respectively, to 100 nM concentration, and then add 30 μl of these to the biochip prepared in Step 1 and hybridize with the biochip. The reaction was measured. After reacting at 37 ° C. for 3 hours, the biochip was washed with washing buffer (0.005% Triton X-100 and 6X SSPE (0.9 M NaCl, 60 mM NaH ₂ PO 4, 6 mM EDTA, pH 7.4)) for 10 minutes, and distilled water. Washed for 10 minutes and then dried.

Afterwards, the degree of hybridization and specificity were confirmed by a scanner. As a result, the target DNA 1 (objective) reacted only to DNA 1 'accurately and did not bind to DNA 2' at all (FIG. 3 (b)). It specifically reacted with DNA 2 ′ (FIG. 3 (c)). As a result, it was confirmed that the target DNA specifically reacted only with the DNA of the gold nanoparticle-DNA complex according to the nucleotide sequence and did not bind nonspecifically to the gold particle or the glass surface.

Example 4 Inspection of Biomaterial Immobilization of Biochips

<Step 1> Biochip manufacturing for biomaterial immobilization

     Two biochips having poor spots and good spots were prepared among the biochips prepared in Step 1 of Example 3 (FIG. 4A).

<Step 2> Measurement of Immobilization Status by Silver Nanoparticle Amplification

After preparing a mixed solution of solution A (silver salt, Sigma, USA) and B (hydroquinone initiator, Sigma, USA) for amplifying silver nanoparticles, poor spots and good spots in the biochip prepared in step 1 of Example 3 The two biochips and the mixed solution were each reacted for 15 minutes at room temperature. As a result, it was confirmed that the results of silver nanoparticle amplification did not come out properly in the case of defective spots (FIG. 4B), and that of the nanoparticles amplification result in the case of good spots (FIG. 4C). .

Comparative Example 1: Comparison of DNA Immobilization Ratio with or Without Gold Nanoparticles

Samples prepared by mixing DNA 1, immobilization buffer, and DMSO (dimethyl sulfoxide) in Table 1, each of which is substituted with amine and Cy5 at both ends, and the gold nanoparticle-DNA 1 complex of Example 2 were prepared. Each of the nanoparticle-DNA 1 complexes was spotted on an aldehyde glass substrate using a microarray spotter and then reacted for 12 hours. The glass substrates to which the sample and the gold nanoparticle-DNA complex were respectively spotted were sequentially washed with sodium dodecyl sulfate (SDS) solution and distilled water, and then covalently bonded with NaBH ₄ . After washing with SDS and distilled water sequentially and dried to prepare a DNA chip and gold nanoparticle-DNA chip, the degree of immobilization of the DNA chip and the gold nanoparticle-DNA chip with a fluorescence scanner to measure the fluorescence of C.y5 Measured by intensity.

As a result of comparing the DNA immobilization ratio between the DNA chip and the gold nanoparticle-DNA chip, the immobilization ratio of the gold nanoparticle-DNA chip immobilized with the DNA using the gold nanoparticles was high, and the gold nanoparticle of the three-dimensional structure was found. It can be seen that due to the binding of DNA to the particle surface, a larger amount of DNA is immobilized (FIG. 5A).

Comparative Example 2: Comparison of Hybridization Ratio with and Without Gold Nanoparticles

After preparing a sample in which DNA 1 'of Table 2, an immobilization buffer, and DMSO mixed with an amine at one end and gold nanoparticle-DNA 1' of <Step 1> of Example 3 were prepared, the sample and the gold nanoparticles- Each DNA 1 ′ complex was spotted on an aldehyde glass substrate using a microarray spotter, and then reacted for 12 hours. The glass substrate onto which the sample and the gold nanoparticle-DNA 1 ′ complex were respectively spotted was sequentially washed with SDS (Sodium dodecyl sulfate) solution and distilled water, and then covalently bonded with NaBH ₄ . Then, washed sequentially with SDS and distilled water and dried to prepare a DNA chip and gold nanoparticle-DNA chip, and then hybridization reaction was measured.

After diluting Cy3 fluorescently labeled target DNA 1 to a concentration of 100 nM, 30 μl of this was reacted with the prepared DNA chip and gold nanoparticle-DNA chip at 37 ° C. for 3 hours, and then the DNA chip and gold nanoparticle- The DNA chip was washed with washing buffer (0.005% Triton X-100 and 6X SSPE (0.9 M NaCl, 60 mM NaH 2 PO 4, 6 mM EDTA, pH 7.4)) for 10 minutes, washed with distilled water for 10 minutes, and dried.

As a result of comparing the DNA hybridization ratio of the DNA chip and the gold nanoparticle-DNA chip, the hybridization ratio of the gold nanoparticle-DNA chip immobilized with the DNA using the gold nanoparticles was high (FIG. 5B). .

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

As described above, according to the present invention, due to the three-dimensional structure, the gold nanoparticles can be combined with more biomaterials to increase the immobilization capacity of the biomaterials compared to the conventional method, and the gold nanoparticles with the substrate Due to the bonding by adsorption, there is an effect that the immobilization is formed uniformly and stably compared to the existing covalent bond. In addition, the immobilized state of the biochip manufactured according to the present invention can be easily examined within a short time by amplification of silver nanoparticles.

<110> Korea Advanced Institute of Science and Technology <120> Method for Immobilizing Bio-molecules on Substrate Using Gold          Nanoparticles <130> P07-B134 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 1 tccagctacc agaaaaaaaa aa 22 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 2 gctaccagtc caaaaaaaaa aa 22 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 3 aaaaaaaaaa gaccatcgac ct 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 4 aaaaaaaaaa acctgaccat cg 22 <210> 5 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 5 aggtcgatgg tc 12 <210> 6 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 6 cgatggtcag gt 12  

Claims (16)

A method of immobilizing a biomaterial onto a substrate using gold nanoparticles, comprising the following steps: (a) mixing gold nanoparticles with a biomaterial to produce a gold nanoparticle-biomaterial composite; And (b) spotting the composite onto a substrate to immobilize the substrate of the gold nanoparticle-bio material composite through adsorption of the gold nanoparticles with the substrate. The method of claim 1, wherein the biomaterial is selected from the group consisting of DNA, RNA, PNA and protein. The method of claim 2, wherein the DNA or RNA is characterized in that the amine group introduced. The method of claim 1 wherein the substrate is modified with an aldehyde group or an amine group. The method of claim 1, wherein the biomaterial is further fixed by covalent bonding with the substrate modified with the aldehyde group. The method of claim 1, wherein the biomaterial is further secured by crosslinking with the substrate modified with the amine group. A biochip manufactured by the method of any one of claims 1 to 3, wherein the gold nanoparticle-bio material complex is fixed to the substrate by adsorption of the gold nanoparticles and the substrate. The method of claim 5, wherein the gold nanoparticles and the biomaterial complex is fixed to the substrate by adsorption of the gold nanoparticles and the substrate, and at the same time, the biomaterial is covalently bonded to the substrate modified with an aldehyde group Biochip. The method of claim 6, wherein the gold nanoparticles and the biomaterial complex is fixed to the substrate by adsorption of the gold nanoparticles and the substrate, and at the same time the biomaterial is cross-linked to the substrate modified with an amine group Biochip. A method of measuring the immobilization state of a biomaterial in a biochip, comprising the following steps: (a) mixing a solution containing silver ions with a solution for reducing silver ions to prepare a mixed solution for amplifying silver nanoparticles; (b) amplifying the silver nanoparticles by reacting the biochip of claim 7 with the mixed solution of step (a); And (c) determining the immobilization state of the biomaterial by measuring the silver nanoparticle amplification result. The method of claim 10, wherein the solution containing silver ions is a silver acetate solution, and the solution for reducing the silver ions is a hydroquinone solution. A method of measuring the immobilization state of a biomaterial in a biochip, comprising the following steps: (a) mixing a solution containing silver ions with a solution for reducing silver ions to prepare a mixed solution for amplifying silver nanoparticles; (b) amplifying the silver nanoparticles by reacting the biochip of claim 8 or 9 with the mixed solution of step (a); And (c) determining the immobilization state of the biomaterial by measuring the silver nanoparticle amplification result. The method of claim 12, wherein the solution containing silver ions is a silver acetate solution, and the solution for reducing silver ions is a hydroquinone solution. A method for detecting a hybridization reaction, characterized by using a DNA chip in which a gold nanoparticle-amine group-introduced DNA complex is immobilized on a substrate by adsorption of gold nanoparticles and a substrate. The method of claim 14, wherein the DNA chip is characterized in that the amine group introduced DNA is further covalently bonded to the substrate modified with an aldehyde group. The method of claim 14, wherein the DNA chip is characterized in that the amine group introduced DNA is further cross-linked to the substrate modified with the amine group.

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