KR20090127745A - Methods for preparing nano-bioplatforms - Google Patents

Abstract

PURPOSE: A method for manufacturing a nano-bioplatform is provided to prepare effectively a nano-bioplatform I where the uniform nanosized micropattern is formed by simple process. CONSTITUTION: A method for manufacturing a nano-bioplatform comprises the steps of forming an alumina coating on a substrate by anodizing an aluminum substrate; removing the alumina coating from the aluminum substrate to obtain aluminum substrate where a concave shape is formed on the surface; coating a biomolecule compatible substance on the aluminum substrate; and removing the aluminum substrate to obtain the biomolecule compatible substance where the micropattern of the convex sides of biomolecule compatible substance.

Description

Method for Preparing Nano-Bioplatforms {Methods for Preparing Nano-Bioplatforms}

The present invention relates to a method for producing a nano-bio platform, a nano-bio platform prepared by the method and a biochip comprising the nano-bio platform.

With the development of nanoscience and nanotechnology, researches on new bioelectronic devices fused with nanotechnology are progressing not only in the fields of materials, electronics and energy, but also in biotechnology such as biotechnology, new drug development and medicine. Biochip is a bioelectronic device made by integrating and combining materials derived from organisms such as DNA, proteins, or cells on inorganic substrates such as semiconductors at high density, and is used for monitoring biochips for diseases such as diseases and monitoring of agricultural, food or environmental pollutants. do.

In the conventional protein chip, since the size of the detector is limited to micro units, the activity and integration of molecules, the type and range of measurement are limited. In addition, since the measurement system uses conventional optical measurement technology, there is a need to overcome disadvantages such as the need for expensive optical equipment or fluorescent labels, which are limitations of the method.

The limitation of measurement sensitivity is to overcome the limitations by forming complexes with nanoparticles at the nanoscale to achieve signal amplification, and to measure miniaturized nanomaterials by incorporating new biomaterial immobilization techniques and nanofluid control techniques into protein chips. You need nanoscale protein chips.

Unlike the conventionally developed DNA chip or protein chip, the cell chip has a feature that the data in the cell can be obtained in real time under the physiological activity. As a result, pharmaceutical companies are increasing the demand for complex bioinformation such as measurement of cell physiological activity and drug side effects on drugs needed to develop anticancer drugs or antibiotics, as well as growing, detecting, and separating cells on a chip. As it can be used as a therapeutic tool, it is necessary to develop cell chip technology considering the size of the pharmaceutical and medical markets in the bio market.

However, in contrast to the increased demand for cell chip technology, the absence of technology such as nano-level immobilization techniques that enable the preservation of cell physiological activity using microstructured surface treatment and nanomaterials becomes an obstacle to the development of highly sensitive standardized cell chip. have.

In order to solve this problem, in order to immobilize cells on a substrate without the complicated process of chemical surface treatment, a physical surface preparation approach is required in addition to the above chemical surface treatment. Considering the weight of the cell, there is a limit in constructing a three-dimensional structure using only a protein or peptide chain, so it is necessary to make a nano-level pattern on the substrate to fix the cell. Therefore, the development of a cell-fixing nanoplatform that combines the nano-level pattern that allows these cells to grow well and the peptide chain required for cell immobilization can simplify cell culture and make it possible to culture cells more efficiently.

The bioplatform, in which the nanomolecules are applied at the nano level and orderly fixed at the nano level, is preserved in the activity of biomolecules and can perform a specific function for each molecule. It can reduce the amount of sample required for drug development, and can be used as a biosensor with good sensitivity and good selectivity to detect minute amounts. Thus, it is possible to construct a multi-component high density biochip with improved sensitivity and selectivity, which is a technical problem of existing biochips, and to be used as a source technology in the next generation measurement field.

In order to implement such nanobiochips, technologies for making nanometer-sized integrated circuit devices and for patterning biomaterials on a substrate, such as protein patterns and antibodies for antigen-antibody reactions, are essential technologies.

Various methods have been attempted to directly pattern biomaterials on a substrate, and research has been attempted to fabricate bioplatforms by finely processing or patterning inorganic substrates using conventional lithography patterning techniques. However, nanobioplatforms implemented only with such patterning techniques have been attempted. Has many problems in terms of resolution, high cost, large area limitation, or reproducibility.

Throughout this specification many patent documents are referenced and their citations are indicated. The disclosures of the cited patent documents are incorporated by reference herein in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present inventors have tried to develop a nano-bioplatform capable of effectively immobilizing biomaterials (eg, proteins, nucleic acid molecules and cells). As a result, a metal is formed on a concave-shaped fine pattern formed on the surface of anodized aluminum, or a biomolecule-affinity material is applied and aluminum is removed, and then a fine pattern of convex surfaces is formed. When the nano-bioplatform composed of biomolecule-affinity material is manufactured, the present invention has been completed by confirming that the bio-molecule, particularly protein and cells, can be used as a biochip by showing excellent immobilization ability.

Accordingly, it is an object of the present invention to provide a method for producing a nano-bio platform.

Another object of the present invention is to provide a nano-bioplatform prepared by the above method.

Still another object of the present invention is to provide a biochip including the nanobioplatform.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for manufacturing a nano-bioplatform having a metal micropattern having a concave shape formed, comprising the following steps:

(a) anodizing the aluminum substrate to form an alumina film;

(b) removing the alumina film from the aluminum substrate on which the alumina film is formed to obtain an aluminum substrate having a concave shape formed on a surface thereof; And

(c) depositing a metal on the aluminum substrate having the concave shape.

According to another aspect of the present invention, the present invention provides a method for producing a nano-bio platform showing the micropattern of the biomolecule-affinity material comprising the following steps:

(a) anodizing the aluminum substrate to form an alumina film;

(b) removing the alumina film from the aluminum substrate on which the alumina film is formed to obtain an aluminum substrate having a concave shape formed on a surface thereof;

(c) applying a biomolecule-affinity material to the concave shaped aluminum substrate; And

(d) removing the aluminum substrate to obtain a biomolecule-affinity material having a fine pattern of convex surfaces of the biomolecule-affinity material.

The present inventors have tried to develop a nano-bioplatform capable of effectively immobilizing biomaterials (eg, proteins, nucleic acid molecules and cells). As a result, a metal is formed on a concave-shaped fine pattern formed on the anodized aluminum surface, or a biomolecule-affinity material is applied and aluminum is removed, and then the fine patterns of convex surfaces are formed. When the nano-bioplatform composed of biomolecule-affinity material was prepared, it was confirmed that the biomolecule, particularly, can be used as a biochip by exhibiting excellent immobilization ability on biomaterials, particularly proteins and cells.

As used herein, the term “bioplatform” means a solid substrate used in a biochip or a substrate on which the solid substrate is bonded, or a substrate on which the biomaterial is directly bonded or indirectly bonded through a linker to immobilize the biomaterial. .

Detailed step-by-step description of the method of the present invention for producing a nano-bio platform as follows:

Formation of Alumina Film

According to the method of the present invention, an aluminum substrate is first anodized to form an alumina film.

As used herein, the term “anodic oxidation” refers to the formation of a nanoporous aluminum oxide film that forms a regular array on the surface of aluminum when an appropriate voltage is applied by connecting aluminum to the anode and connecting the platinum (Pt) electrode to the cathode in an acidic electrolyte solution. It means the process.

Acidic electrolyte solutions used in the present invention include various acidic electrolyte solutions known in the art. Preferably, it is chromic acid, phosphoric acid, sulfuric acid, oxalic acid, or a mixed solution thereof, more preferably sulfuric acid or oxalic acid, and most preferably oxalic acid.

According to a preferred embodiment of the present invention, the anodization is carried out by applying a voltage of 24-60 V.

Obtaining an Aluminum Substrate with Concave Shapes

Removal of the alumina film in the present invention is carried out by various methods known in the art, preferably by etching in the presence of an acidic electrolyte solution.

The acidic electrolyte solution used to remove the alumina film in the present invention includes various known acidic electrolyte solutions. Preferably, it is chromic acid, phosphoric acid, sulfuric acid, oxalic acid, or a mixed solution thereof, more preferably chromic acid, phosphoric acid or a mixed solution thereof, and most preferably a mixed solution of chromic acid and phosphoric acid.

If the alumina film is removed as described above, an aluminum substrate having a concave shape formed on the surface can be obtained.

When the aluminum used in the present invention is applied with the voltage of the optimum condition according to the electrolyte to form a nanoporous alumina layer having a regular arrangement to remove the alumina oxide film, a pattern of regular nano-sized concave shape is formed on the aluminum surface. It is possible to obtain an alumina film which is a kind of inorganic film having nanopores. As a representative feature of nanoporous alumina, a high aspect ratio (length to diameter), which is difficult to obtain in lithography pattern technology, can be obtained, and the aspect ratio can be adjusted according to anodization conditions. That is, depending on the type of electrolyte, temperature, concentration, current density, and voltage, the pore size, cell size (cavity distance), barrier layer thickness, and pore size of the nanoporous alumina can be determined. Eggplant can control the thickness of the alumina layer (porous alumina), the pore density, and the like can be used to control the pattern of the aluminum surface.

Deposition of Metals on Aluminum Substrates

According to the method of the present invention, a nano-bioplatform having a metal micropattern can be manufactured by removing an alumina film, depositing a metal on an aluminum substrate having a concave shape, and forming a concave shape on the surface of the deposited metal layer.

The method of depositing a metal on an aluminum substrate having a concave shape by the method of the present invention may be carried out using a conventional physical evaporation method, preferably thermal evaporation, spin coating. coating or sputtering, and more preferably thermal evaporation.

According to the method of the present invention, the upper surface of the deposited metal may also form a concave shape to fix the biomaterial in three dimensions. The reason for forming the surface of the three-dimensionally deposited metal is to increase the surface area to immobilize the biomaterial at various positions in three dimensions.

The metals used in the methods of the present invention include various metals known in the art for effectively immobilizing biomaterials (eg proteins, nucleic acid molecules and cells), preferably the metals are gold (Au), silver ( Ag), platinum, nickel, copper, titanium, palladium, iridium, rhodium, ruthenium, osmium, zirconium, cadmium, tungsten, gallium or indium, more preferably gold (Au), silver (Ag), platinum, nickel, Copper or titanium, most preferably gold (Au) or silver (Ag).

According to a preferred embodiment of the invention, the metal is deposited to a thickness of 10-100 nm.

The method of manufacturing a nano-bioplatform of the present invention includes using a biomolecule-affinity material in addition to the method of depositing a metal.

Application of biomolecule-affinity materials

According to the method of the present invention, instead of removing the alumina film and depositing a metal on the concave-shaped aluminum substrate, a biomolecule-affinity material may be applied to prepare a nano-bioplatform.

According to the present invention, by applying a biomolecule-affinity material to the aluminum substrate from which the alumina film is removed, a fine pattern of convex surfaces is formed on the surface of the biomolecule-affinity material.

Biomolecule-affinity materials for use in the present invention include various polymeric materials known in the art. Preferably dextran, heparan, heparin, hyuronic acid, alginic acid, agarose, carrageenan, amylopectin, amylose, glycogen, starch, cellulose, chitin, agglutinin, collagen, gelatin, albumin, keratin, decorin, agar Glycans, glycoproteins, antigens, antibodies, receptors, streptavidin, avidin, aptamers, lectins, DNA, RNA, antisense oligonucleotides or ligands, more preferably dextran, hyuronic acid, Alginic acid, agarose, chitin, agglutinin, collagen, gelatin, albumin, antigen, antibody, streptavidin, avidin, aptamer, lectin, DNA, RNA or antisense oligonucleotides. Most preferably the biomolecule-affinity material is dextran, hyuronic acid, alginic acid, agarose, chitin, agglutinin, collagen or gelatin.

Obtaining a Biomolecule-affinity Material with a Fine Pattern of Convex Faces

When the biomolecule-affinity material is used in the present invention, the nano-bioplatform is characterized by not including an aluminum substrate. Therefore, when manufacturing a nano-bio platform using a biomolecule-affinity material, it includes the step of removing the aluminum substrate. Removal of the aluminum substrate can be carried out by various methods known in the art, preferably supersaturated HgCl ₂ The aluminum can be dissolved and removed by immersion in the solution for a certain time.

When the aluminum substrate is removed in the method of the present invention, the biomolecule-affinity material applied to the surface on which the concave shape of the substrate is formed forms a fine pattern of the convex surfaces. By forming a fine pattern of the convex surfaces, it is possible to fix the biomaterial in three dimensions. The reason for forming a fine pattern of three-dimensionally convex surfaces is to fix the biomaterial at various positions in three dimensions by increasing the surface area.

Only the biomolecule-affinity material in which the fine patterns of the convex surfaces are formed is used as a nano-bio platform, and is used in a biochip by immobilizing a biomaterial on the uniformly arranged convex surfaces.

According to another aspect of the present invention, the present invention provides a nano-bioplatform prepared by the method of the present invention described above.

Since the nano-bioplatform of the present invention is produced by the above-described method of the present invention, the contents common between the two are omitted in order to avoid excessive complexity of the specification according to the repetitive description.

According to another aspect of the present invention, there is provided a biochip or a biosensor comprising the nano-bioplatform of the present invention described above.

In the biochip of the present invention, the biomaterial is directly bonded to the nano-bioplatform of the present invention or indirectly immobilized through a linker. Binding biomaterials include various biomaterials known in the art and include, for example, nucleic acid molecules (eg, DNA, RNA and uptamers), proteins, peptides, peptide nucleic acids (PNA), lectins, avidin (or streptavidin). ), But is not limited to single cells or cell populations.

The biochip of the present invention is preferably a DNA microarray, a protein chip or a cell chip. General descriptions applicable to the biochip of the present invention are U.S. Pat. , 5,631,734, 5,795,716, 5.83107 million, 5,837,832, 5,856,101, 5,858,659, 5,889,165, 5,936,324, 5,959,098, 5.96874 million, 5,974,164, 5,981,185, 5,981,956, 6,025,601, 6.03386 million, 6,040,193, 6,090,555, 6,136,269, 6,147,205, 6,262,216, 6,269,846, 6,310,189 and 6,428,752 hoge specifically described It is.

General information that can be applied to the biosensor of the present invention, Myska, J. Mol . Recognit , 12: 279-284 (1999); J. Dubendorfer et al. Journal of Biomedical Optics , 2 (4): 391-400 (1997); And O'Brien et al. Biosensors & Bioelectronics , 14: 145-154 (1999).

According to a preferred embodiment of the present invention, the biochip is a protein chip. The protein chip of the present invention may comprise a protein indirectly immobilized on a solid substrate via a linker. In the present invention, the linker may be any compound used as a linker, and the linker forms a self-assembled monolayer (SAMs) on the nano-bioplatform of the present invention. Allow to be immobilized on a solid substrate.

Examples of proteins to be immobilized include hormones, hormone analogs, enzymes, inhibitors, signaling proteins or portions thereof, antibodies (monoclonal antibodies and polyclonal antibodies) or portions thereof, single chain antibodies, binding proteins or binding domains, peptides, Antigens, adhesion proteins, structural proteins, regulatory proteins, toxin proteins, cytokines, transcriptional regulators, blood clotting factors, and plant biodefense inducing proteins.

According to another preferred embodiment of the present invention, the biochip is a cell chip. Cells patterned on the cell chip of the present invention include a variety of cells known in the art that can be immobilized on the nano-bio platform of the present invention. For example, floating cells and adherent cells, such as hematopoietic and lymphocytic cells, may be used, including microorganisms or animal and plant cells, and particularly, for animal cells.

Examples of adherent cells include endothelial cells, such as hepatocytes, Kupffer cells, vascular endothelial cells and corneal endothelial cells, fibroblasts, osteoblasts, periodontal-derived cells, epithelial keratinocytes, which are parenchymal cells of the liver. Epithelial cells, organ epidermal cells such as the digestive tract, epidermal cells such as cervical epidermal cells and corneal epithelial cells, muscle cells such as mammary gland cells, pericytes, smooth muscle cells and cardiomyocytes, kidney cells, iserangerhans islet cells Nerve cells such as peripheral nerve cells and optic nerve cells, chondrocytes, and bone cells.

In addition, the cells may be undifferentiated cells such as embryonic stem cells, pluripotent stem cells having pluripotent differentiation capacity, such as mesenchymal stem cells, single differentiation stem cells having single differentiation capacity, such as vascular endothelial progenitor cells, or differentiated cells. . In addition, one cell or at least two cells may be cultured.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a nano-bioplatform capable of effectively immobilizing biomaterials (eg proteins, nucleic acid molecules and cells).

(b) The present invention is configured by depositing a metal on a fine pattern having a concave shape formed on the aluminum surface by anodizing aluminum, or applying a biomolecule-affinity material and removing aluminum, and then forming a micro pattern of convex surfaces. Molecular-affinity materials are prepared on a nano-bioplatform.

(c) When the biomolecule-affinity material is used in the present invention, the aluminum substrate is not included in the nano-bioplatform.

(d) The production method of the present invention can more easily and efficiently manufacture the nano-bio platform.

(e) The nano-bioplatform prepared by the method of the present invention can be applied to biochips.

As described in detail above, the present invention provides a method for producing a nano-bio platform, a nano-bio platform prepared by the method and a biochip including the nano-bio platform. The manufacturing method of the present invention is constructed by depositing a metal on a fine pattern in which aluminum is anodized to form a concave shape on an aluminum surface, or applying a biomolecule-affinity material, removing aluminum, and then a convex surface. The biomolecule-affinity material in which the micropatterns are formed is prepared as a nano-bio platform. By such a simple process it is possible to efficiently produce a nano-bio platform formed with a uniform, nano-sized fine pattern. The present invention can effectively immobilize biomaterials (eg proteins, nucleic acid molecules and cells). In addition, in the case of using the biomolecule-affinity material in the present invention, the aluminum substrate is not included in the nano-bio platform. In addition, the nano-bioplatform prepared by the method of the present invention can be applied to biochips.

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 in accordance with the gist of the present invention. .

Example

Example  1: gold ( Au Fabrication of Deposition Nano-Bioplatforms

In an 0.3 M oxalic acid solution at 3 ° C., aluminum was positively connected to the anode and Pt electrodes were connected to the cathode, and a voltage of 40 V was applied to a power supply (power supply UDP-500, Unicon). Porous alumina coating was formed by the anodization (FIG. 1A). Subsequently, the formed alumina film was immersed in a mixed solution of chromic acid (0.2 M) and phosphoric acid (0.4 M) at 65 ° C. for at least 4 hours to dissolve the alumina film. Through the dissolution process, a concave micropattern was formed on the aluminum surface (FIG. 1B).

Subsequently, gold (Au) was thinly deposited to 40 nm or less by thermal evaporation on a nano-sized concave patterned aluminum surface (FIG. 1C). Deposition rate was 0.1 Å / s was deposited at a very low deposition rate and the pressure was maintained at 10 ^-6 Torr. Deposition of gold at very low deposition rates is important for uniform deposition of gold on nanoscale concave patterned aluminum surfaces.

As a result of observing the aluminum surface (FIG. 1B) on which the concave micropattern was formed (SEM), as shown in FIG. 2A, the gap between the pupil and the pupil of the concave surface was 105 (± 5) nm and the density of the pupil was ^{1.0 (± 0.2) x10 10 cm} - ² was confirmed that a fine concave pattern is formed uniformly in the nanoscale. In addition, as can be seen in the SEM photograph of Figure 2b it can be seen that the aluminum surface pattern formed after the gold deposition is also uniformly formed.

Example  2: biomolecule-affinity material Applied  Fabrication of Nano-Bio Platform

In the same manner as in Example 1, an aluminum substrate having a concave micropattern was formed. Then collagen or gelatin was applied (FIG. 1C-1) and then supersaturated HgCl ₂ By soaking in the solution for about 3 hours, the aluminum substrate was removed to form a convex face pattern (FIG. 1D-1).

Biomolecule-affinity materials (collagen or gelatin) with convex sides were used as nano-bioplatforms.

As can be seen in the Atomic Force Microscope (AFM) photograph of FIG. 3, it can be seen that the biomolecule-affinity material was formed in a three-dimensional fine convex pattern.

Example  3: by nano-bioplatform of the present invention Azurin ( Azurin A) immobilization of protein

In order to confirm the degree of protein immobilization, the azurin protein with Cys group (prepared by genetic recombination method in Sogang University, bioelectronics and bioinformatics laboratory) was prepared using HEPES (4- (2-hydroxyethyl) -1-pipe Raginethane sulfonic acid, pH5.4) dissolved in a buffer solution at a concentration of 0.100 mg / ml, then dropped onto the nano-bioplatform prepared in Example 1 and washed with PBS-Tween after 2 hours to fix Components other than lean protein were removed.

As a result of confirming the degree of immobilization of the azurin protein on the nano-bioplatform prepared according to Example 1, as shown in FIG. 4, the present invention, rather than immobilizing the azurin protein on a gold substrate (FIG. 4 (a)), When the Azurin (Azurin) protein is fixed to the nano-bio platform prepared by Example 1 (Fig. 4 (b)) it can be seen that uniformly well fixed because of the micro-pattern formed on the nano-bio platform of the present invention have.

Example  4: by nano-bioplatform of the present invention HeLa  Immobilization of cells

To confirm cell immobilization, HeLa cells (Korea Cell Line Bank), a human epithelial tumor cell line, were prepared using DMEM (Dulbecco's modified Eagle's medium, Gibco) containing 10% FBS (fetal bovine serum, Gibco) and 1% antibiotic (Gibco). ) 5% CO ₂ as a culture And cultured in a bacterial incubator at 37 ° C. The incubator was replaced once every two days and the surface of the micropatterned substrate with gold was washed with alcohol and then immersed in the culture solution containing the cultured HeLa cells. Cultures were removed for SEM measurement, washed with PBS and dried.

As a result of confirming the degree of fixation of HeLa cells (Korea Cell Line Bank) on the nano-bioplatform prepared by Example 1, as shown in FIG. It can be seen that. FIG. 5A is a SEM photograph showing HeLa cells immobilized on a Petri dish, and FIG. 5B on a nano-bioplatform prepared by Example 1. FIG.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

1 is a schematic diagram showing a process of manufacturing a nano-bio platform by the method of the present invention. A series of processes of (a), (b) and (c) of FIG. 1 is embodiment 1, and a series of processes of (a), (b), (c-1) and (d-1) of FIG. Shows the process by which the nano-bio platform is prepared by Example 2.

FIG. 2 (a) is a scanning electron microscope (SEM) photograph showing an aluminum surface obtained by dissolving an oxide film of nanoporous alumina prepared by applying a voltage of 40 V in a 0.3 M oxalic acid solution in a chemical solution.

FIG. 2 (b) shows the surface of aluminum patterned as a concave surface using physical evaporation such as thermal evaporation, spin coating, or sputtering. SEM photograph showing the surface on which is deposited).

3 is an atomic force microscope (AFM) photograph showing a nano-bioplatform prepared by Example 2 of the present invention.

Figure 4 is a SEM photograph showing the degree of immobilization of Azurin protein. Figure 4 (a) shows the azurin protein immobilized on the gold substrate and Figure 4 (b) shows the Azurin protein immobilized on the nano-bio platform prepared by Example 1 of the present invention.

Figure 5 (a) is a SEM photograph of 1000 times showing the HeLa cells attached to the Petri dishes and Figure 5 (b) shows that the HeLa cells are fixed on the nano-bio platform prepared by Example 1 of the present invention SEM picture of 1,000 times showing.

6 is a SEM photograph showing that HeLa cells are fixed to the nano-bioplatform prepared by Example 1 of the present invention. 6 (a) shows a 5,000 times and FIG. 6 (b) shows a SEM picture of 10,000 times.

Claims (12)

A method of manufacturing a nano-bioplatform having a metal micropattern having a concave shape including the following steps: (a) anodizing the aluminum substrate to form an alumina film; (b) removing the alumina film from the aluminum substrate on which the alumina film is formed to obtain an aluminum substrate having a concave shape formed on a surface thereof; And (c) depositing a metal on the aluminum substrate having the concave shape. Method for producing a nano-bio platform showing the micropattern of the biomolecule-affinity material comprising the following steps: (a) anodizing the aluminum substrate to form an alumina film; (b) removing the alumina film from the aluminum substrate on which the alumina film is formed to obtain an aluminum substrate having a concave shape formed on a surface thereof; (c) applying a biomolecule-affinity material to the concave shaped aluminum substrate; And (d) removing the aluminum substrate to obtain a biomolecule-affinity material having a fine pattern of convex surfaces of the biomolecule-affinity material. The method according to claim 1 or 2, wherein the anodization is carried out in the presence of an acidic electrolyte solution. The method of claim 3, wherein the acidic electrolyte solution is chromic acid, phosphoric acid, sulfuric acid, oxalic acid or a mixed solution thereof. The method according to claim 1 or 2, wherein the anodization is carried out by applying a voltage of 24-60 V. The method according to claim 1 or 2, wherein the removal of the alumina coating is performed by etching in the presence of an acidic electrolyte solution. 7. The method of claim 6, wherein the acidic electrolyte solution is chromic acid, phosphoric acid, sulfuric acid, oxalic acid, or a mixed solution thereof. The method of claim 1, wherein the metal is gold (Au) or silver (Ag). The method of claim 1 wherein the metal is deposited to a thickness of 10-100 nm. The method of claim 2, wherein the biomolecule-affinity material is dextran, heparan, heparin, hyuronic acid, alginic acid, agarose, carrageenan, amylopectin, amylose, glycogen, starch, cellulose, chitin, agglutinin, collagen Gelatin, albumin, keratin, decorin, aglycan, glycoprotein, antigen, antibody, receptor, streptavidin, avidin, aptamer, lectin, DNA, RNA, antisense oligonucleotide and ligand And selected from the group consisting of:        Nano-bio platform prepared by the method of claim 1 or 2. Biochip comprising the nano-bio platform of claim 11.

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