KR100965238B1 - Substrate for immobilizing physiological material, and a method of preparing the same - Google Patents

Abstract

The present invention relates to a substrate; A sensitizing agent layer formed on the substrate; And a gold thin film formed on the sensitizer layer, wherein the gold thin film has a thickness of 30 to 200 nm and exhibits XRD peaks at (111) and (200) planes when the gold thin film is irradiated with an incident angle of 1.5 degrees. Provided is a substrate for fixing. The substrate for fixing the biomaterial is formed by coating a coating solution including a sensitizer on the substrate to form a sensitizer layer; Coating a gold colloidal dispersion on the substrate on which the sensitizer layer is formed to form a seed colloidal catalyst layer; Drying or heat-treating the substrate on which the seed colloidal catalyst layer is formed; And coating the substrate with a coating solution consisting of a mixture of a gold salt-containing aqueous solution and a reducing agent solution to obtain a gold thin film.

Seed colloid, sensitizer, gold thin film, biomaterial, immobilization, biochip

Description

SUBSTRATE FOR IMMOBILIZING PHYSIOLOGICAL MATERIAL, AND A METHOD OF PREPARING THE SAME}

1 is a view showing a manufacturing process step of a substrate for fixing a biomaterial of the present invention.

2 is a view showing the surface plasmon resonance (SPR) effect according to the gold thin film formation reaction time of Example 1 of the present invention.

3A and 3B show XRD analysis results of gold thin films formed by the methods of Example 1 and Comparative Example 1 of the present invention, respectively.

4A and 4B are electron micrographs of a plane and a cross section of a substrate for fixing a biomaterial according to Example 1 of the present invention, respectively.

5A and 5B are electron micrographs of a plane and a cross section of a substrate for fixing a biomaterial of Comparative Example 1;

6a and 6b are electron micrographs of the plane and cross section of the substrate for fixing a biomaterial of Comparative Example 2.

7 is a view showing the transmittance of the substrate for fixing the biological material of Example 1 and Comparative Example 2 of the present invention.

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a substrate for fixing a biomaterial and a method for manufacturing the same, and more particularly, to a substrate for fixing a biomaterial and a method for manufacturing the same having excellent transparency and SPR characteristics.

[Private Technology]

Recently, efforts to discover the activity of biomaterial molecules such as nucleic acids, proteins, enzymes, antigens, antibodies and the like by combining biotechnology and semiconductor manufacturing technology are spreading around the world. By using semiconductor processing technology on small silicon chips, biochemically searching biochips in which desired biomolecule molecules are fixed within a specific area can be easily obtained.

Biochip is a device made in the form of a conventional semiconductor chip by combining biological organisms such as enzymes, proteins, antibodies, DNA, microorganisms, animal and plant cells and organs, nerve cells and organs, neurons derived from living organisms and inorganic materials such as semiconductors. Biochips are largely integrated with DNA probes with fixed DNA probes, protein proteins with enzymes, antibodies, antigens, etc., and sample pretreatment, biochemical reactions, detection, and data analysis. It can be classified into "lab-on-a-chip" having an analysis function.

In order to develop such a biochip, an immobilization technology of biomaterials that effectively forms an interface between the biomaterial and the immobilization substrate and makes the most of the unique functions of the biomaterial is important. Immobilization of biomaterials occurs on slide glass, silicon wafers, microwell plates, tubes, spherical particles, and porous membrane surfaces. In particular, in biochips such as DNA chips and protein chips, it is important to fix related biomaterials in limited areas on a micrometer scale.

Gold, one of the typical substrates used for fixing proteins, has thioctic acid, L-cysteine, and mercapto having functional groups such as sulfide and disulfide, which can form chemical bonds on the surface of gold. Propyl acid (mercaptopropyl acid), paraaminothiophene, cysteamine, etc. are commonly used, and have other functional groups such as -SH and -NH ₂ capable of reacting with gold at one end of the molecule and the other end. Derivatives such as calixarene and cyclodextrine having functional groups such as -OH and -NH ₂ having excellent affinity with silver proteins are included. In addition, poly-L-lysine is used to form -NH ₂ groups as a two-dimensional network through polymers (Biosensors & Bioelectronics, 13, 1213 (1998), Anal Biochem. 272, 66 (1999).

Sputtering or evaporation is the most commonly used method for forming a gold surface capable of protein immobilization on glass, silicon wafers or plastic substrates. However, these methods require expensive and precise vacuum facilities and large-scale investment is inevitable in mass production.

The gold coating technology ("Gold Films", The Glass Industry, May 1959 p.280 and June 1959, p330), published by Samuel Wein in 1960, achieved the desired gold surface by deposition or spray methods. However, the reaction rate is slow and requires a high temperature.

In addition, many studies have been conducted by autocatalytic gold deposition. For example, US Pat. No. 3,700,469 uses gold cyanide complexes and alkali metal borohydride or dimethylamine borane as reducing agents. It was proposed a method of forming a thin film. However, these methods also have to raise the temperature for the hydrolysis reaction of the reducing agent and there is a problem that the sludge is generated by the spontaneous decomposition of the gold solution in the solution.

Recently, patents have been filed using non-cyanide gold complexes that lower the pH of a solution for use in electronics packaging (US Pat. Nos. 4,804,559; 5,198,273; 5,202,151; 5,318,621; 5,470,381; 5,935,306). The methods proposed by the above patents have been developed for use in electronic products such as circuit boards and IC chips. In general, the thickness of the gold thin film is about 0.5 to 2 micrometers.

On the other hand, the analysis device of biochips such as protein chips or DNA chips is a device for analyzing the interaction of biomaterials that bind to the chip. The core technologies are laser emission image analysis technology, electrochemical analysis technology, surface plasma resonance technology (SPR) and Technologies such as Surface-Enhanced Laser Desorption / Ionization (SELDI) TOF are used, and in the case of gold thin film substrates, optical methods such as SPR technology and electrochemical analysis techniques are mainly used. After all, the method according to the patent has a problem that it is difficult to apply such an analysis technology, there is a need for a coating method that can obtain a uniform thickness of 0.1 micrometer or less.

Recently, in US Pat. No. 6,168,825, a thin film of 300 nanometers or less could be manufactured using a gold ion solution and a reducing agent, but the problem of sludge generation due to spontaneous decomposition still remains. Yongdong Jin et al. (Anal. Chem., 2001, vol73, 2843-2849) suggested the possibility of use as an SPR substrate by the above method after forming a gold colloid on an aminosilane-coated substrate. The problem of falling much more than the sputtering gold substrate appeared.

In order to solve the problems of the prior art as described above, the present invention is to provide a substrate for fixing a biological material excellent in transparency and SPR characteristics.

Another object of the present invention is to provide a method for manufacturing a substrate for fixing a biomaterial having excellent transparency and SPR characteristics.

In order to achieve the above object, the present invention is a substrate; A sensitizing agent layer formed on the substrate; And a gold thin film formed on the sensitizer layer, wherein the gold thin film has a thickness of 50 nanometers or less, and exhibits XRD peaks at (111) and (200) planes when irradiated with an incident angle of 1.5 degrees to the gold thin film sample. Provided is a substrate for fixing substances.                     

In another aspect, the present invention is to form a sensitizer layer by coating a coating liquid containing a sensitizer on a substrate for fixing a biomaterial; Coating a gold colloidal dispersion on the substrate on which the sensitizer layer is formed to form a seed colloidal catalyst layer; Drying or heat-treating the substrate on which the seed colloidal catalyst layer is formed; And coating a substrate with a coating liquid consisting of a mixture of a gold salt-containing aqueous solution and a reducing agent solution to obtain a gold thin film.

The present invention also provides a biochip comprising a biomaterial fixed to the substrate for fixing the biomaterial.

Hereinafter, the present invention will be described in more detail.

The substrate for fixing a biomaterial of the present invention includes a sensitizer layer formed on the substrate and a gold thin film formed thereon. The substrate may use either a transparent solid substrate or an opaque solid substrate such as a silicon wafer. Preferably, environmentally stable or chemically resistant glass, polycarbonate, polyester, polyethylene (PE), polypropylene (PP), wafer, or the like is possible, but is not limited thereto.

Examples of the sensitizer material used in the sensitizer layer include tin, indium, palladium, germanium, aluminum, lead, titanium, and gallium. , Salts thereof, mixtures and the like can be used. Preferred examples of the salt include chlorides such as PdCl ₂ , PbCl ₂ GeCl ₂ , InCl ₃ , AlCl ₃ , GaCl ₃ , TiCl, SnCl ₂ , or fluorides, among which tin chloride, such as SnCl ₂ , is most preferred.

The thickness of the gold thin film is 30 to 200 nm, preferably 30 to 70 nm, more preferably 30 to 50 nm, in terms of (111) and (200) when irradiating the gold thin film sample with an incident angle of 1.5 degrees. XRD peaks are shown. XRD measurements are preferably carried out by forming a thin film of gold on the substrate surface and using a Cu target and scanning at a scan rate of 0.02 degrees / second.

The substrate for immobilizing the biomaterial of the present invention is thioctic acid (thioctic acid), L-cysteine (L-cysteine), mercaptopropyl acid (mercaptopropyl acid), paraaminothiophene (paraaminothiophene), cysteamine (cysteamine), etc. The biomaterial may be fixed to the substrate using the biochip analysis method such as fixing of the biomaterial and interaction of the biomaterials by SPR or electrochemical method.

In the present invention, the term "biomaterial" includes all of those derived from an organism, equivalent thereto, or manufactured in vitro, and examples thereof include enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, nerve cells, DNA, RNA, and the like. it means. More preferably, it may be DNA, RNA or protein, wherein the DNA comprises cDNA, genomic DNA, oligonucleotide, the RNA comprises genomic RNA, mRNA, oligonucleotide, examples of the protein include antibodies, Antigens, enzymes, peptides and the like.

The biomaterial may be fixed to the immobilized substrate through a photolithography method, a piezoelectric printing method such as an inkjet printer, micro pipetting, spotting, or the like.

A schematic diagram of the manufacturing process of the substrate for fixing a biomaterial of the present invention is shown in FIG. 1. First, the cleaned substrate 1 is coated with a coating liquid in a slurry state containing a sensitizer to form a sensitizer layer 2. The material used as the sensitizer is as described above, and the coating liquid is prepared by adding the sensitizer to the diluent solvent. The diluting solvent may be water or a mixed solvent of water and an organic solvent. The organic solvent is preferably selected from the group consisting of methanol, ethanol, propanol, cellulose solvents, and dimethylformaldehyde.

The concentration of the sensitizer contained in the coating liquid is preferably 0.01% by weight to 50% by weight. If the concentration of the sensitizer is lower than 0.01% by weight, no effect as a sensitizer is exhibited. If the concentration of the sensitizer is higher than 50% by weight, the substrate becomes uneven. As the coating method, a wet coating method such as dipping, spraying, spin coating, printing, or the like may be used, and among these, dipping or spray coating is preferable. The deposition time during the deposition coating should be at least 1 minute. The coating is then cleaned with demineralized water to remove excess sensitizer on the substrate. It is then dried to form a sensitizer layer.

The substrate on which the sensitizer layer 2 is formed is coated with a gold colloidal dispersion to form a seed colloidal catalyst layer 3. The seed colloid catalyst layer 3 includes a gold colloid having a particle diameter of 5 nm to 500 nm.

The gold colloidal dispersion contains a gold salt, a reducing agent, a stabilizer and a solvent. As the gold salt, gold chlorides such as HAuCl ₄ and NaAuCl ₄ may be used, but are not limited thereto. The concentration of the gold salt is preferably 0.001 to 10% by weight and more preferably 0.005 to 1% by weight based on the gold colloidal dispersion. If the concentration of the gold salt is higher than 10% by weight, the monodispersibility of the colloidal particles is lowered, which is not preferable. If the concentration of the gold salt is lower than 0.001% by weight, it is not sufficient to form the seed colloidal catalyst layer.

The reducing agent may be NaBH ₄ , thiocyanate, potassium carbonate, trisodium citrate or hydrates thereof, tannic acid, hydroxyamine or salts thereof, or mixtures thereof. The concentration of the reducing agent is preferably 0.01 to 10% by weight and more preferably 0.1 to 5% by weight based on the gold colloidal dispersion.

As a stabilizer, sodium citrate etc. can be used. The solvent is selected from the group consisting of water, methanol, ethanol, propanol, cellulose solvents, and dimethylformaldehyde.

As the coating method, a wet coating method such as a deposition method, a spray method, a spin coating method, a printing method, or the like may be used, and among these, the deposition method is preferable. In immersion coating, immersion time of 1 minute or more is sufficient.

The seed colloid is adsorbed to dry or heat-treat the substrate on which the seed colloid catalyst layer 3 is formed. Then, a gold thin film 4 is formed by using an autocatalytic deposition method to prepare a substrate for fixing a biomaterial. In forming the spontaneous catalyst thin film, the coating solution is a mixture of a gold salt-containing aqueous solution and a reducing agent solution. The gold salt-containing aqueous solution and the reducing agent solution are separately prepared and then mixed and used immediately before coating. The gold salt is the same as the gold salt used in preparing the coating liquid for forming the seed colloid catalyst layer. The concentration of the gold salt may be used in an amount of 0.01% by weight to 20% by weight with respect to the gold salt-containing aqueous solution, and more preferably used in a concentration of 0.1% by weight to 10% by weight. If the concentration of the gold salt is lower than 0.01% by weight, the desired thickness cannot be obtained. At a concentration of 20% by weight or more, the thickness of the thin film is uneven and expensive gold salt is consumed excessively.

The reducing agent may be NaBH ₄ , thiocyanate, potassium carbonate, trisodium citrate or hydrates thereof, tannic acid, hydroxyamines or salts thereof, or mixtures thereof, among others hydroxyamine or It is preferable because the salt can obtain a uniform thin film. The concentration of the reducing agent solution may be used in a concentration of 0.01mM to 1M, preferably 0.01mM to 100mM. If the concentration of the reducing agent is 0.01mM or less, the desired gold thin film thickness cannot be obtained and the time is consumed excessively. In addition, the concentration of 1M or more is fast reaction rate is less easy to control the thickness of the gold thin film.

As a coating method for forming the gold thin film, a plating method is used, and in particular, an electroless plating method is used. That is, the gold salt-containing aqueous solution and the reducing agent solution are mixed well in a reaction vessel, and the substrate on which the seed colloidal catalyst layer is formed is deposited and then stirred to form a gold thin film. Since the thickness and the reaction time of the gold thin film have a linear relationship, it is possible to form a gold thin film having a desired thickness by appropriate time deposition, and it is preferable to react for about 10 minutes to obtain the SPR characteristics. By using this plating method, the thickness of the thin film can be easily adjusted to the desired nano-thickness. In addition, it is possible to manufacture large substrates necessary for mass production without expensive equipment investment such as vacuum deposition equipment.

Hereinafter, preferred embodiments of the present invention will be described. However, the following examples are intended to illustrate the invention and the present invention is not limited to the following examples.

EXAMPLE

Example 1

Preparation of Gold Colloidal Dispersion

To 100 ml of pure water, 1 ml of 1% HAuCl ₄ .3H ₂ O aqueous solution was added, followed by vigorous stirring. After 1 minute, 1 ml of 1% aqueous sodium citrate solution was added while stirring. After 1 min 1 ml of 1% sodium citrate solution containing 0.075% NaBH ₄ was added. After further stirring for 5 minutes, it was stored at 4 ° C.

Preparation of Coating Liquid for Gold Thin Film Formation

HAuCl ₄ .3H ₂ O was added to the pure water to prepare an aqueous gold chloride solution at a concentration of 1% by weight. A reducing agent solution was prepared by adding 8 mM NH ₂ OH.HCl to pure water as a reducing agent.

Preparation of Biomaterial Fixing Substrate

The washed slide glass (25x75mm) was immersed in a 1% aqueous solution of SnCl ₂ .2H ₂ O for 10 minutes, stirred and washed for 5 minutes in pure water, and dried. This substrate was deposited for 1 hour with a gold colloidal dispersion to form a seed colloidal catalyst layer. The substrate on which the seed colloidal catalyst layer was formed was deposited on a reaction vessel in which 1 ml of an aqueous gold chloride solution and 40 ml of a reducing agent solution were mixed to form a thin gold film.

In order to obtain a gold thin film substrate having a thickness suitable for the substrate for fixing the biomaterial, the reaction time was clearly expressed using an SPR spectrometer (Optrel GBR, FRG). The results are shown in FIG. As shown in FIG. 2, it can be seen that as the reaction time increases, the SPR peak appears more clearly.

In addition, it is shown in Table 1 to measure the electrical resistance value of the substrate according to the reaction time.

TABLE 1

Reaction time
(min) 2 4 6 8 10 12 Resistance over ^* 7.5E + 01 1.2E + 00 6.4E-1 2.6E-01 2.1E-01

* over: exceeds the limit of 10E7 of surveying equipment

As shown in Table 1, the longer the reaction time, the lower the resistance value of the thin film. The low resistance value of the thin film means that the coating is dense.

Comparative Example 1

A glass substrate on which a gold thin film was formed was prepared by using SRVAC-820 sputtering apparatus of ULVAC.

Comparative Example 2

10 g of tin (II) chloride-2H ₂ O and 40 mL of 37.3% HCl were mixed with 1 L of water and diluted. After the substrate was dipped in this slurry solution for 2 minutes, the reducing agent solution and the gold salt solution were sprayed to form a desired gold thin film before the surface dries. In this case, the reducing agent solution was prepared by mixing 20 ml of 3% hydrogen peroxide in 1 L of water and 1.2 g of sodium carbonate in 1 L of water. Gold salt solution was prepared by dissolving 1.5 g of sodium tetrachlorolaurate and 1.5 g of sodium carbonate in 1 L of water. XRD analysis was performed on a gold thin film formed by the method of Example 1 and Comparative Example 1 at a scanning speed of 0.02 degrees / second using a copper target. The resolution of the detector was 0.037 degrees and CuKa was used for X-ray irradiation. The results are shown in FIGS. 3A and 3B.

As shown in FIG. 3A, it can be seen that in the thin film according to Example 1, crystal phases preferentially grow in (111) and (200) planes. In contrast, in the thin film of Comparative Example 1 shown in FIG. 3B, the crystal phase was grown on the (220) plane, and thus, the thin film of Comparative Example 1 showed a different crystal phase from the thin film of Example 1.

4A and 4B show electron micrographs (SEM) of planes and cross sections of the substrate for fixing a biomaterial according to Example 1. FIG. In addition, electron micrographs of the plane and the cross-section of the substrate for fixing a biomaterial of Comparative Example 1 are shown in FIGS. 5A and 5B. 6A and 6B illustrate electron microscope photographs of planes and cross-sections of the substrate for fixing a biomaterial according to US Pat. No. 6,168,825.

As shown in FIGS. 4A and 4B, the gold thin film according to the present invention can be seen that the gold thin film is formed by the seed colloid to form grains around the seed colloid. On the other hand, in the gold thin film of Comparative Example 1 shown in FIGS. 5A and 5B, such grains were not observed. In Comparative Example 2 shown in FIGS. 6A and 6B, the grain size was very large and rough, and the surface pores were large. It can be seen that.

The transmittances of the biomaterial-fixing substrate of Example 1 and the substrate for-fixing the biomaterial of Comparative Example 2 (US Pat. No. 6,168,825) were measured and shown in FIG. 7. As shown in FIG. 7, Example 1 and Comparative Example 2 exhibited opposite transmittances. The substrate of Example 1 had a golden reflective color and was dark purple when viewed in transmission color. On the other hand, the substrate of Comparative Example 2 had a golden reflective color, but the transmission color was dark purple. In addition, the substrate of Example 1 showed a specific absorption peak in the visible region, while the substrate of Comparative Example 2 did not show a specific absorption peak in the visible region and the transmission color was also opaque.

The substrate for fixing a biomaterial of the present invention can produce a large substrate at a low cost, which is essential for mass production, without expensive equipment investment such as vacuum deposition equipment.

Claims (24)

Board; A sensitizing agent layer formed on the substrate; And a gold thin film formed on the sensitizer layer, The gold thin film has a thickness of 30 to 200 nm, and when the gold thin film sample is irradiated with an incident angle of 1.5 degrees exhibits an XRD peak at (111) and (200) plane, The substrate for fixing the biomaterial Coating a coating solution including a sensitizer on the substrate to form a sensitizer layer; Coating a gold colloidal dispersion on the substrate on which the sensitizer layer is formed to form a seed colloidal catalyst layer; Drying or heat-treating the substrate on which the seed colloidal catalyst layer is formed; And A substrate for fixing a biomaterial, which is prepared by coating a substrate with a coating solution consisting of a mixture of a gold salt-containing aqueous solution and a reducing agent solution to obtain a gold thin film. The substrate of claim 1, wherein the substrate is selected from the group consisting of glass, polycarbonate, polyester, polyethylene (PE), polypropylene (PP), and wafers. The method of claim 1, wherein the sensitizer used in the sensitizer layer comprises tin, indium, palladium, germanium, aluminum, lead, titanium, A substrate for fixing a biomaterial, which is selected from the group consisting of gallium, salts thereof, and mixtures thereof. The substrate of claim 3, wherein the salt is chloride or fluoride. The substrate of claim 4, wherein the chloride is selected from the group consisting of PdCl ₂ , PbCl ₂ GeCl ₂ , InCl ₃ , AlCl ₃ , GaCl ₃ , TiCl, and SnCl ₂ . A biochip comprising a biomaterial immobilized on a substrate according to any one of claims 1 to 5. The biochip of claim 6, wherein the biomaterial is selected from the group consisting of enzymes, proteins, DNA, RNA, microorganisms, animal and plant cells and organs, and neurons. Coating a coating solution including a sensitizer on the substrate to form a sensitizer layer; Coating a gold colloidal dispersion on the substrate on which the sensitizer layer is formed to form a seed colloidal catalyst layer; Drying or heat-treating the substrate on which the seed colloidal catalyst layer is formed; And Coating a substrate with a coating solution consisting of a mixture of a gold salt-containing aqueous solution and a reducing agent solution to obtain a gold thin film The method of manufacturing a substrate for fixing a biomaterial according to claim 1. The method of claim 8, wherein the sensitizer is tin, indium, palladium, germanium, aluminum, lead, titanium, gallium, A method for producing a substrate for fixing a biomaterial, which is selected from the group consisting of salts thereof and mixtures thereof. The method of claim 9, wherein the salt is chloride or fluoride. The method of claim 8, wherein the sensitizer is used in an amount of 0.01 wt% to 50 wt% with respect to the coating liquid. The method of claim 8, wherein the coating method of the sensitizer layer is selected from the group consisting of a dipping method, a spraying method, a spin coating method, and a printing method. The method of claim 8, wherein the seed colloidal catalyst layer comprises a gold colloid having a particle diameter of 5 nm to 500 nm. The method of claim 8, wherein the gold colloidal dispersion comprises a gold salt, a reducing agent, a stabilizer, and a solvent. The method of claim 14, wherein the gold salt is selected from the group consisting of HAuCl ₄ , NaAuCl _4, and mixtures thereof. The method of claim 14, wherein the reducing agent is selected from the group consisting of NaBH ₄ , thiocyanate, potassium carbonate, trisodium citrate or hydrates thereof, tannic acid, hydroxyamine or salts thereof and mixtures thereof. At least one method for manufacturing a substrate for fixing a biomaterial. The method of claim 14, wherein the stabilizer is sodium citrate. The method of claim 8, wherein the seed colloid catalyst layer coating method is selected from the group consisting of a dipping method, a spray method, a spin coating method, and a printing method. The method of claim 8, wherein the gold salt used in preparing the gold salt-containing aqueous solution is selected from the group consisting of HAuCl ₄ , NaAuCl _4, and mixtures thereof. The method of claim 8, wherein the gold salt-containing aqueous solution contains 0.01 wt% to 20 wt% gold salt. The method of claim 8, wherein the reducing agent used in the preparation of the reducing agent solution is hydroxyamine or a salt thereof. The method of claim 8, wherein the concentration of the reducing agent solution is 0.01mM to 1M. The method of claim 22, wherein the concentration of the reducing agent solution is 0.01mM to 100mM. The method of claim 8, wherein the coating method for forming the gold thin film is a plating method.

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