KR20110055904A - Substrate for supporting bioactive materials, fabrication method thereof, and biochip comprising the same - Google Patents

Abstract

PURPOSE: A substrate for fixing bioactive materials and a method for manufacturing the same are provided to produce cheap plastic biochips with high reliability. CONSTITUTION: A substrate for fixing bioactive materials comprises: a plastic substrate(10); a silicon thin film(3) of silicon nitride, silicon oxy-nitride, or silicon oxide thin film; a self-assembled thin film(5) having a hydrophilic group. The thickness of the silicon thin film is 1-100nm. The hydrophilic group contains a hydroxyl group, amine group, aldehyde group, carboxyl group, or mixture thereof. A method for manufacturing the substrate comprises: a step of forming the silicon film on the plastic substrate; a step of performing oxygen plasma process to enhance hydroxyl group density on the surface of the silicon thin film; and a step of forming the self-assembled thin film on the silicon thin film.

Description

Substrate for supporting bioactive materials, fabrication method including, and biochip comprising the same}

The present invention relates to a substrate for fixing a biomaterial suitable for mass production of a plastic biochip that can be fixed at a high density and improves a signal-to-noise ratio, can be analyzed with high reliability, and is inexpensive, and a method for manufacturing the same and a biochip having the same. .

Biochips are integrated circuits made up of tiny electrical elements, and have a structure in which genes of living organisms, DNA, proteins, antibodies, etc., which make up these genes are attached to small thin films made of inorganic materials. The biochip has a high density of biomolecules (genetic materials) integrated on a solid surface, so that a large amount of bioinformation can be searched in a short time.

The types of biochips are divided into DNA chips, protein chips, and cell chips according to the types of biomolecules attached to the solid, and related to antibodies, receptors, ligands, nucleic acids, and carbohydrates that can react with these DNA, proteins, or cells. The material is immobilized at high density on a single chip to find information about DNA, proteins or cells.

Currently, biochips are used for various clinical diagnosis such as blood test and blood sugar analysis, and are also used for genetic analysis and new drug development. Furthermore, it can be applied to water and marine pollution measurement, pollutant detection, biochemical weapon detection, and biometric recognition system.

The substrate material of the biochip may be an optically transparent substrate which immobilizes the biomaterial at a high density and forms a stable bond between the substrate and the biomaterial.

As a substrate material, a glass material such as soda lime is most widely used. The glass substrate has an advantage of easy surface modification, excellent optical properties, and smooth flow of fluid in the channel, thereby facilitating various types of analysis. However, the micro process is very difficult compared to silicon, and the process (cleaning, photoresist coating, photolithography, etching, cleaning) is very expensive. In addition, since the solution used for etching during the process is a strong acid, a strong base, there are various problems such as environmental pollution, such as waste generation, it is not easy to mass-produce biochips using glass substrates.

In order to replace the glass substrate, various materials such as metal, inorganic materials, or plastic materials have been proposed as substrate materials.

In particular, plastic materials have many advantages such as processability, optical properties, ease of processing, and mass production cost reduction, and thus, many studies have been conducted as commercial materials. When plastic is used as a substrate, a variety of methods such as injection molding, laser ablation, imprinting, hot embossing are available, thereby increasing the choice of processing. In addition, it has the advantage of being able to form a surface favorable to the fluid flow, inexpensive and relatively free to choose a process.

However, even with this advantage, plastic substrates have a hydrophobic surface as a hydrophobic functional group in the molecular structure (76 ° contact angle for PMMA), and this surface property causes low wettability of aqueous solution, resulting in a problem that fluid flow is not smooth. do. In addition, most biomaterials may cause nonspecific adsorption due to hydrophobic surfaces, hydrophobic bonds, and van der Waals bonds, which may degrade performance when used as a fluid chip. Moreover, it is difficult to fix the biomaterial on the surface of the plastic substrate, and as a result, the reliability of the analysis result is greatly reduced.

In order to solve this problem, various modification methods have been proposed to increase the wettability or adhesion of the substrate surface by introducing a polar functional group to the surface of the plastic substrate.

Modification of plastic substrates is largely divided into physical surface modification method and chemical surface modification method.

Physical surface modification methods include polymer surface treatment using flame; Corona Discharge Treatment; Plasma Treatment; Ablation and Transfor-mation; There is a method such as ultraviolet irradiation. In the case of a microfluidic channel made of PMMA or PC, which is a representative plastic substrate, a method of first introducing COOH and OH functional groups onto a surface through ultraviolet / ozone treatment is known. However, this physical surface modification method has a problem in that the treatment process is simple, but the surface modification of the plastic is temporary and converted back to hydrophobic nature when used for a long time, or in some cases, the apparatus is expensive.

Chemical surface modification methods also include metal deposition (Metalization); Plasma Polymerization Using Plasma; Polymer grafting; Hydrolysis; Self-assembled thin film deposition method (SAM deposition) and the like. The chemical surface modification method has the advantage that the modification is permanent, but in the case of self-assembled thin film and the like has a low adhesion to the substrate, there is a limit of mass production application due to the sensitivity of the process and waste.

In order to solve the above problem, the present inventors have conducted various studies on the method of immobilizing a biomaterial using a plastic material as a substrate and mass production of biochips, and as a result, an appropriate combination of physical and chemical surface modification methods of plastics The present invention was completed by finding out that the hydrophilic modification of the plastic substrate is permanent and harmless to the environment, and mass production is possible.

Accordingly, an object of the present invention is to provide a substrate for fixing a biomaterial in which a hydrophilic functional group is present at a high density so that the biomaterial can be fixed at a high density.

In addition, another object of the present invention is to provide a method for manufacturing a substrate for fixing a biomaterial, in which surface modification of the substrate is maintained for a long time and mass production is applicable.

In addition, another object of the present invention is to provide a biochip that can be analyzed with high reliability through the fixing of various biological materials including DNA, proteins and antibodies.

In addition, another object of the present invention is to provide a biochip that can be easily immobilized in a variety of biological materials including DNA, proteins and antibodies, and can be analyzed with high reliability through hydrophilic high wettability that facilitates fluid flow. .

It is another object of the present invention to improve the signal to noise ratio by preventing the biomaterial from non-specific adsorption by the hydrophobic surface and the hydrophobic bond and van der Waals bond through hydrophilic modification of the surface.

In order to achieve the above object,

Plastic substrates;

At least one silicon thin film selected from silicon nitride, silicon oxynitride, or silicon oxide thin films; And

Provided is a substrate for fixing a biomaterial having a structure in which a self-assembled thin film having a hydrophilic group is sequentially stacked.

In addition,

Forming one silicon thin film selected from silicon nitride, silicon oxynitride, or silicon oxide thin film on the plastic substrate;

Performing an oxygen plasma treatment to increase the density of hydroxyl (-OH) on the silicon thin film surface; And

Forming a self-assembled thin film having a hydrophilic group on the silicon thin film

It provides a method of manufacturing a substrate for fixing a biological material comprising a.

The present invention also provides a biochip in which a biomaterial is fixed to the substrate for fixing the biomaterial.

The substrate for fixing a biomaterial according to the present invention modifies the surface of the hydrophobic plastic substrate with hydrophilicity, and the hydrophilicity is permanently maintained to enable high density of biomaterial immobilization.

The substrate for fixing the biomaterial may be applied to various biochips such as an array chip or a fluid chip using a protein, DNA, RNA, antigen, antibody, or enzyme.

The biochip can be fixed at a high density of biomaterials, so that the signal-to-noise ratio is improved and can be analyzed with high reliability. These biochips enable the production of low-cost plastic biochips due to various process selections using plastic substrates.

The present invention is described in more detail below.

1 is a view showing a substrate for fixing a biomaterial according to an embodiment of the present invention. Referring to FIG. 1, the biomaterial fixing substrate 10 may include a plastic substrate 1; Silicon thin film 3; And a self-assembled thin film 5 having a hydrophilic group R sequentially stacked.

The plastic substrate 1 is a transparent polymer and includes one material selected from the group consisting of ester polymers, silicone polymers, acrylic polymers, olefin polymers, and copolymers thereof. Preferably, polyethylene terephthalate, polybutylene terephthalate, polysilane, polydimethylsiloxane, polysilazane, polyacrylate, polymethyl methacrylate, polyethyl acrylate, polyethyl methacrylate, polyethylene naphthalate, inter Click olefin polymers, polyethylene, polypropylene, polyimide, polystyrene, polyacetal, polyetheretherketone, polyestersulfone, polytetrafluoroethylene, polyvinylchloride, polyvinylidene fluoride, perfluoroalkyl polymers, and these One selected from the group consisting of a combination is possible. More preferably, as the plastic substrate 1, polymethyl methacrylate (PMMA), cyclic olefin polymer (COC) or polyethylene terephthalate (PET) is used.

In this case, the plastic substrate 1 is shown as a sheet-like substrate in the drawing, but has a variety of forms and structures in which a flexible film, a sheet-type with a plurality of microwells, or a pattern such as a flow channel is formed. In one example, the microwells are arranged vertically and horizontally at the same interval on the plastic substrate 1, so that the microwells can be applied to the array chip. The microwells have various shapes of cylindrical, polyhedron, inverted cone, or inverted pyramidal shape, and 10-10000 are formed on the substrate, and the number and size of the microwells may vary depending on the application.

However, since the plastic substrate 1 exhibits hydrophobicity and is not easy to fix the biomaterial, the silicon thin film 3 is formed on the plastic substrate 1 so that the surface of the substrate 10 for fixing the biomaterial is hydrophilic. To be modified. The silicon thin film 3 may be one selected from silicon nitride, silicon oxynitride, or silicon oxide thin film, and preferably silicon oxide.

In the case of PMMA, a typical plastic substrate material, the contact angle is hydrophobic at 76 °, whereas in the case of silicon oxide thin film, hydroxyl (-OH) is formed on the surface by contact with air, and the contact angle is 0 ° by these hydroxyl groups, resulting in extremely hydrophilicity. Have However, the hydroxyl group formed on the surface of the silicon thin film has a low density, making it difficult to effectively fix the biomaterial. Therefore, in the present invention, as described later, the density of the hydroxyl group (-OH) on the surface of the silicon thin film 3 is increased by 2 to 10 times through oxygen plasma treatment. This high density hydroxyl group can further increase the density of the self-assembled thin film formed subsequently, and as a result, it is possible to further increase the fixed density of the biomaterial combined with the hydrophilic group in the self-assembled thin film. Preferably, it has extremely hydrophilicity at 0 degrees even after the oxygen plasma treatment.

The thickness of the silicon thin film 3 according to the present invention is not particularly limited in the present invention, but it is preferable to have a thickness of 1 to 100 nm, preferably 5 to 50 nm so as to sufficiently modify the hydrophobic property of the plastic substrate 1. Do. If the thickness is less than the above range, the hydrophilic modification of the substrate is insufficient. If the thickness exceeds the above range, problems such as cracking occur, so that the thickness is appropriately formed within the above range.

The self-assembled thin film 5 having a hydrophilic group is formed on the silicon thin film 3 to be bonded to a biomaterial. The self-assembled thin film 5 has a relatively long alkyl group in the molecular structure, molecules having a functional group capable of covalent bonding by interacting with a plurality of hydroxyl groups (-OH) present on the surface of the silicon thin film 3 at the end thereof. The monolayer is formed through a self-assembly phenomenon which is two-dimensionally aligned on the surface of the silicon thin film 3. The self-assembled thin film 5 (SAM) has a hydrophilic group at one end that does not bond with the silicon thin film 3 (-OH), an amine group (-NH ₂ ), and an aldehyde group (-C (= O) as a hydrophilic functional group. H), a carboxyl group (-C (= 0) OH) and combinations thereof. In the case of proteins or nucleic acids, the main component is an amino acid, and biomaterials such as proteins introduced into a sample to be analyzed are stably fixed to the substrate 10 due to the hydrophilic functional groups present on the surface of the self-assembled thin film 5.

The self-assembled thin film 5 according to the present invention has a very high bonding force and has a stable and uniform surface to easily bond with the bioactive material to fix the bioactive material, and is formed to a thickness of 1 to 10 nm.

The material constituting the self-assembled thin film 5 is not particularly limited in the present invention and may be any material as long as the material can form the self-assembled thin film. Preferably, the self-assembled thin film 5 may be a material represented by the following Chemical Formula 1.

[Formula 1]

R ^1- (CH ₂ ) n-Si- (R ² ) ₃

(Wherein R ¹ = OH, NH ₂ , COH, or COOH, R ² is Cl or alkoxy of OC _m H _{2m + 2} , n is 1 to 6, m is 1 to 6)

The compound of Formula 1 may be trichlorosilane or trialkoxysilane, more preferably, aminopropyltriethoxysilane (3-aminopropyltriethoxysilane), aminoethylaminopropyltrimethoxysilane (N- (2-aminoethyl)- 3-aminopropyltrimethoxysilane), (3-aminopropyl) trimethoxysilane, 3-Aminopropyl (diethoxy) methylsilane, triethoxysilylpropyl essenionic hydride ((3-triethoxysilylpropyl 1) in the group consisting of succinic anhydride) and combinations thereof.

The compound of Chemical Formula 1 is fixed vertically to the silicon thin film 3 through chemical bonding with OC _m H _{2m + 2} , which is a Cl or alkoxy group in the molecule, and a hydroxyl group (-OH) of the silicon thin film 3, and is not fixed. The hydroxyl group (-OH), the amine group (-NH ₂ ), the aldehyde group (-C (= O) H), and the carboxyl group (-C (= O) OH) are exposed to the substrate surface to impart hydrophilicity to the substrate. .

In addition, it is possible to modify the surface of the self-assembled thin film 5 with other suitable hydrophilic functional groups according to the object to be analyzed. For example, in the exemplary embodiment of the present invention, a hydrophilic thin film having an amine group on the surface of aminopropyltriethoxysilane may be treated with glutaaldehyde to modify the surface to have an aldehyde group by reacting the amine group with glutaaldehyde. The material for the surface treatment is not particularly limited in the present invention, and any material may be used as long as it is a material known in the art such as glutaraldehyde, acetic acid, and the like.

The modified biomaterial-fixing substrate 10 has a hydrophilic characteristic of a contact angle of 25 to 48 °, thereby facilitating the smooth flow of fluid on the plastic channel and the fastening of biomaterials such as proteins and DNA on the surface, and being permanent. Surface modification is possible. In addition to this, in addition to the plastic substrate, it is possible to modify the surface of all materials regardless of the material to a hydrophilic surface having excellent bio stability.

The preparation of the substrate for fixing the biomaterial as described above is performed through performing various deposition and coating processes, that is, a dry process and a wet process.

Substrate for fixing a biomaterial according to an embodiment of the present invention

Forming one silicon thin film selected from silicon nitride, silicon oxynitride, or silicon oxide thin film on the plastic substrate;

Performing an oxygen plasma treatment to increase the density of hydroxyl (-OH) on the silicon thin film surface; And

It is prepared through the step of forming a self-assembled thin film having a hydrophilic group on the silicon thin film.

Hereinafter, each step will be described in more detail with reference to the accompanying drawings. 2 is a view showing a manufacturing procedure of a substrate for fixing a biomaterial.

First, a hydrophobic plastic substrate 1 is prepared to manufacture a substrate for fixing a biomaterial (FIG. 2A). If necessary, the plastic substrate 1 performs an IPA cleaning process using ultrasonic waves to remove foreign substances or impurities before use.

The plastic substrate 1 uses various forms that can be applied to a biochip such as a fluid chip or an array chip such as a sheet, a film, a microwell, a substrate on which a channel channel is formed.

Next, a silicon thin film 3 such as silicon nitride (SiN), silicon oxynitride (SiON) and silicon oxide (SiO) is formed on the surface of the plastic substrate 1 (FIG. 2B).

Formation of the silicon thin film 3 may be a conventional dry deposition method and wet deposition method, for example, dry deposition such as sputtering, ion beam deposition, chemical vapor deposition, and plasma deposition, or sol-gel coating, dip coating, doctor A wet coating method such as a blade method, spin coating, or spray coating is possible, preferably plasma deposition (PECVD).

Formation of the silicon thin film 3 by plasma deposition method includes positioning the plastic substrate 1 in the deposition process chamber, performing a plasma treatment process on the plastic substrate 1, and a silicon-containing gas, NH ₃ ,. It consists of a continuous step of depositing the silicon thin film 3 on a substrate with a gas selected from the group consisting of nitrogen containing gas, oxygen containing gas, and combinations thereof at a temperature of 100 ° C. or less.

At this time, it is possible to form the silicon thin film 3 having various compositions by adjusting the plasma processing gas type, the plasma processing gas flow rate, the plasma power level, the plasma pressure, and the plasma processing time.

In the case of forming the silicon oxide with the silicon thin film 3, the plasma is treated with an inert gas including Ar, He, Xe, Kr, and a mixed gas thereof, followed by RF power of 5 to 1000 W and 0.1 to 5.0 Torr. 40-200 ° C. under pressure (process temperature is related to the Tg point of the plastic), preferably at 80 ° C., a silicon-containing gas such as SiH _{4 at} a flow rate of 50-600 sccm and an oxygen-containing gas such as N ₂ O Can be deposited by flowing at a flow rate of 50-1000 sccm.

The silicon nitride (SiN) thin film is formed by applying RF power of 10 to 1000 W and injecting a silicon-containing gas such as SiH _{4 at} a flow rate of 100 to 500 sccm and a nitrogen-containing gas such as NH _{3 at} a flow rate of 100 to 500 sccm.

In addition, the silicon oxynitride (SiON) thin film has an RF power of ₅ to 600 W, a silicon containing gas such as SiH _{4 at} a flow rate of 10 to 500 sccm, an oxygen containing gas such as N ₂ O at a flow rate of 10 to 1000 sccm, and / Alternatively, vapor deposition is possible by flowing a nitrogen-containing gas such as N _{2 at} a flow rate of 10 to 1000 sccm.

Since the silicon thin film 3 has good adhesion to the plastic substrate 1 and is deposited on the order of tens of nanometers, the transparency of the plastic substrate 1 may be maintained to some extent.

Next, the silicon thin film 3 is subjected to an oxidative plasma treatment to increase the density of hydroxyl (—OH) in the surface of the silicon thin film 3 (FIG. 2C).

Oxidized plasma treatment is not particularly limited in the present invention, it is possible to perform a known method, it is preferable to perform at room temperature and normal pressure in consideration of using a plastic material as a substrate.

For example, after placing the plastic substrate 1 on which the silicon thin film 3 is formed in the chamber of the plasma processing apparatus, an inert gas and an active gas are applied by applying a voltage to generate a plasma to generate a hydroxyl group in the surface of the silicon thin film 3. -OH) is introduced.

The inert gas uses argon (Ar) or helium (He), and the active gas may be oxygen to form a hydroxyl group, these are inert gas and functional group-containing gas in 99: 1 to 95: 5 volume ratio Is preferably injected into the plasma generating apparatus at a flow rate of 1-20 L / min.

The applied voltage at the time of plasma generation is preferably performed at 100 to 600 W, preferably at 300 W, and the plasma treatment is preferably performed at 0.1 to 10 mm / sec for 0.1 to 5 minutes. In this case, if the plasma treatment speed or treatment time is less than the above range, sparks are generated at the electrode, and if the plasma exceeds the above range, plasma is not effectively generated.

According to this process, a hydroxyl group, which is a polar functional group, is introduced on the surface of the silicon thin film 3 to further increase the hydrophilicity of the silicon thin film 3. In particular, as the present invention performs the oxygen plasma treatment process, the density of the self-assembled thin film to be formed later is higher by about 2 to 10 times higher than the density of hydroxyl groups present on the silicon thin film formed by simple PECVD or general wet coating. It can be raised further.

Next, the self-assembled thin film 5 is formed on the silicon thin film 3 on which the high-density hydroxyl group (-OH) group is formed (FIG. 2D).

The self-assembled thin film 5 is formed using a self-assembled thin film method using a solution containing one hydrophilic group selected from the group consisting of a hydroxyl group, an amine group, an aldehyde group, a carboxyl group and a combination thereof.

If necessary, as shown in Fig. 3, the self-assembled thin film 5 is further modified to have a specific modified hydrophilic group R '. At this time, the modified hydrophilic group R 'may be OH, NH ₂ , COH, COOH, -CONH-, -SO ₃ H -OSO ₃ H -PO (OH) ₂ -NH- and the like.

The self-assembled thin film 5 includes a specific hydrophilic group required in the field in which the biochip is to be used.

Specifically, the silane compound of Formula 1 is added to a diluting solvent to prepare a coating composition, and then coated on the silicon thin film 3 using a wet coating method, followed by drying to form a thin film.

The diluent solvent may be water, an organic solvent or a mixed solvent thereof, and the organic solvent may be alcohol such as methanol, ethanol, propanol, isopropanol, butanol, methyl cellulsolve, ethyl cellulsolve, dimethylformamide, dimethylformacetate, and the like. , Acetone and the like are possible.

The density of the functional group of the self-assembled thin film is treated with dyeing materials such as isothiocyanate, fluorescein isothiocyanate (FITC) activated with succinimide ether, tetraethylrhodamine isothiocyanate (STN-TMR), and SIE-succinimide (tetramethylrhodamine succinimide). By labeling the laser beam continuously irradiated to distinguish the light caused by the dye material. Density analysis results (surface component analysis) It was confirmed that the substrate for fixing the biomaterial prepared according to the present invention can form a very stable immobilized functional group at a high density uniformly.

In addition, the present invention provides a biochip comprising a biomaterial fixed to the substrate for fixing the biomaterial.

The biochip may be applied to various fields, such as IT (Information Technology), BT (Bio Technology), ET (Environment Technology), NT (nano technology), AT (Agriculture Technology) or a fusion thereof. For example, the high sensitivity array chip can be used for analyzing biomolecules such as proteins and nucleic acids.

That is, the biomaterial may be any one that can be fixed by reacting with a hydrophilic functional group present on the surface of the self-assembled thin film, that is, amine, carboxylic acid, or the like. For example, it may be a biochip including an array chip or a fluid chip which is a protein, peptide, DNA, RNA, antigen, antibody, enzyme, microorganism, animal or plant cells and organs, nerve cells.

Such a biochip improves signal to noise ratio by preventing the biomaterial from non-specific adsorption by hydrophobic and hydrophobic bonds and van der Waals bonds through hydrophilic modification of the surface.

In the case of biomaterials that can directly react with functional groups on the surface of the self-assembled thin film, the biomaterials are immobilized by stirring in the PBS buffer solution for 1 to 24 hours at room temperature. However, if there is no functional group that reacts directly, an additional modification process is performed. For example, glutaaldehyde is added to an amino group-bonded biomaterial-fixing substrate, stirred for 1 to 24 hours, washed to introduce an aldehyde group, and then a biomaterial. It is placed in this containing PBS buffer solution and fixed by stirring for 1 to 24 hours at room temperature.

The biomaterial can be fixed to the substrate with high density. In addition, in the case of scanning and analyzing the entire chip by maintaining the flatness of less than a micrometer unit, the same measurement as in the case of using a conventional planar chip can be performed.

In addition, an expensive arrangement device is not required, and since the biomaterial can be immobilized easily in a desired pattern, a user-friendly platform can be manufactured.

Hereinafter, preferred examples and experimental examples of the present invention are presented. However, the following examples are only preferable examples of the present invention, and the present invention is not limited to these examples.

Experimental Example 1

Manufacture of Biomaterial Substrate

The PMMA substrate was pretreated with isopropyl alcohol and ultrasonic waves and then mounted in a PECVD chamber. 5% SiH ₄ / He was injected with 150sccm (process conditions) and 700sccm of N ₂ O gas, and then reacted at 70 ° C., 1000 mtorr, and 30 W for 2 minutes and 20 seconds to deposit a 2 nm thick silicon oxide thin film.

Subsequently, oxygen plasma treatment was performed by injecting oxygen in the chamber at 100 sccm and 300 W for 1 minute under normal temperature and atmospheric pressure. Next, the APTES (aminopropyltriethoxysilane) solution (3% and 6%, respectively) was nitrogen-dried using a liquid self assembled monolayer (L-SAM) to form a self-assembled thin film having a thickness of 2 nm to form PMMA / SiO _2. A substrate for fixing a biomaterial composed of a / amine SAM layer was prepared (see FIG. 4A).

Protein fixation

The substrate for fixing the biomaterial consisting of the PMMA / SiO ₂ / amine SAM layer was immersed in glutaaldehyde solution (25%) for 24 hours, washed with distilled water, and modified with an aldehyde group present on the surface with an aldehyde group (FIG. 4B). Reference).

Subsequently, a solution containing BSA protein (CY5-BSA) labeled with CY5 fluorescent chromosome in PBS buffer solution was prepared, and the substrate was immersed therein, followed by stirring at room temperature for 1 hour to fix the labeled protein on the substrate.

XPS Analysis

XPS analysis (X-ray photonelectron spectroscopy) was performed to determine the chemical bonding state of the SAM interface in the prepared biomaterial fixing substrate.

Figure 5 (a) is PMMA, PMMA / SiO ₂ , the XPS graph of the substrate for fixing the biomaterial (APTES 3%), the substrate for fixing the biomaterial (APTES 6%) and (b) is an enlarged view thereof. Referring to Figure 5, it can be seen that there exists a nitrogen element except the hydrogen element of low mass. In addition, the enlarged view, the content of N atoms derived from APTES is 4.52 ~ 4.54% level, it can be seen that formed as a monolayer (SAM) on the SiO ₂ thin film.

Experimental Example 2

Contact angle measurement

When manufacturing a substrate for fixing a biomaterial, APTES concentrations were prepared at 3% and 6%, respectively, and then the substrates were immersed for 10 minutes to 120 minutes to confirm the change in contact angle over time to examine the change in hydrophilicity.

6 is an image showing a change in contact angle according to the concentration and processing time of the APTES solution, it can be seen that the contact angle of the substrate has a contact angle in the hydrophilic range of 27 ° to 48 °.

Fluorescence intensity measurement

The prepared biomaterial-fixing substrate was labeled with a fluorescent dye CY5 to irradiate a laser beam, and the emitted light was detected using a Scanarray (fluorescence scanner) to measure the fluorescence intensity.

7 is an image showing the change in fluorescence intensity with the concentration of APTES solution and the treatment time. At this time, the higher the value of the fluorescence intensity shown in Figure 6 is the number of amine groups fixed on the surface, the presence of such a plurality of amine groups means that the presence of a large number of OH groups on the surface of the bottom SiO ₂ .

As shown in FIG. 7, the hydrophilicity of the substrate is improved as the processing time is longer than the concentration of APTES, and the amine group, which is an active group, is stably maintained at high density as compared with 560 of PMMA alone and 840 of SiO ₂ of Comparative Example 1. Able to know.

Experimental Example 3

In Experimental Example 1, the stability test of the functional group of the substrate prepared by treating with APTES (3%) for 120 minutes was performed. Table 1 below shows the change in contact angle after being left for 2 weeks after substrate manufacture.

1 day 3 days 7 days 14 days Contact angle 43 ° 47 ° 46 ° 46 °

As shown in Table 1, it can be seen that even after two weeks, the contact angle does not show a large change, so that the amine group on the surface is stably fixed on the SiO ₂ thin film, so that the hydrophilic property is maintained permanently instead of temporarily.

Au nanoparticle test

Figure 8 is a photograph of the Au nanoparticle test was performed after 30 days. As shown in FIG. 8, it can be seen that even after 30 days, the amine group is stably fixed to the substrate due to the bonding force due to the electrical charge (charge interaction) between Au and the amine surface.

Experimental Example 4

Fluorescence intensity measurement

The oxygen plasma treated / untreated substrate was treated with an APTES solution of 3% for 1 hour to form a self-assembled thin film, and labeled with a fluorescent dye CY5 to irradiate a laser beam and emit light with a Fluorescence Scanner. The fluorescence intensity was measured after detecting the fluorescence image.

As a result, the fluorescence intensity au was 11561 for the oxygen plasma-treated substrate, and was measured at 984 for the untreated substrate. This value means that a high density hydroxyl group is formed on the SiO ₂ thin film through oxygen plasma, and the number of amine groups bonded to the formed hydroxyl group is high. The oxygen plasma treatment improves the hydrophilicity of the substrate and improves the sensitivity of the biochip. It can be seen that even higher.

Experimental Example 5

A fluid chip was manufactured using the substrate for fixing a biomaterial according to the present invention, and the fluid flowability thereof was measured. At this time, the manufacture of the fluid chip was carried out in cooperation with SD Co., Ltd. as a subject of the Defense Science Research Institute.

FIG. 9 is a view showing the flowability of a fluid chip made of PMMA only and a fluid chip made of a substrate on which a PMMA / SiO ₂ / amine SAM layer is deposited.

Referring to FIG. 9, it can be seen that the flowability of the fluid chip modified with the SiO ₂ / amine SAM layer according to the present invention is very good, and the flowability of the unmodified fluid chip is greatly reduced.

Experimental Example 6

Instead of PMMA, PET and COC were used as a plastic substrate to prepare a substrate for fixing a biomaterial, and then, a BSA protein (CY5-BSA) labeled with a CY5 fluorescent chromosome was fixed on the surface thereof. Table 2 below is an image showing the fluorescence intensity of the Bare substrate on the PMMA, COC and PET substrate and the substrate modified with SiO ₂ / amine SAM layer.

Fluorescence intensity (a.u) PMMA COC PET Unmodified substrate 560 3402 785 Modified substrate 13412 12376 14787

Referring to Table 2, it can be seen that in the case of the surface-modified substrate, the luminescence intensity is much superior, so that a larger amount of protein is fixed to the substrate. This result means that the hydrophobicity of the plastic substrate can be modified more effectively with hydrophilicity by increasing the density of hydroxyl groups through the formation of SiO ₂ thin film and oxygen plasma treatment.

The substrate for fixing a biomaterial according to the present invention may be used as a biochip to analyze biomolecules such as proteins or nucleic acids.

1 is a three-dimensional cross-sectional view showing a substrate for fixing a biomaterial according to an embodiment of the present invention.

2 (a) to (d) is a flow chart showing a manufacturing procedure of the substrate for fixing a biomaterial.

Figure 3 is a three-dimensional cross-sectional view showing a substrate for fixing a biomaterial according to another embodiment of the present invention.

Figure 4 (a) is an image showing the manufacturing procedure of the substrate for fixing the biomaterial in Experimental Example 1, (b) is a view showing the fixing of the protein.

Figure 5 (a) is PMMA, PMMA / SiO ₂ , the XPS graph of the substrate for fixing the biomaterial (APTES 3%), the substrate for fixing the biomaterial (APTES 6%) and (b) is an enlarged view thereof.

6 is an image showing a change in contact angle with the concentration of APTES solution and the treatment time.

7 is an image showing the change in fluorescence intensity with the concentration of APTES solution and the treatment time.

Figure 8 is a photograph of the Au nanoparticle test was performed after 30 days.

FIG. 9 is a view showing the flowability of a fluid chip made of PMMA only and a fluid chip made of a substrate on which a PMMA / SiO ₂ / amine SAM layer is deposited.

Claims (13)

Plastic substrates; At least one silicon thin film selected from silicon nitride, silicon oxynitride, or silicon oxide thin films; And A substrate for fixing a biomaterial having a structure in which self-assembled thin films having a hydrophilic group are sequentially stacked. The method of claim 1, The plastic substrate may be polyethylene terephthalate, polybutylene terephthalate, polysilane, polydimethylsiloxane, polysilazane, polyacrylate, polymethylmethacrylate, polyethylacrylate, polyethyl methacrylate, polyethylene naphthalate, Cyclic olefin polymers, polyethylene, polypropylene, polyimide, polystyrene, polyacetal, polyetheretherketone, polyestersulfone, polytetrafluoroethylene, polyvinylchloride, polyvinylidene fluoride, perfluoroalkyl polymer, and Substrate for fixing a biomaterial that comprises one selected from the group consisting of these. The method of claim 1, The silicon thin film is a substrate for fixing a biomaterial will have a thickness of 1 to 100nm. The method of claim 1, Wherein the hydrophilic group is a substrate for fixing a biological material containing one species selected from the group consisting of hydroxyl group, amine group, aldehyde group, carboxyl group, and combinations thereof. The method of claim 1, The self-assembled thin film is R ^1- (CH ₂ ) n -Si- (R ² ) ₃ (wherein R ¹ = OH, NH ₂ , COH, or COOH, R ² is Cl or OC _m H _{2m + 2} And alkoxy, n is 1 to 6, and m is 1 to 6), which is trichlorosilane or trialkoxysilane. The method of claim 1, The self-assembled thin film is pentyl trichlorosilane (PETCS), phenyltrichlorosilane (Phenyltrichlorosilane (PTCS), benzyltrichlorosilane (BZTCS), toytrichlorosilane (Tolyltrichlorosilane (TTCS), 2-[( Trimethoxysilyl) ethyl] -2-pyridine (2-[(trimethoxysilyl) ethl] -2-pyridine (PYRTMS)), 4-biphenylyltrimethowysilane (BPTMS), octadecyltrichloro Silane (Octadecyltrichlorosilane (OTS), 1-naphthyltrimehtoxysilane (NAPTMS), 1-[(trimethoxysilyl) methyl] naphthalene (1-[(trimethoxysilyl) methyl] naphthalene: MNATMS) and (9-methylanthracenyl) trimethoxysilane (MANTMS) comprising a substrate selected from the group consisting of biomaterials for fixing. Forming one silicon thin film selected from silicon nitride, silicon oxynitride, or silicon oxide thin film on the plastic substrate; Performing an oxygen plasma treatment to increase the density of hydroxyl (-OH) on the silicon thin film surface; And Forming a self-assembled thin film having a hydrophilic group on the silicon thin film Method of manufacturing a substrate for fixing a biological material comprising a. The method of claim 7, wherein The silicon thin film may be formed by dry deposition including sputtering, ion beam deposition, chemical vapor deposition, and plasma deposition, or by wet coating including sol-gel coating, dip coating, doctor blade method, spin coating, or spray coating. Method of manufacturing a substrate for fixing a biological material that is carried out by one of the coating method. The method of claim 8, The plasma deposition method is a step of performing a plasma treatment process using an inert gas containing Ar, He, Xe, Kr or a mixture of these on a plastic substrate, and silicon-containing gas, NH ₃ , nitrogen-containing gas, oxygen-containing A method of manufacturing a substrate for fixing a biomaterial, wherein a silicon thin film is deposited on a substrate at a temperature of 100 ° C. or less with a gas mixture selected from the group consisting of a gas and a combination thereof. The method of claim 7, wherein The oxygen plasma treatment is performed by using an inert gas of Ar, He, Xe, Kr at 20 to 100 ° C., and then injecting oxygen at a flow rate of 50 to 5000 sccm under a pressure of 0.5 to 5 torr for 1 minute. Method for producing a substrate for fixing a phosphorous biomaterial. The method of claim 8, The self-assembled thin film is a method of manufacturing a substrate for fixing a biomaterial, which is prepared by coating and drying a solution containing one hydrophilic group selected from the group consisting of a hydroxyl group, an amine group, an aldehyde group, a carboxyl group, and a combination thereof. Claim 1 substrate for fixing the biomaterial, and Biochip comprising a biomaterial fixed to the substrate. The method of claim 12, The biomaterial is a protein, DNA, RNA, antigen, antibody or enzyme that is a biochip.

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