KR20050018028A - A method of preparing substrate for immobilizing physiological material - Google Patents

Abstract

PURPOSE: A method for preparing a substrate for immobilizing physiological materials is provided, thereby uniformly distributing the amine immobilizing functional groups on the substrate by pretreatment with strong basic amine compounds, so that immobilization efficiency of physiological materials can be improved. CONSTITUTION: The method of preparing a substrate for immobilizing physiological materials comprises the steps of: pretreating a substrate with strong basic amine compounds; and treating the pretreated substrate with aminosilane compounds, wherein the substrate is glass, wafer, polycarbonate, polyethylene, polypropylene polyester, polystyrene or polyurethane; the strong basic amine compound is primary, secondary or tertiary amine represented by formula (1), NHmRn, wherein R is alkyl, m+n is 3 and n and m are in the range of 0 to 3; the aminosilane compound is represented by formula (2), NH2-R1-Si(OR2)3, wherein R1 is alkyl, aromatic carbohydrate, ether, ester or amine, and R2 is hydrogen, C1-C20 alkyl or phenyl; and the physiological material is enzyme, protein, DNA, RNA, microorganism, cell and organ of animal and plant, and nerve cell.

Description

A method of manufacturing a substrate for fixing a biomaterial {A METHOD OF PREPARING SUBSTRATE FOR IMMOBILIZING PHYSIOLOGICAL MATERIAL}

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a method for manufacturing a substrate for fixing a biomaterial, and more particularly, to a method for manufacturing a substrate for fixing a biomaterial having excellent immobilization efficiency due to uniform distribution of amine-immobilized functional groups.

[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. The biosensor may be classified into a biosensor capable of detecting and analyzing various biochemicals such as a lab-on-a-chip having an analytical 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, 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.

Various methods have been proposed to induce binding between the substrate surface and the probe DNA, and the method of immobilizing biomaterials using a silane compound or aminosilane oligomer having an amine function is most widely applied.

U.S. Patent No. 3,519,538 introduced a method of bonding a biomaterial to a support using a silane coupling agent0 as a coating material of a substrate for immobilizing a biomaterial, and then a self-assembled monolayer of amino silane was introduced. Patents related to the method of forming have been filed and widely used in the manufacture of functional thin films.

According to U.S. Patent No. 5,985,551, immobilization of DNA using polyacrylamide gel increases the chance of hybridization and facilitates analysis. The gel has amino groups or aldehyde groups and forms stable covalent bonds with DNA. According to US Pat. No. 5,869,272, a method of finding bacterial antigens is analyzed by changing the optical properties (colors) of the optically active surfaces constituting the immobilization layer and the protein layer. In this case, the immobilization layer is composed of dendrimers, star polymers, molecular self-assembly polymers, polysiloxanes, latexes, and the like. The forming method is to form amino silane on a silicon wafer by spin coating. Way. U. S. Patent No. 5,919, 523 discloses a method of forming an immobilized layer, which is treated with glycine or serine on an amino silane treated substrate, or a method of coating an amine-based, imine-based, amide-based organic polymer. In addition, WO 02072887 uses hetero-poly-functional saccaride (dextran) to bind primary amines on the surface of the substrate and make strong covalent bonds with the amine groups of DNA and oligonucleotides.

In the above patents, the amino silane compound is a representative example of a compound capable of forming an immobilization layer having a relatively high functional density without generating acid by-products. Much research is underway to form well-defined homogeneous monolayers having high functional density using aminosilane compounds (Abraham Ulman, Chem. Rev., 96, 1533-1554 (1996)). These studies have established a method for forming a silane monolayer on the surface of a substrate. However, it is difficult to obtain a uniform amine functional density, so that the immobilization efficiency is low or inconsistent. In other words, the amine-immobilized functional group of the aminosilane compound has a disadvantage in that the amine-immobilized functional group capable of chemically bonding with the surface of the substrate may react with the probe material and the surface distribution becomes uneven.

In order to solve the problems of the prior art as described above, the present invention is to provide a method for manufacturing a substrate for fixing a biological material excellent in the immobilization efficiency is uniformly distributed amine immobilized functional groups.

Another object of the present invention is to provide a substrate for coating an aminosilane-immobilized compound in which the amine-immobilized functional groups can be uniformly distributed.

In order to achieve the above object, the present invention comprises the steps of pre-treating a strong basic amine compound on the substrate; It provides a method for producing a substrate for fixing a biological material comprising the step of forming an immobilization layer by treating the aminosilane compound on the pre-treated substrate.

The present invention also provides a substrate for coating an aminosilane-immobilized compound in which a strong basic amine compound is treated on a substrate.

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

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

In the present invention, in order to prepare a substrate for fixing a biomaterial to improve the immobilization efficiency is uniformly distributed amine immobilization functional groups pre-treated with a strong amine compound on the substrate and then treated with an aminosilane compound to form an immobilization layer to form a substrate Manufacture.

As a substrate used as a solid support, glass or silicon wafers are commonly used, but hydroxyl groups on the surface of the substrate form covalent bonds with an alkoxy group of the aminosilane compound to form an amine-immobilized layer capable of immobilizing a biomaterial. . The more amine immobilization functional groups, the higher the immobilization efficiency. However, some amine-immobilized functional groups may form hydrogen bonds with hydroxyl groups of the substrate instead of alkoxy groups, thereby reducing the amine-immobilized functional groups capable of reacting with biological materials, resulting in poor immobilization efficiency. In the present invention, the strong base amine compound is first pretreated to a solid substrate to allow the strong base amine compound to react with the alkoxy group of the aminosilane compound, thereby preventing the amine immobilized function from reacting with the hydroxyl group of the substrate, thereby preventing the immobilization ability of the amine immobilized functional group. Improve.

The manufacturing process of the substrate for fixing a biomaterial of the present invention is shown in FIG. 1. First, the strong basic amine compound is reacted with the substrate 1 to be pretreated. As the substrate, an opaque solid substrate such as a transparent solid glass substrate or a silicon wafer which has been cleaned to remove surface organic matter and ensure uniform hydrophilicity, in particular, an environmentally stable or chemically resistant glass or wafer may be preferably used. Polycarbonate, polyethylene (PE), polypropylene (PP) polyester, polystyrene, polyurethane and the like can also be used. It may also be in the form of a microwell plate, tube, spherical particles, porous membrane. Of course, the substrate of the present invention is not limited to that illustrated above.

The strong basic amine compound may be a primary, secondary, or tertiary amine represented by the following Chemical Formula 1.

NH _m R _n (1)

Wherein R is alkyl, preferably methyl, ethyl, propyl, butyl, m + n = 3, n and m are in the range of 0-3.

Preferred specific examples of the strong basic amine compound include N, N-isopropylethylamine, ethylamine, dimethylamine, diethylamine, triethylamine, dipropylamine, and the like, of which N, N-isopropylethylamine, Triethylamine can be preferably used.

Since the strong basic amine compound has a high water solubility, it is preferable to maintain the reactivity of the amine compound to the cleaning substrate by performing the process in a relative humidity environment or a nitrogen environment of less than 25%.

As the coating method of the strong basic amine compound, a deposition method (self-assembled thin film coating method), a spin method, a spray method, a printing method, a CVD (chemical vapor deposition method), etc. can be used.

When the coating method is a wet method, it is preferable to disperse the strong basic amine compound in an organic solvent and coat the substrate. The concentration of the amine compound in the coating composition is preferably 0.1 to 10% by weight, and 0.1 to 3% by weight. More preferred. If the concentration of the amine compound is less than 0.1% by weight, the coating effect does not appear effectively, and when it is more than 10% by weight, it is not preferable because it inhibits uniform surface formation. As the organic solvent, alcohols such as ethanol, ethanol, and the like can be used, such as cellosolves such as methyl cellosolve, dimethylformamide, acetone, toluene or a mixed solvent thereof.

After the coating process, hydrogen bond with the substrate on the surface of the substrate is used. Excess amine compound, physically weakly bound amine compound, and impurities are again removed by washing the substrate with a diluting solvent to remove the hydroxyl groups and the strong substance on the substrate. A uniform bonding layer of the amine of the basic amine compound is formed (step S1 of FIG. 1). Immersion method (self-assembled thin film coating method) is possible if the reaction time of the substrate and the amine compound is 1 minute or more time, spin coating method is the time of 10 seconds or more in the range of 300 rpm ~ 1000 rpm Uniform bonding of amine groups is possible. In addition, washing with a diluting solvent is preferably repeated two or more times to remove the uneven layer.

As described above, the substrate pretreated with the strong basic amine compound may be used as a substrate for coating the aminosilane compound. The surface state of the resulting substrate can be determined by analyzing the light emitted from the dyeing material by continuously irradiating a laser beam after fluorescence isothiocynate treatment.

The strong basic amine compound blocks the amine immobilization functional group from reacting with the hydroxyl group of the substrate and reacts with the alkoxy group of the aminosilane to form an immobilization layer (S2). The amine-immobilized functional group of the substrate for immobilizing the biomaterial according to the present invention does not react with the hydroxyl group of the substrate so that the amine-immobilized functional group is uniformly distributed. Thus, an immobilization layer having a high immobilization capability and a uniform distribution of immobilization functional groups is formed on the substrate.

The aminosilane compound has an amine-based functional material may be represented by the following formula (2). In FIG. 1, R in step S2 may vary depending on the aminosilane compound, and R ¹ of Formula 2 may be used.

NH ₂ -R ¹ -Si (OR ² ) ₃ (2)

Wherein R ¹ is selected from the group consisting of alkyl, aromatic hydrocarbons, ethers, esters, and imines, preferably methyl, ethyl, propyl, or butyl. R ² is selected from the group consisting of hydrogen, an alkyl group having 1 to 20 carbon atoms or a phenyl group.

Preferred examples of the aminosilane compound include 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 2-aminoundecyltrimethoxysilane, aminophenyltrimethoxy Silane (aminophenyltrimethoxysilane), bis (trimethoxysilylpropyl) amine (bis (trimethoxysilylpropyl) amine), N- (2-aminoethylaminopropyl) trimethoxysilane (N- (2-aminoethylaminopropyl) trimethoxysilane), and the like.

In addition, in order to adjust the hydrophobicity of the immobilization layer, a hydrophobic silane compound of the following formula (3) or a silane alkoxide of the following formula (4), or both thereof, may be used in combination with the aminosilane compound to increase the binding force.

(3)

Wherein R ³ is a group consisting of an alkyl group having 1 to 14 carbon atoms, an aromatic group having 6 to 12 carbon atoms or a substituted aromatic group (wherein the substituent is preferably methyl, ethyl or propyl) and CX ₃ (X is halogen) Is preferably methyl, ethyl or propyl, and R ⁴ and R ⁵ are each independently selected from the group consisting of alkoxy, acetoxy, hydroxy and halogen groups having 1 to 14 carbon atoms, preferably methoxy, Oxy, acetoxy or chlorine, R ⁶ is selected from the group consisting of hydrogen, an alkyl group having 1 to 14 carbon atoms and an aromatic group having 6 to 12 carbon atoms, preferably methyl or ethyl, and k is an integer of 1 to 15.

M (OR ⁷ ) _n (4)

Wherein M is at least one element selected from the group consisting of Group 4B, 3A, 4A, and 5A elements of the periodic table, and preferably selected from the group consisting of Si, Zr, Ti, Al, Sn, In and Sb At least one element, R ⁷ is hydrogen, an alkyl group of 1 to 20 carbon atoms or an aromatic group of 6 to 12 carbon atoms, preferably hydrogen, methyl, ethyl, propyl, butyl or phenyl, n is determined according to M It is in the range of 3-4.

Using the hydrophobic silane compound of Formula 3 together with the amine coating material can control the hydrophilicity of the amine layer. Examples of such hydrophobic silane compounds include methyltrimethoxysilane, propyltriacetoxysilane, and the like.

When used together with the silane alkoxide of the formula (4) can increase the binding strength with the substrate. Preferred examples of the compound having the formula (4) include silicon tetraalkoxide such as tetraethylorthosilicate, aluminum tributoxide, zirconium tetrabutoxide and the like.

The amine coating material and the compound of Formula 3 or 4 are preferably used in a weight ratio of 0.01: 99.99 to 100: 0, and more preferably in a weight ratio of 50:50 to 95: 5.

The aminosilane compound may be prepared by coating a silane compound further comprising a compound of Formula 2, optionally a compound of Formula 3 or 4, by adding alcohols such as water, ethanol, methanol, etc. to prepare a coating solution; Stirring the coating solution to condensate the silane compound to obtain an oligomeric hydrate present in the solution, and then by firing the amine coating film obtained by coating it on a substrate pretreated with a strong base amine compound at a temperature of about 100 to 300 degrees 3 An immobilization layer may be formed by forming a dimensional network structure layer.

In addition, the solution containing the aminosilane compound is coated with a wet coating method such as an immersion coating method (self-assembled thin film coating method), a spin coating method, a spray method, a printing method, etc., and then washed with a solvent used in the coating material to clean the surface of the substrate. A method of forming an amine monomolecular film on a substrate by removing impurities and physically weakly bound aminosilane compounds present in the substrate may also be used. Immersion coating method (self-assembled thin film coating method) is possible if the immersion time more than 1 minute and spin coating method is possible in the range of 300 rpm ~ 1000 rpm. In addition, the immobilization layer may also be formed by the CVD method.

Provided is a biochip manufactured by reacting and binding a biomaterial activated to have a biomaterial or a functional group to a substrate for fixing a biomaterial formed as described above, and then cleaning the unreacted biomaterial to form a pattern.

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.

Briefly describing a method of manufacturing a DNA chip in a biochip, a biological material, i.e., an oligonucleic acid, is immobilized on the surface of a substrate by one of the above methods, and the target DNA and a target solution labeled with fluorescent material therefor are 1 to 24 hours. Reaction is performed under a certain degree of accuracy, and the emitted light is signaled by irradiating a laser beam.

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 Substrate Pretreated with Strong Basic Amine Compound

40 ml of N, N-diisopropylethylamine was added to 360 ml of ethanol to prepare a coating solution. Clean soda-lime glass (sodalime glass) was immersed in the coating solution. Coating reaction time was 15 minutes at room temperature. The coated glass was washed with 400 ml of ethanol for 1 minute, and then washed again with 400 ml of ethanol and dried. The pretreated substrate was measured using a laser scanner (packard / scanarray5000. Axon / genefix4000), and the fluorescence was measured after FITC treatment. It can be seen that the amine group is bonded to the glass surface.

Example 2: Preparation of Substrate Pretreated with Strong Basic Amine Compound

A pretreated substrate was prepared in the same manner as in Example 1, except that triethanolamine was used instead of N, N-diisopropylethylamine. The obtained pretreated substrate was measured using a laser scanner (Axon / genefix4000, packard / scanarray5000) and measured the fluorescence intensity after self-luminescence and FITC treatment. It can be seen that the amine group is bonded to the glass surface.

Example 1 (N, N-diisopropylethylamine pretreatment) Example 2 (triethanolamine pretreatment) Cleaning glass (not pretreated) Self-luminous intensity (a.u.) 121.8 123.3 48.4 FITC fluorescence intensity (a.u.) 30591 31824 10015

As shown in Table 1, it can be seen that the amine concentration of the substrate pretreated with the strong basic compound is excellent.

Preparation of DNA Chip and Measurement of Nonspecific Binding Strength

An aminosilane compound was coated on the substrates of Examples 1 and 2 and the substrates of unpretreated cleaning glass (Comparative Example 1) to form an immobilization layer. The aminosilane-immobilized layer was formed by immersing the substrates of Examples 1, 2 and Comparative Example 1 in a coating solution in which 0.5 g of aminopropyltrimethoxysilane was added to 500 ml of methanol, followed by drying at 120 degrees for 1 hour. After cooling to room temperature, a DNA chip was prepared by immobilizing the probe DNA by a spotting method in which the DNA was contacted. The target DNA labeled here was hybridized to analyze a scanned image with an Axon scanner, and the results are shown in FIGS. 2A to 2C. The intensity of the spot was measured in a laser scanner (Axon / genefix4000) and is shown in Table 2 below.

Example 1 Example 2 Comparative Example 1 Spot intensity (a.u.) 4286 5468 2433

As shown in FIGS. 2A to 2C and Table 2, the strengths of the substrates of Examples 1 and 2 treated with the strong basic amine compound were higher than those of Comparative Example 1. This shows that the target DNA binding ability of the substrate pretreated with the strong base amine compound is better, which means that the accuracy of the biomaterial analysis is so high.

In the present invention, by pretreating the strong base amine compound before forming the immobilization layer of the substrate for immobilizing the biomaterial, the amine immobilization functional group can be uniformly distributed, thereby improving the immobilization efficiency.

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

Figures 2a to 2c is a view showing a scan image photograph after the hybridization reaction of the DNA chip prepared using the substrate prepared according to Example 1, 2 and Comparative Example 1, respectively.

Claims (16)

Pretreating a strongly basic amine compound on the substrate; A method of manufacturing a substrate for fixing a biomaterial, comprising: forming an immobilization layer by treating an aminosilane compound on the pretreated substrate. The method of claim 1, wherein the substrate is glass, a wafer, polycarbonate, polyethylene (PE), polypropylene (PP) polyester, polystyrene, or polyurethane. The method of claim 1, wherein the strongly basic amine compound is a primary, secondary, or tertiary amine represented by the following Chemical Formula 1. NH _m R _n (1) Wherein R is alkyl, m + n = 3, n and m are in the range of 0-3. The biomaterial of claim 3, wherein the strongly basic amine compound is selected from the group consisting of N, N-isopropylethylamine, ethylamine, dimethylamine, diethylamine, triethylamine, dipropylamine, and mixtures thereof. Method of manufacturing a substrate for fixing. The method of claim 1, wherein the strong base amine compound is coated by a deposition method (self-assembled thin film coating method), spin method, spray method, printing method, or CVD (chemical vapor deposition) method Way. The method of claim 1, wherein the aminosilane compound is a compound of Formula 2 below. NH ₂ -R ¹ -Si (OR ² ) ₃ (2) Wherein R ¹ is selected from the group consisting of alkyl, aromatic hydrocarbons, ethers, esters, and migration, R ² is selected from the group of hydrogen, carbon atoms consisting of 1 to 20 alkyl group or a phenyl group. According to claim 6, wherein the aminosilane compound is 3-aminopropyltrimethoxysilane (aminopropyltrimethoxysilane), 3-aminopropyltriethoxysilane (aminopropyltriethoxysilane), 2-aminoundecyltrimethoxysilane (aminoundecyltrimethoxysilane), aminophenyl Trimethoxysilane, bimethoxy (trimethoxysilylpropyl) amine, N- (2-aminoethylaminopropyl) trimethoxysilane (N- (2-aminoethylaminopropyl) trimethoxysilane), and Method for producing a substrate for fixing a biomaterial that is selected from the group consisting of a mixture thereof. The method of claim 1, wherein the immobilization layer further comprises a compound selected from the group consisting of an aminosilane compound and a hydrophobic silane compound of formula (3), a silane alkoxide of formula (4), and mixtures thereof Method for producing a substrate for use. (3) Wherein R ³ is selected from the group consisting of an alkyl group having 1 to 14 carbon atoms, an aromatic group having 6 to 12 carbon atoms or a substituted aromatic group (wherein the substituent is methyl, ethyl or propyl) and CX ₃ (X is halogen) R ⁴ and R ⁵ are each independently selected from the group consisting of alkoxy, acetoxy, hydroxy and halogen groups having 1 to 14 carbon atoms, and R ⁶ is hydrogen, an alkyl group having 1 to 14 carbon atoms and an aromatic group having 6 to 12 carbon atoms. Selected from the group consisting of: k is an integer from 1 to 15; M (OR ⁷ ) _n (4) Wherein M is at least one element selected from the group consisting of 4B, 3A, 4A, and 5A group elements of the periodic table, R ⁷ is hydrogen, an alkyl group having 1 to 20 carbon atoms or an aromatic group having 6 to 12 carbon atoms, n Depends on M and is in the range of 3 to 4. The method of claim 1, wherein the strong basic amine compound is coated with a coating composition dispersed in an organic solvent, the organic solvent is ethanol, alcohols such as ethanol, cellosolves such as methyl cellosolve, dimethylformamide, acetone, Method for producing a substrate for fixing a biomaterial that is selected from the group consisting of toluene and mixtures thereof. A biochip comprising a biomaterial immobilized on a substrate prepared according to any one of claims 1 to 9. The biochip of claim 10, wherein the biomaterial is selected from the group consisting of enzymes, proteins, DNA, RNA, microorganisms, animal and plant cells and organs, and neurons. A biosensor comprising the biochip of claim 10. A substrate for coating an aminosilane compound pretreated with a strong basic amine compound on the substrate. The substrate of claim 13, wherein the substrate is glass, wafer, polycarbonate, polyethylene (PE), polypropylene (PP) polyester, polystyrene, or polyurethane. The substrate of claim 13, wherein the strongly basic amine compound is a primary, secondary, or tertiary amine represented by Formula 1 below. NH _m R _n (1) Wherein R is alkyl, m + n = 3, n and m are in the range of 0-3. The substrate of claim 15, wherein the strongly basic amine compound is selected from the group consisting of N, N-isopropylethylamine, ethylamine, dimethylamine, diethylamine, triethylamine, dipropylamine, and mixtures thereof.