KR101050690B1 - Multifunctional polymer membrane and its use - Google Patents

Abstract

The present invention relates to a process for preparing a solid substrate comprising a reactor coated with a polymeric film on a surface and exhibiting anti-biofouling properties and capable of forming covalent bonds: (a) (i) ) A first monomer of a polymer comprising a surface-anchoring site bonded to a surface of the solid substrate, (ii) a second monomer to which a polymer exhibiting anti-biofouling properties is bound, and (iii) Reacting with a third monomer comprising a reactive group capable of reacting with the first monomer to produce a polymer comprising a surface-anchor moiety, a protein-resistant moiety and a reactive moiety; And (b) coating the surface of the solid substrate with the polymer.

Substrate, biochip, biosensor, self-assembled monolayer

Description

{Multi-Functional Polymeric Layers and Their Uses}

The present invention relates to a method for producing a solid substrate, a solid substrate, a biochip or a biosensor and a novel polymer used for coating a solid substrate.

It is of great interest to generate biologically active substances (e.g., biotin, DNAs, saccharides, peptides and proteins) ¹ and cells on a solid phase substrate ² because such patterns are useful for biosensor ³ It is used for high-speed screening analysis system and has excellent applicability ⁴ . In particular, SiO ₂ that is currently being used for the study or diagnosis of diseases of the proteome - Building a protein-based substrate on the microarray is an area that is larger attention ^5. In general, two functionalities are required for the successful construction of a protein array: i) a bioactive surface to immobilize the probe protein; And ⅱ) low signal-to-noise ratio (S / N ratio) Surface biologically inactive (anti-bio-fouling) to minimize nonspecific adsorption of unwanted proteins that cause ^6. Conventional strategies for achieving this objective have been based on the discovery that one of the conventional strategies for achieving this goal is to use a surface reactive group (i.e., trimethoxy or trichlorosilane) at one end and a functional group (i.e., amine, alcohol, oligethylene glycol) at the other end Self-assembled monolayers (SAMs) formed by mono-balant compounds. ⁷ . However, that SAMs are different from those ⁸ directly formed by using a mono-balance agent silicone compound forming the SAMS on a SiO ₂ substrate on the gold surface, and often as well to generate aggregates (polymer hwadon siloxane) on the surface and it is causing a lot of shortcomings, which leads to poor reproducibility of results ^9. Therefore, it is very important to develop a new platform capable of specifically immobilizing a biological material on a SiO ₂ substrate while suppressing nonspecific adsorption.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made an attempt to improve the substrate surface coating which is an underlying technology of biosensors and biochips. Particularly, efforts are being made to provide a biosensor and a biochip with excellent analytical ability and reproducibility by effectively immobilizing biomolecules on a substrate surface while suppressing nonspecific adsorption, and significantly improving signal-to-noise. Respectively. As a result, a polymer was prepared using three types of monomers each having surface-anchoring, anti-biofouling, and reactive sites, coated on a solid substrate, and immobilized on a biomolecule to confirm excellent properties. It was completed.

Accordingly, an object of the present invention is to provide a method for producing a solid substrate comprising a reactor coated with a polymer film on a surface thereof and exhibiting anti-biofouling properties and capable of forming a covalent bond.

It is another object of the present invention to provide a dual functional solid substrate comprising surface-anchoring, anti-biofouling and reactive sites.

It is still another object of the present invention to provide a biochip or a biosensor.

It is another object of the present invention to provide a novel polymer which is used for coating a solid substrate.

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

According to one aspect of the present invention, the present invention provides a method for producing a solid substrate comprising a reactor coated with a polymer film on a surface including the following steps and exhibiting anti-biofouling properties and capable of forming a covalent bond to provide:

(i) a first monomer of a polymer comprising a surface-anchoring site bonded to a surface of the solid substrate, (ii) a second monomer to which the polymer exhibiting anti-biofouling properties is bound, and (iii) Reacting a third monomer comprising a reactor capable of forming a covalent bond to produce a polymer comprising a surface-anchor site, a protein-resistant site, and a reactive site; And

(b) coating the surface of the solid substrate with the polymer.

According to another aspect of the present invention, the present invention provides a dual functional solid substrate coated with a polymer formed by the polymerization of the following monomers and comprising surface-anchoring, anti-biofouling and reactive sites: (I) a first monomer comprising a surface-anchoring moiety attached to the surface of the solid substrate, (ii) a second monomer to which the polymer exhibiting anti-biofouling properties is bound, and (iii) ≪ / RTI >

The present inventors have attempted to improve the substrate surface coating, which is a base technology of biosensors and biochips. Particularly, efforts are being made to provide a biosensor and a biochip with excellent analytical ability and reproducibility by effectively immobilizing biomolecules on a substrate surface while suppressing nonspecific adsorption, and significantly improving signal-to-noise. Respectively. As a result, polymers were prepared using three types of monomers having surface-anchoring, anti-biofouling, and reactive sites, respectively, and the polymer was coated on a solid substrate and immobilized with biomolecules.

Biomolecules (proteins, peptides, nucleotides or sugars) or chemicals are attached to the surface of a solid substrate used in a biosensor or a biochip for analysis. TECHNICAL FIELD The present invention relates to a technique for attaching biomolecules or the like to the surface of such solid substrate, and is carried out by coating a polymer on the substrate surface.

The polymers of the present invention are prepared using three monomers. The three monomers include (i) a surface-anchoring site, (ii) an anti-biofouling site, and (iii) a reactive site through which the dual functionality, i.e., anti-biofouling and attachment of biomolecules .

Of the three monomers, the first monomer comprises a surface-anchoring moiety that is bonded to the surface of the solid substrate.

What is available as the first monomer is that various monomers of the polymer may be used and include, for example, acrylic acid, acrylonitrile, allylamine, methacrylic acid, alkyl acrylate, alkyl methacrylate, butadiene, (Meth) acrylate of polydimethylsiloxane, ethylene, ethylene glycol, propylene glycol, (ether) urethane, urethane, vinyl chloride, vinyl alcohol, maleic anhydride, cellulose chloride, vinyl alcohol But are not limited to, cellulose nitrate, carboxymethyl cellulose, dextran, dextran sulfate, propylene, ester, carbonate, ether, butene, maleic acid, fluoropolymer monomer units, unsaturated polymer monomers, isoprene, melamine, Protein, gelatin, protein, gelatin, elastin, Butyl methacrylate, butyl methacrylate, hydroxyethyl methacrylate, polyethylene glycol dimethacrylate, polypropylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, N-acryloxy succinimide, glycidyl methacrylate, Dicyclohexyl methacrylate, diallyl methacrylate, hexamethylene diisocyanate, acrolein, glycerol monomethacrylate, heparin methacrylate, methacryloylethylphosphoryl choline, butyl acrylate, polyethylene glycol monomethacrylate, isobutyl methacrylate, Ethyl acrylate, 2-ethylhexyl methacrylate, ethyl methacrylate, methyl acrylate, hexadecyl methacrylate, octadecyl methacrylate, styrene, methyl Styrene, vinyl toluene, and tert-butyl acrylate Hydroxybutyrate, 3-hydroxyvalerate, and 3-hydroxypyrrolidone, such as, for example, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ≪ / RTI > hydroxyhexanoate, and the like.

Preferably, the first monomer of the present invention is an acyl or methacrylic monomer, and may be, for example, methacrylic acid, acrylic acid, alkyl methacrylates (e.g., methyl methacrylate, ethyl methacrylate, butyl methacrylate (E.g., methyl acrylate, ethyl acrylate and tert-butyl acrylate), aryl acrylate (e.g., methyl acrylate, ethyl acrylate and butyl acrylate), isobutyl methacrylate, hexadecyl methacrylate, octadecyl methacrylate and cyclohexyl methacrylate) (E.g. benzyl acrylate), aryl methacrylate (e.g., benzyl methacrylate), 2-ethylhexyl methacrylate, lauryl methacrylate, hydroxyl ethyl methacrylate, PEG acrylate, PEG methacrylate Acrylate, hydroxypropyl methacrylate, hydroxypropyl methacrylamide, 3-trimethylsilylpropyl methacrylate Acrylonitrile, acrylate of polydimethylsiloxane, methacrylate of polydimethylsiloxane, glycidyl methacrylate, glycerol monomethacrylate, heparin methacrylate, glycerol monomethacrylate, But are not limited to, polyethylene glycol monomethacrylate, 2-hydroxyethyl acrylate, and 2-ethylhexyl methacrylate.

According to a preferred embodiment of the present invention, the first monomer of the present invention includes a substituted or unsubstituted silane group, a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group as a surface-anchoring site.

The substituted or unsubstituted silane group in the first monomer forms a covalent bond with the hydroxyl group of the solid phase substrate (e.g., Si / SiO ₂ wafer) to anchor the polymer film to the solid phase substrate.

The first monomer may have an unsubstituted silane group, i.e., SiH ₄ , which is anchored to the solid substrate surface. Preferably, the first monomer has a substituted silane group, through which the polymer is anchored to the solid substrate surface.

When the surface-anchoring site in the first monomer is a substituted silane group, it is preferably a silane group substituted with an alkoxy group (i.e., an alkoxysilyl group), more preferably a silane group substituted with a C ₁ -C ₆ alkoxy group. The term alkoxy in the present specification means an -O alkyl group. The term alkoxysilyl means an alkoxy substituted silyl group, preferably trimethoxysilyl and triethoxysilyl, more preferably trimethoxysilyl.

The first monomer may have a silane group substituted with a hydroxy group (i.e., a hydroxysilyl group). The term hydroxysilyl means a hydroxy-substituted silyl group, preferably a trihydroxysilyl group.

According to a preferred embodiment of the present invention, the first monomer may comprise a substituted or unsubstituted alkyl group. Such alkyl groups impart hydrophobicity to the first monomer such that the polymer is anchored to the solid substrate (e.g., a polystyrene substrate having a hydrophobic surface) through hydrophobic interactions and / or dipole interactions.

Preferably, the alkyl group in the first monomer is a C _{6 -30} alkyl group, more preferably a C _{8 -30} alkyl group. The term alkyl means a straight or branched saturated hydrocarbon group having the designated carbon number. When the alkyl group is substituted, it is preferably F, Cl or Br, more preferably F.

According to a preferred embodiment of the present invention, the first monomer may comprise a substituted or unsubstituted aryl group. This aryl group allows the polymer to be anchored to a solid substrate (e.g., a polystyrene substrate having a hydrophobic surface) via pp interaction. The term aryl means a substituted or unsubstituted monocyclic or polycyclic carbon ring which is totally or partially unsaturated and is preferably monoaryl or biaryl. The monoaryl preferably has from 5 to 6 carbon atoms, and the biaryl preferably has from 9 to 10 carbon atoms. Most preferably, the aryl is substituted or unsubstituted phenyl. When monoaryl, such as phenyl, is substituted, substitution can be made by various substituents at various positions, but is preferably selected from halo, hydroxy, nitro, cyano, C ₁ -C ₄ Substituted or unsubstituted straight or branched alkyl, C ₁ -C ₄ straight or branched chain alkoxy, alkyl substituted sulfanyl, phenoxy, C ₃ -C ₆ cycloheteroalkyl or substituted or unsubstituted amino groups .

The basic structure of the second monomer used in the present invention is the same as that of the first monomer. Preferably, the second monomer is a methacrylate-based or acrylate-based monomer. The description of the methacrylate-based or acrylate-based monomer cites the description of the first monomer.

The second monomer has a polymer that exhibits anti-biofouling properties. Preferably, the polymer exhibiting anti-biofouling properties is selected from the group consisting of polyethylene glycol (PEG), polyalkylene oxide (e.g., polyoxyethylene, polyoxypropylene or copolymers thereof such as polyethylene oxide-polypropylene oxide- (Methoxyethyl methacrylate), poly (methacryloylphosphatidylcholine), perfluorinated polyether, dextran, or the like), polyphenylene oxide, copolymers of PEG and polyalkylene oxide, poly Polyvinylpyrrolidone, more preferably PEG, a polyalkylene oxide or a copolymer of PEG and polyalkylene oxide, and most preferably PEG.

The basic structure of the third monomer used in the present invention is the same as that of the first monomer. Preferably, the second monomer is a methacrylate-based or acrylate-based monomer. The description of the methacrylate-based or acrylate-based monomer cites the description of the first monomer.

The third monomer is coupled with a reactor. The reactor is capable of reacting with proteins, peptides, nucleotides (DNA or RNA), sugars or chemicals to form covalent bonds. Thus, the reactor in the third monomer comprises various reactors. For example, the reactor in the third monomer can be an aldehyde; Epoxy; Haloalkyl; Primary amine; Thiol; Maleimide; Esters (preferably N-hydroxysuccinimide ester functional groups); And activated reactors such as a carboxyl group (activated by hydroxy-succinimide ester formation) and a hydroxyl group (activated by cyanogen bromide). Most preferably, the third monomer comprises a carboxyl group as a reactor.

When the third monomer comprises a carboxyl group as a reactor, preferably the carboxyl group is selected from the group consisting of succinimide, succinamidyl ester, sulfo-succinamidyl ester, 2,3,5,6-tetrafluorophenol ester, 4 - sulfo-2,3,5,6-tetrafluorophenol esters, aldehydes, acid anhydrides, azides, azolides, carboyimides, epoxides, esters, glycidyl ethers, halides, imidazoles or imidates . Most preferably, the carboxyl group as the reactor is activated by succinimide or succinimidyl ester.

According to a preferred embodiment of the present invention, the polymer coated on the solid substrate is represented by the following formula:

Wherein R ₁ is selected from the group consisting of a silylalkyl group, an (alkoxysilyl) alkyl group, a (hydroxysilyl) alkyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, A substituted alkaryl group; R ₂ is at least one selected from the group consisting of PEG (polyethylene glycol), polyalkylene oxide, polyphenylene oxide, a copolymer of PEG and polyalkylene oxide, poly (methoxyethyl methacrylate), poly (methacryloylphosphatidylcholine) Lt; / RTI > polyether, dextran or polyvinylpyrrolidone; R ₃ is an aldehyde, an epoxy, a haloalkyl, a primary amine, a thiol, a maleimide, an ester, a carboxyl group or a hydroxyl group; R ₄ , R ₅ and R ₆ independently of one another are H or a C ₁ -C ₅ alkyl group; X, Y and Z are independently of each other an oxygen, sulfur or nitrogen atom; l, m and n are independently integers from 1 to 10,000.

The term silyl alkyl used in the definition of R ₁ in formula (I) refers to an alkyl group substituted with a substituted silyl group. For example, silylalkyl includes silylmethyl, silylethyl, silylpropyl, and silylbutyl. The term (alkoxysilyl) alkyl means an alkoxy-substituted silylalkyl group. For example, (alkoxysilyl) alkyl is an alkoxysilyl group such as trimethoxysilylethyl, trimethoxysilylpropyl, trimethoxysilylbutyl, trimethoxysilylpentyl, triethoxysilylethyl, triethoxysilylpropyl, triethoxysilylbutyl, Trimethoxysilylpentyl, methyldimethoxysilylethyl, methyldimethoxysilylpropyl, dimethylmethoxysilylethyl, and dimethylmethoxysilylpropyl. The term (hydroxysilyl) alkyl means a hydroxyl-substituted silylalkyl and includes, for example, trihydroxysilylethyl, trihydroxysilylpropyl, trihydroxysilylbutyl and trihydroxysilylpentyl.

The term " aralkyl " means an aryl group bonded to the structure by one or more alkyl groups, such as a benzyl group and a phenylpropyl group. The term " alkaryl " means an alkyl group attached to the structure consisting of one or more aryl groups.

Preferably, R ₁ in the formula (1) is an (alkoxysilyl) alkyl group, a substituted or unsubstituted C ₆ -C ₃₀ alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, group-, more preferably (C _{1 -6} alkoxysilyl) C _{1 -10} as the alkyl group, substituted or unsubstituted C ₆ a _-30 alkyl group, a substituted or unsubstituted phenyl group, a substituted or unsubstituted aralkyl group unsubstituted C _{1 - 5} alkyl group, or a substituted or unsubstituted alkaryl group in which a C _{1 -5} alkyl group is bonded to a phenyl group, most preferably a (C _{1 -3} alkoxysilyl) C _{1 -5} alkyl group, a substituted or unsubstituted a phenyl group as an unsubstituted C _{8 -20} alkyl groups, substituted or unsubstituted phenyl group, a substituted or unsubstituted aralkyl group being bonded to the phenyl group in C _{1 -5} alkyl group, or a substituted or unsubstituted alkaryl group C _{1 - 5} < / RTI >

The coating of the polymer prepared by the three monomers described above on the solid substrate surface can be carried out through various methods known in the art. For example, a solution of the polymer can be coated by impregnation of the solid substrate for a suitable period of time.

According to a preferred embodiment of the present invention, the polymer coated on the solid substrate is in the form of a self-assembled monolayer on a solid substrate.

The self-assembled monolayer formed on the solid substrate has a thickness of 0.8-2 nm, preferably 0.9-1.3 nm.

According to a preferred embodiment of the present invention, the method comprises the steps of (c) adding a chemical substance, a protein, a peptide, a nucleotide (DNA or RNA), a peptide nucleic acid 0.0 > (PNA) < / RTI > or sugar.

Binds a protein, a peptide, a nucleotide (DNA or RNA), a sugar or a chemical substance to a reactor derived from a third monomer of a polymer film coated on a solid substrate.

For example, when the reactor is an activated carboxyl group, it can be covalently bonded through an amine group such as a protein. When the reactor is an amine group, it can be covalently bonded through a carboxyl group such as a protein.

In addition, proteins, peptides, nucleotides and sugars can be indirectly bound to the reactor. For example, a substance having affinity to a protein, a peptide, a nucleotide or a sugar is first bound to a reactor, and then a protein (e.g., streptavidin or avidin) is bonded to an affinity substance (biotin) Covalently bonded) to the reactor.

When a biomolecule or the like is immobilized on a solid substrate through a self-assembled membrane, the biomolecule can be immobilized by the following method: contactless printing such as contact pin printing, ink jet printing and aerosol printing, capillary printing, micro contact printing , Pad printing, screen printing, silk screening, microwaving or spraying.

Any solid substrate that can be used in the present invention can be used regardless of the surface properties. For example, the solid substrate is a metal (e.g., alloys of gold, gold and copper, aluminum num), metal oxides, glass, ceramic, quartz, silicon, semiconductor, Si / SiO ₂ wafer, germanium, gallium arsenide, But are not limited to, carbon, carbon nanotubes, polymers (e.g., polystyrene, polyethylene, polypropylene, polyacrylamide), sepharose, agarose, and colloids.

When the surface-anchoring site in the first monomer is a substituted or unsubstituted silane group, it is preferred to use glass, metal oxide, ceramic, quartz, silicon, semiconductor and Si / SiO ₂ wafer substrates. When the surface-anchoring site in the first monomer is a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group, the carbon, the carbon nanotubes and the polymer (e.g., polystyrene, polyethylene, polypropylene, polyacrylamide) Is preferably used.

According to the present invention, it is possible to stably coat a polymeric self-assembled monolayer on a solid substrate and suppress adsorption of molecules other than a desired biomolecule due to the anti-biofouling property, thereby increasing the signal-to- The biomolecule of interest can be immobilized to the solid substrate in a stable and patterned form through the reactor in the polymer.

According to another aspect of the present invention, there is provided a biochip or a biosensor including the above-described solid substrate of the present invention.

In the biochip or biosensor of the present invention, the biomolecule is immobilized on the solid substrate through the reactor of the self-assembled monolayer polymer.

The biochip of the present invention is preferably a DNA microarray or a protein chip. A general description applicable to the biochip of the present invention can be found in U.S. Patent Nos. 5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783, 5,412,087, 5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839, 5,599,695, , 5,631,734, 5,795,716, 5.83107 million, 5,837,832, 5,856,101, 5,858,659, 5,889,165, 5,936,324, 5,959,098, 5.96874 million, 5,974,164, 5,981,185, 5,981,956, 6,025,601, 6.03386 million, 6,040,193, 6,090,555, 6,136,269, 6,147,205, 6,262,216, 6,269,846, 6,310,189 and 6,428,752 hoge specifically described .

The general contents applicable to the biosensor of the present invention are described in Myska, J. Mol . Recognit , 12: 279-284 (1999); J. Dubendorfer et al. Journal of Biomedical Optics , 2 (4): 391-400 (1997); And O'Brien et al. Biosensors & Bioelectronics, 14: 145-154 (1999).

According to another aspect of the present invention, the present invention provides a polymer represented by the following formula (1): < EMI ID =

Formula 1

In the above formula, R ₁ is a silylalkyl group, an (alkoxysilyl) alkyl group, a (hydroxysilyl) alkyl group, a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group; R ₂ is at least one selected from the group consisting of PEG (polyethylene glycol), polyalkylene oxide, polyphenylene oxide, a copolymer of PEG and polyalkylene oxide, poly (methoxyethyl methacrylate), poly (methacryloylphosphatidylcholine) Lt; / RTI > polyether, dextran or polyvinylpyrrolidone; R ₃ is an aldehyde, an epoxy, a haloalkyl, a primary amine, a thiol, a maleimide, an ester, a carboxyl group or a hydroxyl group; R ₄ , R ₅ and R ₆ independently of one another are H or a C ₁ -C ₅ alkyl group; X, Y and Z are independently of each other an oxygen, sulfur or nitrogen atom; l, m and n are independently integers from 1 to 10,000.

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

(a) The invention uses polymers with three functionalities, surface-anchoring, anti-biofouling and reactive sites.

(b) According to the present invention, a polymeric self-assembled monolayer can be stably coated on a solid substrate.

(c) The present invention has excellent anti-biofouling properties and can greatly improve the signal-to-noise.

(d) According to the present invention, a desired biomolecule can be fixed to a solid substrate in a stable and patterned form through a reactor of a polymer used.

(e) According to the present invention, by controlling the amount of the three monomers used, bioreactive functionality and anti-biofouling functionality for binding of biomolecules can be controlled.

(f) According to the present invention, the SAM can be formed on the surface of the solid substrate irrespective of the type of the substrate. In particular, the SAM can be formed with excellent efficiency on the surface of the polymer substrate.

(g) According to the present invention, it is possible to provide a biosensor or a biochip with greatly improved signal-to-noise.

As described in detail above, the present invention utilizes polymers with three functionalities, surface-anchoring, anti-biofouling and reactive sites. According to the present invention, a polymeric self-assembled monolayer can be stably coated on a solid substrate, and excellent anti-biofouling properties can be obtained, thereby greatly improving signal-to-noise. According to the present invention, desired biomolecules can be fixed to a solid substrate in a stable and patterned form through a reactor of a polymer to be used, and by controlling the amount of the three monomers used, bioreactive functionality, and anti-biofouling functionality. In addition, according to the present invention, a SAM can be formed on the surface of a solid substrate regardless of the type of the substrate, and SAM can be formed with excellent efficiency, particularly on the surface of the polymer substrate. According to the present invention, it is possible to provide a biosensor or a biochip with greatly improved signal-to-noise.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

Experimental material

N-Acryloxy succinamide (NAS 99%) was purchased from ACROS Organics (Noisy-le-Grand, France). 3- (trimethoxysilyl) propyl methacrylate, poly (ethylene glycol) methyl ether methacrylate (average Mn = ca. 475), dodecyl methacrylate, Zonyl ^TM TM fluoro-monomer (average Mn = ca. 534), benzyl methacrylate, methacrylic acid and 2,2'-azobisisobutyronitrile (AIBN) were purchased from Aldrich Chemical Co. (Milwaukee, Wis.). (+) - all were purchased bond streptomycin from Pierce (Rockford, IL) - biotinyl-tree -3,6,9- oxa undecane diamine (biotin -NH _2), and fluorescein. All organic solvents were used as purchased without further purification. Si / SiO ₂ All substrates used in experiments such as wafers, poly (dimethyl siloxane) (PDMS) and glass slides were cleaned with detergent and then washed several times with deionized water and methanol. Prior to forming the polymer film, all substrates were treated with O ₂ plasma for 1 minute to clean the surface and form an -OH group.

Measure

¹ H NMR (300 MHz) spectrum was recorded with JEOL JNM-LA300WB FT-NMR (Tokyo, Japan). Waters 1515 series isocratic pump, and a Rheodyne Model 7725 injector with a 100 [mu] l injection loop at a flow rate of 0.4 mL / min were subjected to organic phase gel permeation chromatography (GPC). The thickness of the monolayer film was measured at a 70 ^° incident angle using a Gaertner L116A ellipsometer (Gaertner Scientific Corporation, IL). A refractive index of 1.46 was used for all films, and thickness was calculated using a three-phase model. The contact was measured using a phoenix300 equipped with a video camera and monitor, and a contact angle & surface tension analyzer (Surface electro optics, Kyonggi, Korea). X-ray photoelectron spectroscopy (XPS) spectra were obtained using a Kratos AXIS Ultra Imaging X-ray photoelectron spectrometer with a monochromatinated Al K x-ray source.

Of poly (TMSMA-r-PEGMA-r-NAS)  synthesis

Prior to the polymerization reaction, the knitted PEGMA was passed through an inhibitor removal column (Aldrich Chemical Co.). To a vial containing PEGMA (1.425 g, 3 mmol, 1 equiv) and TMSMA (0.744 g, 3 mmol, 1 equiv), NAS (0.507 g, 3 mmol, 1 equiv), and AIBN (16.5 mg, 0.1 mmol, 0.01 equiv) And dissolved in tetrahydrofuran (anhydrous, inhibitor-free, 99.9%, 10 mL). The mixture was degassed from the mixture using an Ar gas stream for 20 minutes and then the vial was sealed with a Teflon-lining screw-cap. Polymerization was carried out at 70 DEG C for 24 hours. After evaporation of the solvent under vacuum, the polymer was obtained in an adhesive liquid state. Copolymers with different molar ratios of the bioactive groups (NAS: 33%, 20%, and 10%) were prepared. The initial feeding ratios of the three monomers were 1: 1: 1, 2: 2: 1 and 4: 5: 1 for poly 1, poly 2 and poly 3, respectively. To calculate the substantial proportion of the three polymers present in the final copolymer, the integrated number of peaks at δ = 4.13 (CO ₂ -C H ₂ at PEGMA) in the ¹ H NMR spectrum, δ = 3.92 (CO2-C H ₂ at TMSMA) was compared to the integrated value of the peak at δ = 2.82 (CO-C H ₂ -C H ₂ -CO at NAS). ¹ H NMR of the polyester _{1 (300 MHz, CDCl 3)} : δ = 4.13 (br, 2H, CO 2 -C H 2 of PEGMA), 3.92 (br, 2H, CO 2 -C H 2 of TMSMA), 3.66 ( s, 30H), 3.63-3.55 (s , 9H; m, 2H), 3.40 (s, 3H), 2.82 (br, 4H, CO-C H 2 C H 2-CO of NAS) 2.0-1.71 (br, 6H), 1.04 (br, 2H), 0.87 (br, 4H), 0.66 (br, 2H). Poly 1 (Mn = 15784 with Mw / Mn = 1.58), Poly 2 (Mn = 17245 with Mw / Mn = 1.71) and Poly 3 (Mn = 16571 with Mw / Mn = 1.67).

Bipolar ( amphipathic ) Synthesis of polymer

Poly - 4 was synthesized by using dodecyl methacrylate at the anchoring position. Dithyl methacrylate (4 mmol, 1.016 g, 2 equiv), PEGMA (4 mmol, 1.9 g, 2 equiv) and methacrylic acid (2 mmol, 0.172 g, 1 equiv) - lack). The mixture was degassed by bubbling with N ₂ for 15 minutes. 0.1 mmol AIBN (16.4 mg, 0.05 equiv) was added as a radical initiator and the vial was sealed with Teflon-lining screw-cap. Polymerization was carried out at 70 DEG C for 24 hours. The final product was stored at 4 ° C. Poly 5 and poly 6 were prepared by substituting dodecyl methacrylate at the anchor position using fluoromonomer and benzyl methacrylate. (Mn = 19581 with Mw / Mn = 1.88), poly-5 (Mn = 21831 with Mw / Mn = 1.97) and poly-6 (Mn = 16255 with Mw / Mn = 1.91). ^{1 H NMR (300.40 MHz, CDCL} 3): poly-4 δ = 4.05 (br, 2H, CO2-CH2 of PEGMA), 3.82 (br, 2H, CO 2 -CH 2 of dodecylMA), 3.66 (s, 30H) , 3.40 (s, 3H), 2.0-1.71 (br, 10H), 1.5-1.1 (br, 20H), 0.87 (br, 3H); poly-6? = 7.30 (s, 5H), 4.93 (br, 2H, CO ₂ -CH ₂ of benzylMA), 4.05 (br, 2H, CO 2 -CH 2 of PEGMA), 3.66 (s, 30H), 3.40 (s, 3H), 2.0-1.71 (br, 10H), 0.87 (br, 3H), 0.72 (br, 4H).

Bipolar ( amphipathic ) Polymer Polystyrene series  Deformation of plastic surface

THF was evaporated using a vacuum pump. After evaporation, a viscous liquid polymer was obtained. The poly 4-6 polymer is well soluble in water because a large number of PEG groups are present. The polystyrene substrate was impregnated with an aqueous solution of bipolar polymer (20 mg / mL) at ambient temperature and washed with pure water to form pSAMs of the bipolar polymer on the polystyrene-based substrate.

Polymer coated plastic surface anti- Biofouling  effect

Nonspecific binding of proteins on plastic surfaces occurs mainly by hydrophobic interactions. In order to evaluate the protein-resistance of the polymer (poly-4, poly-6) coating, the polymer coated plastic surface was impregnated with bovine serum albumin (BSA) solution (0.25 mg / mL in PBS, pH 7.4) for 2 hours . The degree of nonspecific adsorption of proteins on the plastic surface was analyzed by high resolution N (1s) XPS spectra.

EDC / NHS Activation of polymer coated surface using

For specific immobilization of biomolecules, polymer coated plastic surfaces were activated. The carboxylic acid group of poly 4-6 was converted to the NHS ester group using EDC / NHS. A 1: 1 mixture of EDC (0.4 M) and NHS (0.1 M) in distilled water was applied for 20 minutes to activate the polymer coated plastic surface COOH groups.

Biological Ligand  Micro contact printing ( Microcontact printing : μ CP )

After inking, the amine-terminal biotin ligand (biotin-NH ₂ , 10 mM in ethanol) was printed for 60 seconds by contacting the PDMS stamp on PSAMS. The sample was then immediately impregnated with borate buffer (pH 9.0) for 2 hours. After generation of the biotin pattern, the sample was impregnated with a solution of tetramethylrhodamine isothiocyanate (TRITC) -conjugated streptavidin (0.1 mg / mL) in phosphate buffered saline (PBS, pH 7.4) at room temperature. After 60 minutes, the sample was removed and washed several times with PBS and distilled water. (Leica Microsystems AG, Wetzlar, Germany) equipped with a 40, 100, 200 (1.4 NA) oil objective and a TRITC-optimized filter set (Omega Optical Inc, Brattleboro, (? _Ex = 488 nm,? _Em = 520 nm) of the streptavidin was visualized by using a fluorescence microscope. Images were acquired using a CoolSNAPfx CCD camera operated by MetaMorph imaging software (Universal Imaging Co., Downingtown, PA, USA). For poly 4-6, a Leica DMRBE microscope (Leica Microsystems AG, Wetzlar, Germany) equipped with 200x, 400x objective and TRITC-optimized filter sets (Omega Optical Inc, Brattleboro, VT, USA) Pattern (? _Ex = 547 nm,? _Em = 572 nm) was visualized.

Amphibian  Deformation of polymer surface by polymer ELISA  Applications

Surface coating of the amphiphilic polymer was performed using a 96 well flat-bottom assay plate. 100 [mu] L (20 mg / mL) of the amphiphilic polymer in water was added to each well and allowed to stand at ambient temperature for 1 hour. The wells were then washed four times with 150 [mu] l of deionized water and soaked for 30 seconds between each wash. To activate the -COOH group in the reactive site, 100 μl EDC (400 mM) and NHS (100 mM) were added to each well, left at 27 ° C. for 2 hours, and then washed four times. 100 μl of an amine-terminated biotin ligand (biotin-NH ₂ , 10 mg / mL in ethanol) was added to each well, reacted at 27 ° C. for 1 hour and washed three times with 150 μl of ethanol, Soaking process was performed. 150 [mu] l of borate buffer (pH 9.0) was immediately added to each well and left at 27 [deg.] C for 2 hours. After removing the borate buffer, 100 μl (10 μg / ml) of streptavidin in PBS was added to each well, reacted at 27 ° C. for 1 hour, and washed three times with 150 μl PBS. 100 [mu] l Biotin-conjugated mouse anti-rabbit IgG monoclonal antibody was added to each well at different concentrations and reacted at 27 [deg.] C for 1 hour and then 150 [mu] l using Immuno ™ Washers (Nunc A / S, Roskilde, Denmark) And washed three times with PBST (0.1% Tween 20). 100 μl of HRP-conjugated anti-mouse IgG antibody (1 μg / ml) was added to each well, reacted at 27 ° C for 1 hour, and then washed three times. Finally, 100 μl of substrate solution (TMB) was added to each well, and after 30 minutes, 100 μl of 1 M HCl was added to stop the enzyme reaction. The intensity was calculated from the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

Polymer-coated On the surface  Antibody Immobilization with Protein A

An amphiphilic polymer coated plastic surface was prepared by impregnating the substrate with an aqueous solution of amphiphilic polymer (20 mg / mL) at ambient temperature followed by washing with water. Polymer coated plastic surfaces were activated with EDC / NHS reagent. A 1: 1 mixture of EDC (400 mM) and NHS (100 mM) in distilled water was applied for 20 minutes to activate the -COOH group on the polymer-coated plastic surface. Then, μCP was performed to immobilize protein A. Protein A (100 ug / ml in PBS) was printed by contacting the PDMS stamp to the polymer coated plastic surface for 1 hour. The sample was then immediately impregnated in borate buffer (pH 9.0) for 2 hours. After generating a pattern of protein A, the sample was added to the anti-BSA solution (10 g / ml in PBS) at room temperature for 1 hour. After washing with PBS, FITC-labeled BSA (50 μg / ml in PBS) was finally added to the sample coated plastic surface. After 1 hour, the sample was washed several times with PBS. The pattern of BSA (λ _ex = 494 nm, λ) was measured using a Leica DMRBE microscope (Leica Microsystems AG, Wetzlar, Germany) equipped with a 200x, 400x objective and TRITC-optimized filter set (Omega Optical Inc, Brattleboro, _em = 521 nm).

Experiment result

The chemical structure of the triple functional copolymer used in the experiments is shown in Scheme 1 below:

The copolymer named poly (TMSMA-r-PEGMA-r-NAS) was synthesized from three monomers by radical polymerization: (trimethoxysilyl) propyl methacrylate (TMSMA) as the surface-anchor site; Polyethylene glycol methacrylate (PEGMA) as a protein-resistant region; And N-acryloyl succinimide (NAS) as a bioactive site. Since the bioactive and antimicrobial functional surface densities can be controlled by simply changing the initial feeding rates of the respective monomers in the polymerisation reaction, the present inventors have found that three different NAS concentrations A series of random copolymers were synthesized: poly 1 = 1: 1: 1, poly 2 = 2: 2: 1 and poly 3 = 4: 5: 1.

The actual NAS amount in each copolymer obtained based on the integrated value in the ¹ H NMR spectrum (FIG. 2) was analyzed as 35%, 21% and 12% for poly 1, poly 2 and poly 3, respectively. The composition of the copolymer was in good agreement with the initial feed rate. The molecular weight of the three copolymers synthesized was 15,000-17,000 and showed a polydispersity of about 1.6, which was measured by Gel Permeation Chromatography (GPC) as a relative value to the monodisperse polystyrene standards.

Si / SiO ₂ wafers were impregnated with a solution of the copolymer (10 mg / mL in methylene chloride) at ambient temperature for 1 hour and washed with solvent to form polymeric self-assembled monolayers (pSAMs) . Then, by dehydration by curing the pSAMs while at 110 ℃ 5 bun additional covalent bonds between the hydroxyl groups of the substrate is formed after the silane group and an oxygen plasma treatment of the copolymer formed it was ^10.

The self-assembled structure formed on the Si / SiO ₂ surface and the specific immobilization of biomolecules are shown in FIG. The thickness and hydrophobicity of pSAMs formed by poly 1-3 were analyzed by ellipsometry and contact angle measurements, respectively (Table 1).

The thickness and static water contact angle of self-assembled polymeric monolayer built on Si / SiO ₂ wafers measured with ellipsometry and contact angle and surface tension analyzer Analysis item Poly 1 Poly 2 Poly 3 Thickness (Å) 9.9 ± 0.1 10.4 ± 0.1 10.7 ± 0.1 Static CA (°) 35.6 ± 0.4 35.3 ± 0.3 33.7 ± 0.5

Similar to the published prior art with respect to the ¹⁰ pSAMs of the copolymer without the NAS part, ultra-thin film was two days of the copolymer produced over the entire surface, the thickness was approximately 1 nm, which was measured by ellipsometry. This result means that the thin film is present as a polymeric monolayer (not a polymeric multilayer film).

The wettability of pSAMs was analyzed by measuring the static water contact angle at ambient temperature. As the hydrophilic PEG in the copolymer increased, the contact angle decreased. The presence of the polymer film was further confirmed by X-ray photoelectron spectroscopy (FIG. 3 and Table 2).

Element composition measured by XPS of PSAMs of poly 1 on Si / SiO ₂ wafers temperament Element composition (%) C1s N1s O1s Si2p PS 1 of poly 1 22.7 1.21 33.38 42.71

Micropatterns of biomolecules were prepared using a soft lithographic technique, microcontact printing (μCP) ¹² (FIG. 4), to investigate the operability of pSAMs as a dual functional surface (bioactive / bioactive). First, an amine-terminated biotin ink (biotin-NH ₂ , 10 mM in ethanol) was contact-printed onto pSAMs of poly 1 using a positive PDMS stamp with a 50 x 50 탆 circular pattern. Since the amine groups of proteins readily react with the reactive NAS of pSAMs, biotin-patterned pSAMs after stamp removal are impregnated with borate buffer (pH 8.5) for 1 hour to hydrolyze non-reactive NAS on the surface, followed by TRITC - labeled streptavidin solution (0.1 mg / mL PBS buffer, pH 7.4). Figure 4 is a fluorescence image for a pattern of TRITC-labeled streptavidin. As expected, streptavidin bound only to the biotin-functional site, and this result shows a specific interaction between streptavidin and biotin. Interestingly, the nonspecific adsorption of streptavidin was not detected at sites where only the hydrolyzed polymer membrane was present without biotin, resulting in a high contrast of signal-to-noise.

Similar results were obtained when a smaller PDMS stamp (25 탆 x 25 탆 having a circular pattern) was used. Although the backprinting ¹³ of PEG ¹³ or the pre-coating ¹⁴ of bovine serum albumin (BSA) is required to minimize nonspecific adsorption of proteins on the surface, the polymer system of the present invention can be used for such additional No process is required.

Hydrolysis pSAMs form of ^protein-15 using, as a model BSA plasma proteins to examine the resistance to evaluate the degree of protein adsorption. The hydrolyzed form of pSAMs on Si / SiO ₂ wafers was treated with BSA solution (0.1 mg / mL in PBS buffer, pH 7.4) for 1 hour, then washed and air-dried. Table 3 is data on the thickness of pSAMs of poly 1-3 measured by ellipsometry before and after BSA adsorption.

Control (unmodified) of pSAM on Si / SiO ₂ wafers before and after adsorption of bovine serum albumin and the thickness of the hydrolyzate Item Control group Poly 1 Poly 2 Poly 3 Thickness (Å) (Before) Can not get
(not available) 10.6 ± 0.6 11.5 ± 0.6 11.9 ± 0.6 Thickness (Å) (After) 44.7 ± 0.1 11.5 ± 0.1 11.6 ± 0.1 11.6 ± 0.1

The thickness of pSAMs after BSA adsorption was almost the same as the initial thickness. However, the thickness of the silicon wafer as a control group was considerably increased. Analysis of the high solubility nitrogen (1s) on the pSAMs measured by X-ray photoelectron spectroscopy and on the control (unmodified Si / SiO ₂ wafer) showed that the pSAMs had a BSO adsorption of dir of 3% 97% resistance). This result clearly shows that NSA-hydrolyzed pSAMs show high resistance to nonspecific protein adsorption. According to all the above-mentioned data, it can be seen that the biomolecule of interest can be immobilized on pSAMs of poly (TMSMA-r-PEGMA-r-NAS), specifically and with little or no nonspecific adsorption.

Since the spacing between the immobilized biomolecule on the surface to affect the interaction of the target molecules, is to control the density of the green reactive groups on the surface for optimization of the bio-chip is very important ^16. Thus, a series of poly (TMSMA-r-PEGMA-r-NAS) copolymers [poly 1 (35%), poly 2 (21%) and poly 3 (12%) with different molar ratios of bioactive groups )] Was used to investigate the spacing effect. 5 is a fluorescence microscope photograph of the pattern of TRITC-labeled streptavidin on pSAM prepared in the same manner as the above-mentioned micro contact printing method. Streptavidin was effectively immobilized on each pSAM to provide a micropattern having a high signal-to-noise ratio with different fluorescence intensities depending on the density of the bioactive groups on the surface. For a more clear comparison, data on the relative signal intensities of the circular regions in the photographs of each pSAM were obtained and are summarized in Table 4.

Item Poly 1 Poly 2 Poly 3 Average fluorescence intensity 231.77 ± 15.04 156.67 ± 45.82 99.01 ± 57

Poly 1 with 35% NAS content showed a fluorescence intensity of 232 ± 15 au and poly 2 and poly 3 showed lower intensity as 157 ± 46 and 99 ± 57, respectively. As the NAS content on the polymer pSAMs increased, the immobilized streptavidin gradually increased. This result shows another advantage of the inventive copolymer system in which the initial feeding ratio of each monomer can be changed to control the simple density of the bioreactive site, which is useful in the optimization of the biochip.

On the other hand, the chemical structure of the synthesized bipolar polymer of the present invention is as follows:

Chemical structure of bipolar polymer

Each polymer was synthesized by radical polymerization from the corresponding three monomers at a molar feed ratio of 2: 2: 2. In poly-4, dodecyl, in poly-5, fluoroalkyl and in poly-6, benzyl was selected as anchoring group for plastic surface. Poly-4 was hydrophobic interaction, poly-6 was hydrophobic interaction and dipole interaction , And poly-6 is anchored to the polystyrene plastic surface by a pi-pi stacking interaction. All three polymers showed good solubility in water, which is attributed to the PEG group.

Poly-4 and poly-6 coated plastic surfaces were analyzed with a static water contact angle analyzer at room temperature to determine the wettability of the plastic surface (Table 5).

Analysis item Control group Poly 4 Poly 6 Static CA (°) 130 ± 7 74 ± 1 79 ± 2

By coating with the amphiphilic polymer for 1 hour, the water contact angle of poly-4 and poly-6 significantly decreased from 130 +/- 7 DEG to 74 +/- 1 DEG and 79 +/- 2 DEG. Significant reduction in the contact angle indicates that the plastic surface has changed to more hydrophilic as the hydrophilic PEG in the copolymer increases or decreases.

To evaluate whether a polymer coated plastic surface could be used as a functional surface, micropatterns of biomolecules were prepared by micro-contact printing. Figure 7 is a fluorescence microscope image of the pattern of TRITC-labeled streptavidin. As a result, streptavidin was efficiently immobilized on each polymer-coated plastic surface to form a micropattern with a high signal-to-noise ratio, in which case the anchoring position of each polymer (poly-4 dodecyl, poly- Benzyl group).

To more accurately compare poly-4 and poly-6, the relative signal intensities of the circle regions in the images of each polymer coated plastic surface were measured (Table 6).

polymer Poly 4 Poly 6 Fluorescence intensity 149.9 ± 24.4 179.7 ± 34.3

Fluorescence intensity of poly - 4 with dodecyl group was 149.9 ± 24.4 au and poly - 6 showed fluorescence intensity of 179.7 ± 34.4 a.u. Poly-4 has an alkyl chain as anchoring position and is bonded to the plastic surface through hydrophobic interaction and van der Waals interaction. On the other hand, poly-6 has an aromatic ring of benzyl group and strongly binds to plastic surface through π-π stacking interactions as well as hydrophobic interactions and van der Waals interactions. Little background signal was observed at sites where biotin was absent and only hydrolyzed polymer surface, resulting in a high signal-to-noise ratio. According to the prior art, backprinting of PEG or precoating step of bovine serum albumin (BSA) is essential to minimize non-specific adsorption of proteins on the surface. However, the polymer surface of the present invention does not require this step.

Antibiotic fouling was analyzed for poly 4-6. The polystyrene substrate coated with poly 4-6 was impregnated with BSA solution (0.25 mg / mL in PBS, pH 7.4) for 2 hours. The degree of nonspecific protein adsorption on pSAMs was obtained from high resolution N (1s) XPS spectra (FIG. 6). As can be seen in FIG. 6, the polymer of the present invention exhibited a degree of BSA adsorption (i.e., 99% resistance) of about 1% as compared to the control group for the coated polystyrene substrate.

Next, the 96-well plate was surface-treated with the polymer of the present invention and subjected to ELISA (FIG. 8A). The polymer-coated streptavidin plate of the present invention was compared with purchased commercially available streptavidin plates. In the case of the purchased streptavidin plate, 100 μl of a milk protein solution as a blocking buffer was treated for 2 hours. However, the polymer-coated streptavidin plate of the present invention did not require this process because the PEG group blocks non-specific binding of the protein. 8B shows an absorption curve according to the concentration of biotin-IgG. The polymer coated streptavidin plates exhibited stronger strength than the conventional streptavidin plates at the same concentration. These results show that the polymer-coated streptavidin plate of the present invention captures biotin-IgG better than conventional plates at the same concentration. The polymer system of the present invention showed similar intensity to the control at a concentration of 0 ng / ml of biotin-IgG, even though the blocking buffer was not treated, which is due to the PEG moiety.

Immobilization of the antibody was carried out using protein A on the polymer coated plastic surface. Protein A is used to immobilize antibodies in many immunoassays. Protein A specifically binds to the Fc region of the antibody to provide orientation of the antibody. Protein A-mediated antibody immobilization does not require modification of the antibody. Therefore, the present invention provides an excellent antibody detection method in comparison with conventional antibody immobilization methods such as covalent binding or physical adsorption. Figure 9a is a schematic diagram of immobilization of anti-BSA antibody using protein A on a polymer coated plastic surface, and Figure 9b is a fluorescence microscope image of FITC-labeled BSA. As expected, FITC-labeled BSA was deposited only at the anti-BSA functionalization site, indicating a specific interaction therebetween. It is a noteworthy result that no nonspecific adsorption of BSA was observed at sites where only the hydrolyzed polymer layer was present without the anti-BSA antibody, resulting in a high signal-to-noise ratio. The relative signal intensities of the circular images were measured in the images of each polymer coated plastic surface. Fluorescence intensity of poly - 4 with dodecyl group was 98.7 ± 15 au and poly - 6 showed fluorescence intensity of 42.6 ± 6.9 a.u. Therefore, the polymer system of the present invention is very advantageous for culturing the antibody immobilization using the protein A on the plastic surface, which can be used for protein chip development.

In conclusion, the present invention facilitates the production of bi-functional surfaces (bioactive and bioactive) on SiO ₂ -based and plastic substrates by forming polymeric self-assembled monolayers (pSAMs) using the novel PEG copolymers Can be done. Biomolecules are not only immobilized effectively on pSAMs with high specificity, but also exhibit minimized nonspecific adsorption. Also, by simply adjusting the initial feeding rate in polymer synthesis, the density of bioerodible and antibiotic fouling functionalities can be controlled. Collectively, the dual function pSAMs platform of the present invention has excellent applicability in the field of biosensors and biochips.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

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BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram showing specifically immobilizing biomolecules on the self-assembled polymer monolayer (PSAM) of the present invention having anti-biofouling properties.

Figure 2 is a ¹ H NMR spectrum of poly 1, poly 2 and poly 3 of the present invention.

3 is a high-resolution C (1s) X-ray photoelectron spectrum for PSA of poly 1 formed on a Si / SiO ₂ wafer.

Figure 4 is a graph showing the effect of TRITC-labeled streptavidin < RTI ID = 0.0 > (TRITC-labeled < / RTI > streptavidin) prepared by micrometric printing of biotin- EO-amines on PSAMs of poly 1 using (a) 50 x 50 mu m and (b) 25 x 25 mu m circular patterned stamp A fluorescence microscope photograph of the pattern.

Figure 5 is a fluorescence micrograph of a TRITC-labeled streptavidin pattern formed on PSAMs of poly 1-3 produced using a 50 x 50 탆 circular patterned stamp

Figure 6 is a high-resolution N (1s) X-ray photoelectron spectrum for poly 4-6 PSAM formed on a polystyrene substrate.

Figure 7 is a fluorescence microscope image of a TRITC-labeled streptavidin pattern formed by microcontact printing of biotin-amine on a polymer coated plastic surface using a 50 x 50 탆 circular patterned stamp.

8A is a schematic diagram of a 96 well plate coated with streptavidin by a polymer of the present invention used to detect biotin-binding mouse anti-rabbit IgG.

Fig. 8B shows the results of EILSA detection of adsorbed-captured biotinylated mouse IgG. Immobilized biotinylated mouse IgG was detected with HRP conjugated anti-mouse IgG.

9A is a schematic diagram of the orientation of anti-BSA antibody using protein A on a polymer coated plastic surface.

Figure 9B is a fluorescence microscope image of a FITC-labeled BSA pattern formed by micro-contact printing of protein A on a polymer coated plastic surface using a 50 x 50 탆 circular patterned stamp and addition of an anti-BSA antibody.

Claims (22)

A process for preparing a solid substrate comprising a reactor coated with a polymeric film on a surface comprising the steps of: forming a covalent bond and exhibiting anti-biofouling properties; (i) a first monomer of a polymer comprising a surface-anchoring site bonded to a surface of the solid substrate, (ii) a second monomer to which the polymer exhibiting anti-biofouling properties is bound, and (iii) Reacting a third monomer comprising a reactor capable of forming a covalent bond to produce a polymer comprising a surface-anchor site, a protein-resistant site, and a reactive site; And (b) coating the surface of the solid substrate with the polymer. The method of claim 1, wherein the first monomer is a methacrylate-based or acrylate-based monomer, and the surface-anchoring site in the first monomer is a substituted or unsubstituted silane group, a substituted or unsubstituted alkyl group, Is a substituted aryl group. The method of claim 2, wherein the substituted silane group is a silane group substituted with an alkoxy group. The method according to claim 2, wherein the alkyl group is a C _{6 -30} alkyl group. 3. The method of claim 2, wherein the substituted alkyl group is substituted with F, Cl or Br. The method of claim 1, wherein the second monomer is a methacrylate-based or acrylate-based monomer, and the polymer exhibiting anti-biofouling properties in the second monomer is selected from the group consisting of PEG (polyethylene glycol), polyalkylene oxide, Or polyvinylpyrrolidone. The method of claim 1, wherein the third monomer is a methacrylate-based or acrylate-based monomer, and the reactor in the third monomer is a carboxyl group. The method of claim 7, wherein the carboxyl group is selected from the group consisting of succinermide, succinamidyl ester, sulfo-succinamidyl ester, 2,3,5,6-tetrafluorophenol ester, 4-sulfo-2,3,5,6 Characterized in that it is activated by a tetrafluorophenol ester, an aldehyde, an acid anhydride, an azide, an azole, a carboimide, an epoxide, an ester, a glycidyl ether, a halide, an imidazole or an imidate. The method according to claim 1, wherein the polymer is represented by the following formula (1): Formula 1

In the above formula, R ₁ is a silylalkyl group, an alkoxysilylalkyl group, a hydroxysilylalkyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, or a substituted or unsubstituted alkaryl group ego; R ₂ is at least one selected from the group consisting of PEG (polyethylene glycol), polyalkylene oxide, polyphenylene oxide, a copolymer of PEG and polyalkylene oxide, poly (methoxyethyl methacrylate), poly (methacryloylphosphatidylcholine) Lt; / RTI > polyether, dextran or polyvinylpyrrolidone; R ₃ is an aldehyde, an epoxy, a haloalkyl, a primary amine, a thiol, a maleimide, an ester, a carboxyl group or a hydroxyl group; R ₄ , R ₅ and R ₆ independently of one another are H or a C ₁ -C ₅ alkyl group; X, Y and Z are independently of each other an oxygen, sulfur or nitrogen atom; l, m and n are independently integers from 1 to 10,000. The method of claim 1, wherein the polymer is present in the form of a self-assembled monolayer on a solid substrate. 9. The method of claim 1 or 8, wherein the method further comprises the step of (c) covalently bonding a chemical, protein, peptide, nucleotide or sugar to a reactor derived from a third monomer of the polymeric membrane coated on the solid substrate ≪ / RTI > A dual functional solid substrate coated with a polymer formed by the polymerization of the following monomers and comprising surface-aqueering, anti-biofouling and reactive sites: (i) a surface-anchoring (Ii) a second monomer to which a polymer exhibiting anti-biofouling properties is bound, and (iii) a reactor capable of forming a covalent bond. 13. The method of claim 12, wherein the first monomer is a methacrylate-based or acrylate-based monomer, and the surface-anchoring site in the first monomer is a substituted or unsubstituted silane group, a substituted or unsubstituted alkyl group, Gt; is a substituted aryl group. The method of claim 13 wherein the alkyl group is a solid phase substrate, characterized in that C _{6 -30} alkyl group. 14. The solid substrate of claim 13, wherein the substituted alkyl group is substituted with F, Cl or Br. The method of claim 12, wherein the second monomer is a methacrylate-based or acrylate-based monomer, and the polymer exhibiting anti-biofouling properties in the second monomer is selected from the group consisting of polyethylene glycol (PEG), polyalkylene oxide, Or polyvinyl pyrrolidone. 13. The solid substrate according to claim 12, wherein the third monomer is a methacrylate-based or acrylate-based monomer, and the reactor in the third monomer is a carboxyl group. 18. The method of claim 17, wherein the carboxyl group is selected from the group consisting of succinermide, succinamidyl ester, sulfo-succinimidyl ester, 2,3,5,6-tetrafluorophenol ester, Characterized in that it is activated by at least one of the following: - tetrafluorophenol ester, aldehyde, acid anhydride, azide, azolide, carboimide, epoxide, ester, glycidyl ether, halide, imidazole or imidate . 13. The solid substrate according to claim 12, wherein the reactive site is bound to a chemical substance, a protein, a peptide, a nucleotide or a sugar. The solid substrate according to claim 12, wherein the polymer is represented by the following formula (1) Formula 1

In the above formula, R ₁ represents a silylalkyl group, an alkoxysilylalkyl group, a hydroxysilylalkyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, or a substituted or unsubstituted alkane Lt; / RTI > R ₂ is at least one selected from the group consisting of PEG (polyethylene glycol), polyalkylene oxide, polyphenylene oxide, a copolymer of PEG and polyalkylene oxide, poly (methoxyethyl methacrylate), poly (methacryloylphosphatidylcholine) Lt; / RTI > polyether, dextran or polyvinylpyrrolidone; R ₃ is an aldehyde, an epoxy, a haloalkyl, a primary amine, a thiol, a maleimide, an ester, a carboxyl group or a hydroxyl group; R ₄ , R ₅ and R ₆ independently of one another are H or a C ₁ -C ₅ alkyl group; X, Y and Z are independently of each other an oxygen, sulfur or nitrogen atom; l, m and n are independently integers from 1 to 10,000. 20. A biochip or a biosensor comprising the solid substrate of any one of claims 12 to 20. A polymer represented by the following formula (1): Formula 1

In the above formula, R ₁ is a silylalkyl group, an alkoxysilylalkyl group, a hydroxysilylalkyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, or a substituted or unsubstituted alkaryl group ego; R ₂ is at least one selected from the group consisting of PEG (polyethylene glycol), polyalkylene oxide, polyphenylene oxide, a copolymer of PEG and polyalkylene oxide, poly (methoxyethyl methacrylate), poly (methacryloylphosphatidylcholine) Lt; / RTI > polyether, dextran or polyvinylpyrrolidone; R ₃ is an aldehyde, an epoxy, a haloalkyl, a primary amine, a thiol, a maleimide, an ester, a carboxyl group or a hydroxyl group; R ₄ , R ₅ and R ₆ independently of one another are H or a C ₁ -C ₅ alkyl group; X, Y and Z are independently of each other an oxygen, sulfur or nitrogen atom; l, m and n are independently integers from 1 to 10,000.

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