CN102912343A - Method for preparing biological molecule pattern on gold sheet - Google Patents
Method for preparing biological molecule pattern on gold sheet Download PDFInfo
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- CN102912343A CN102912343A CN2012103261998A CN201210326199A CN102912343A CN 102912343 A CN102912343 A CN 102912343A CN 2012103261998 A CN2012103261998 A CN 2012103261998A CN 201210326199 A CN201210326199 A CN 201210326199A CN 102912343 A CN102912343 A CN 102912343A
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Abstract
The invention discloses a method for preparing a biological molecule pattern on a gold sheet, which comprises the following steps: (1) preparing a perforated polymer honeycomb ordered porous membrane; (2) covering the perforated polymer honeycomb ordered porous membrane on a gold sheet; (3) adding a polyfunctional group organic substance solution with sulfhydryl group into the gold sheet surface, and selectively introducing functional groups into the membrane pores on the gold sheet surface to form a patterned monomolecular layer surface; (4) soaking the gold sheet in a releasing agent remove the perforated polymer honeycomb ordered porous membrane; and (5) dropwisely adding a biological molecule solution onto the patterned gold sheet surface with fixed sulfhydryl compounds, and cleaning to obtain the biological molecule pattern. The method disclosed by the invention is independent of the existing patterning techniques, such as photoetching, electron-beam etching and the like, is a cheap, simple, convenient and effective method for preparing the biological molecule pattern, and has wide application prospects in the fields of biological sensing, biological analysis and the like.
Description
Technical field
The present invention relates to the materials chemistry field, be specifically related to a kind of method for preparing the biomolecules pattern on the gold plaque surface.
Background technology
The patterning of biomolecules is a kind of specified location that biomolecules is fixed on solid phase surface, and avoids the microarray technology of other regional non-specific adsorption.This technology provides powerful for the repercussion study of the biomolecules such as protein, enzyme, sugar, nucleic acid and its acceptor, in field extensive application such as biosensor, biological medicine instrument, micro-fluidic chip and organizational projects.Gold plaque is one of solid phase base material of commonly using.
The now widely used method for preparing the biomolecules pattern mainly comprise photolithography, mechanical little point sample method and soft quarter method.Wherein, photolithography and mechanical little point sample method have been used the photoelectron lithographic technique, need photomask, electron beam emitter and skilled operator, and the human and material resources, the financial resources that expend are higher, the biomolecule arrays size that template obtains after determining is relatively fixing, is difficult for realizing dynamic adjustments.Soft quarter, method used polydimethylsiloxane (PDMS) as seal usually, take make up biomolecule arrays at base material as template, compare photolithography convenient.A kind of macromolecular method of fixed biologically that is total to patterning at the inorganic silicon material surface is disclosed among the Chinese invention patent application CN1425920A, to carry out the aldehyde radical processing after the amination of inorganic silicon material surface, inorganic silicon material surface at aldehyde radical forms little pattern with PDMS seal micro-contact printing biomacromolecule, in patterning immobilization the inorganic silicon material surface of a kind of biomacromolecule drip the solution of another kind of biomacromolecule, make the latter be fixed on the former unlapped zone, namely get two kinds of biomacromolecules after flushing and the ultrasonic cleaning and fix at the common patterning on same surface.Chinese invention patent application CN02160335.9 discloses the little Transfer Technology of a kind of usefulness in the macromolecular method of polymer active patterned surface fixed biologically, first the biomacromolecule selectivity " to be packed into " in little well of polydimethylsiloxane seal, form one deck water of condensation on its surface again, utilize behind seal and the polymer-modified film compacting water of original water in little well and contact area transverse dispersion that biomacromolecule is dissolved again, thereby with form and the surface group generation chemical reaction of solution, realize at polymer active patterned surface fixed biologically macromole; The method simple and flexible, good reproducibility, the little pattern that obtains has very high contrast gradient and firmness, require lower to seal surface properties and little well depth simultaneously, a kind of macromolecular effective ways of patterning fixed biologically of cheap and simple, be particularly useful for having biomolecules than low reaction activity/active surface system, have a good application prospect in the fields such as biological device structure, organizational project and cytobiology fundamental research based on cell.But the process of preparation PDMS seal relies on the technology such as photoengraving or electron beam lithography equally, has hindered the popularization of method at soft quarter.This shows, the technology of existing preparation biomolecules pattern almost all depends on photoengraving or electron beam lithography, needs large-scale instrument and equipment and complex operation complicated, can not satisfy actual preparation technology's needs fully.
Summary of the invention
The purpose of this invention is to provide the method for preparing the biomolecules pattern on the gold plaque surface that a kind of operating procedure is very simple, reaction conditions is gentle, with low cost, the method does not rely on the technology such as photoengraving or electron beam lithography.
The ultimate principle of the inventive method is that the through polymkeric substance honeycomb-patterned ordered porous-film of employing water droplet template synthesis is template, this template is covered clean gold plaque surface and assembles unimolecular layer, then biomolecules optionally is fixed on the fenestra zone of through polymkeric substance honeycomb-patterned ordered porous-film, makes the biomolecules pattern.
A kind of method for preparing the biomolecules pattern on the gold plaque surface may further comprise the steps:
(1) polymer dissolution is made the polymers soln of homogeneous in solvent, under 15 ℃～30 ℃ with the polymers soln uniform spreading on the ice face, place rapidly the environment with humidity, after solvent evaporates, take out and make through polymkeric substance honeycomb-patterned ordered porous-film; Described polymkeric substance is styrol copolymer;
(2) gold plaque is placed piranha solution soaked 10 minutes～15 minutes, use washed with de-ionized water after taking out, the through polymkeric substance honeycomb-patterned ordered porous-film that makes in gold plaque surface coverage step (1) after nitrogen dries up is as template, and at 80 ℃～85 ℃ thermal treatments 1 minute～5 minutes, the gold plaque after obtaining processing;
(3) gold plaque after processing in the step (2) is immersed in the polyfunctional group organic solution with sulfydryl, envrionment temperature (preferred 15 ℃～30 ℃) reaction 0.5 hour～12 hours, then use successively ethanol, washed with de-ionized water gold plaque, obtain the gold plaque that there is self assembled monolayer on the surface;
(4) surface in the step (3) there is the gold plaque of self assembled monolayer be soaked in the releasing agent 0.2 hour～5 hours, use successively afterwards ethanol, washed with de-ionized water gold plaque surface, remove template, obtain the gold plaque that there is the patterning self assembled monolayer on the surface;
(5) surface in the step (4) there is the gold plaque of patterning self assembled monolayer activate, then place biomolecule solution, 15 ℃～30 ℃ reactions 0.2～2 hour, after finishing, reaction with buffered soln or washed with de-ionized water, makes the biomolecules pattern.
In the step (1), described polymkeric substance is selected styrol copolymer, has suitable styrene units, can access the through polymkeric substance honeycomb-patterned ordered porous-film of aperture homogeneous.
In described styrol copolymer optimization styrene/methacrylic acid hydroxyl ethyl ester segmented copolymer (PS-b-PHEMA), styrene/methacrylic acid dimethylaminoethyl segmented copolymer (PS-b-PDMAEMA), styrene/acrylic segmented copolymer (PS-b-PAA), styrene/isoprene/styrene segmented copolymer (SIS), the styrene/butadiene/styrene block copolymers (SBS) one or more.The molar content of styrene units is preferably 30%～99% in the described styrol copolymer.
Described solvent is selected according to polymkeric substance, and principle is the solvent of selecting saturated vapor pressure high, is preferably dithiocarbonic anhydride, methylene dichloride, chloroform, tetrahydrofuran (THF), benzene or toluene.
Polymer concentration in the described polymers soln is preferably 0.1mg/mL～5.0mg/mL.
Described humidity is selected according to polymkeric substance, is preferably relative humidity 60%～90%.
The consumption of described polymers soln is selected according to the membrane area of the through polymkeric substance honeycomb-patterned ordered porous-film of required preparation, the polymers soln consumption is more, membrane area is also larger, and the present invention is take the film that forms 2 square centimeters of left and right areas as example, and the polymers soln consumption is 0.1mL.
In the step (2), described piranha solution can adopt the commercially available prod, also can adopt existing method preparation, is generally 30% (mass percent) H
2O
2With 98% (mass percent) H
2SO
4Be mixing in 1: 3 by volume.
In the step (2), described template only allows the gold plaque surface in fenestra zone to contact with other materials, and hides the gold plaque surface in non-fenestra zone.
In the step (3), described polyfunctional group organism with sulfydryl forms self assembled monolayer on the gold plaque surface.
Described polyfunctional group organic solution with sulfydryl is that 3-thiohydracrylic acid (MPA) concentration is that ethanolic soln or 11-sulfydryl undecanoic acid (MUA) concentration of the 3-thiohydracrylic acid of 0.1mmol/L～20mmol/L is the ethanolic soln of the 11-sulfydryl undecanoic acid of 0.1mmol/L～20mmol/L.
In the step (4), described releasing agent is preferably dithiocarbonic anhydride, methylene dichloride, chloroform, tetrahydrofuran (THF), benzene or toluene.
In the step (5), described biomolecules is polypeptide, enzyme, protein biomolecules and derivative thereof, can select a kind of in horseradish peroxidase (HR), glucose oxidase (GOX), catalase (CAT), albumin (BSA), fibronectin (FN), poly-lysine, gsh (GSH), gelatin, the collagen etc.Solvent in the described biomolecule solution is selected the buffer soln of the corresponding pH value of above-mentioned biomolecules or aqueous solution etc., such as phosphate buffered saline buffer (PBS) solution of BSA, the PBS solution of gelatin, the acetic acid aqueous solution (mass percentage concentration of acetic acid is 0.3% in the acetic acid aqueous solution) of collagen etc.
The concentration of biomolecules is preferably 0.001mg/mL～50mg/mL in the described biomolecule solution, can be according to the difference adjustment of biomolecule specy.
The step of described activation comprises: have the gold plaque of patterning self assembled monolayer to be immersed in the phosphate buffer soln of 1-ethyl-3-(dimethyl aminopropyl) phosphinylidyne diimine (EDC) and N-maloyl imines (NHS) on surface in the step (4), envrionment temperature (preferred 15 ℃～30 ℃) reaction 0.2 hour～2 hours; In the phosphate buffer soln of EDC and NHS, the concentration of EDC is 2mg/mL～50mg/mL, and the mol ratio of EDC and NHS is 5/2, and phosphate buffer soln pH is 7.4.
Compared with prior art, the present invention has following advantage:
The inventive method may further comprise the steps: the preparation of (1) through polymkeric substance honeycomb-patterned ordered porous-film; (2) at the through polymkeric substance honeycomb-patterned ordered of gold plaque surface coverage porous-film; (3) in the polyfunctional group organic solution of above-mentioned gold plaque surface adding with sulfydryl, optionally functional group is introduced on the gold plaque surface in fenestra, forms the unimolecular layer surface of patterning; (4) above gold plaque is soaked in the suitable solvent, removes through polymkeric substance honeycomb-patterned ordered porous-film; (5) drip biomolecule solution on the gold plaque surface that patterning has been fixed sulfhydryl compound, make the specific zone of sulfhydryl compound that has been fixed on grafting of biomolecules, namely get biomolecules after the cleaning and fix at the patterning on gold plaque surface.Among the present invention, the biomolecules pattern can pass through the size of the through polymkeric substance honeycomb-patterned ordered porous-films of control such as type of polymer, polymer concentration, relative humidity, polymer solvent, perhaps regulates and control reaction times, temperature of reaction and sulfydryl polyfunctional group organism and biomolecule solution concentration and controls.
The most outstanding advantage of the present invention is not rely on the patterning techniques such as existing photoetching and electron beam lithography, avoided the use of large-scale high price plant and instrument, thereby the method operating procedure is simple, and reaction conditions gentleness, good reproducibility, with low cost, the firmness of fixing biological molecules is high and can realize the dynamic controllable adjustment of patterning size.Prepared biomolecules patterning gold plaque surface is having a good application prospect based on fields such as the bio-sensing of QCM (Quartz Crystal Microbalance) and surface plasma resonance and bioanalysiss.
Embodiment
By following examples the present invention is done more detailed description, but described embodiment is not construed as limiting the invention.
Embodiment 1
PS-b-PHEMA is dissolved in the homogeneous PS-b-PHEMA solution that dithiocarbonic anhydride makes 1mg/mL, the molar content of styrene units is 99% among the PS-b-PHEMA, the PS-b-PHEMA solution that extracts 0.1mL with syringe under the room temperature spreads over equably that (solution usage can change on the ice face, the film forming area of different solutions consumption is different), be placed on rapidly the environment of relative humidity 75%, take out after solvent evaporates and make through PS-b-PHEMA honeycomb-patterned ordered porous-film, its aperture size is 2 μ m; Gold plaque is placed piranha solution (30%H
2O
2/ 98%H
2SO
4, mass percent, v/v=1: soaked 10 minutes 3), after taking out with a large amount of washed with de-ionized water, after nitrogen dries up at the through PS-b-PHEMA honeycomb-patterned ordered of gold plaque surface coverage porous-film as template, and thermal treatment 5 minutes in 80 ℃ of baking ovens; Gold plaque after the above-mentioned thermal treatment is immersed in the ethanolic soln of MPA that MPA concentration is 20mmol/L (mM), and temperature of reaction is 15 ℃, and 0.5 hour reaction times is then successively with ethanol, washed with de-ionized water gold plaque; Gold plaque after the above-mentioned cleaning is soaked in the dithiocarbonic anhydride 0.2 hour, successively with ethanol, washed with de-ionized water gold plaque surface, removes template afterwards; The above-mentioned gold plaque that makes is immersed in the phosphate buffer soln of EDC and NHS, temperature of reaction is 30 ℃, reacts 0.2 hour, wherein, EDC concentration is 2mg/mL, the mol ratio of EDC and NHS is 5/2, and phosphate buffer soln pH is 7.4, then places PBS (pH the is 7.4) solution of the HRP of 0.001mg/mL, reacted 0.2 hour, temperature of reaction is 15 ℃, and reaction is at room temperature cleaned with phosphate buffer soln after finishing, and namely makes the HRP pattern.
Embodiment 2
PS-b-PDMAEMA is dissolved in the homogeneous PS-b-PDMAEMA solution that dithiocarbonic anhydride makes 1mg/mL, the molar content of styrene units is 90% among the PS-b-PDMAEMA, PS-b-PDMAEMA solution with syringe extraction 0.1mL under the room temperature spreads on the ice face equably, be placed on rapidly the environment of relative humidity 80%, take out after solvent evaporates and make through PS-b-PDMAEMA honeycomb-patterned ordered porous-film, its aperture size is 2.5 μ m; Gold plaque is placed piranha solution (30%H
2O
2/ 98%H
2SO
4, mass percent, v/v=1: soaked 10 minutes 3), after taking out with a large amount of washed with de-ionized water, after nitrogen dries up at the through PS-b-PDMAEMA honeycomb-patterned ordered of gold plaque surface coverage porous-film as template, and thermal treatment 1 minute in 80 ℃ of baking ovens; The above-mentioned gold plaque that makes is immersed in the ethanolic soln of the MPA of 0.1mM, temperature of reaction is 30 ℃, and 12 hours reaction times is then successively with ethanol, washed with de-ionized water gold plaque; The above-mentioned gold plaque that makes is soaked in the dithiocarbonic anhydride 5 hours, successively with ethanol, washed with de-ionized water gold plaque surface, removes template afterwards; The above-mentioned gold plaque that makes is immersed in the phosphate buffer soln of EDC and NHS, temperature of reaction is 15 ℃, reacted 2 hours, EDC concentration is 50mg/mL, and the mol ratio of EDC and NHS is 5/2, phosphate buffer soln pH is 7.4, then place PBS (pH the is 7.4) solution of the GOX of 10mg/mL, reacted 2 hours, temperature of reaction is 30 ℃, reaction is at room temperature cleaned with deionized water after finishing, and namely makes the GOX pattern.
Embodiment 3
PS-b-PAA is dissolved in the homogeneous PS-b-PAA solution that methylene dichloride makes 5mg/mL, the molar content of styrene units is 95% among the PS-b-PAA, PS-b-PAA solution with syringe extraction 0.1mL under the room temperature spreads on the ice face equably, be placed on rapidly the environment of relative humidity 90%, take out after solvent evaporates and make through PS-b-PAA honeycomb-patterned ordered porous-film, its aperture size is 3 μ m; Gold plaque is placed piranha solution (30%H
2O
2/ 98%H
2SO
4, mass percent, v/v=1: soaked 10 minutes 3), after taking out with a large amount of washed with de-ionized water, after nitrogen dries up at the through PS-b-PAA honeycomb-patterned ordered of gold plaque surface coverage porous-film as template, and thermal treatment 5 minutes in 80 ℃ of baking ovens; The above-mentioned gold plaque that makes is immersed in the ethanolic soln of the MUA of 0.1mM, temperature of reaction is 30 ℃, and 12 hours reaction times is then successively with ethanol, washed with de-ionized water gold plaque; The above-mentioned gold plaque that makes is soaked in the methylene dichloride 3 hours, successively with ethanol, washed with de-ionized water gold plaque surface, removes template afterwards; The above-mentioned gold plaque that makes is immersed in the phosphate buffer soln of EDC and NHS, temperature of reaction is 20 ℃, reacted 1 hour, EDC concentration is 10mg/mL, and the mol ratio of EDC and NHS is 5/2, phosphate buffer soln pH is 7.4, then place PBS (pH the is 7.4) solution of the CAT of 5mg/mL, reacted 1 hour, temperature of reaction is 20 ℃, reaction is at room temperature cleaned with phosphate buffer soln after finishing, and namely makes the CAT pattern.
Embodiment 4
SIS is dissolved in the homogeneous SIS solution that dithiocarbonic anhydride makes 0.1mg/mL, the molar content of styrene units is 30% among the SIS, SIS solution with syringe extraction 0.1mL under the room temperature spreads on the ice face equably, be placed on rapidly the environment of relative humidity 90%, take out after solvent evaporates and make through SIS honeycomb-patterned ordered porous-film, its aperture size is 8 μ m; Gold plaque is placed piranha solution (30%H
2O
2/ 98%H
2SO
4, mass percent, v/v=1: soaked 10 minutes 3), after taking out with a large amount of washed with de-ionized water, after nitrogen dries up at the through SIS honeycomb-patterned ordered of gold plaque surface coverage porous-film as template, and thermal treatment 1 minute in 80 ℃ of baking ovens; The above-mentioned gold plaque that makes is immersed in the ethanolic soln of the MUA of 20mM, temperature of reaction is 15 ℃, and 2 hours reaction times is then successively with ethanol, washed with de-ionized water gold plaque; The above-mentioned gold plaque that makes is soaked in the dithiocarbonic anhydride 5 hours, successively with ethanol, washed with de-ionized water gold plaque surface, removes template afterwards; The above-mentioned gold plaque that makes is immersed in the phosphate buffer soln of EDC and NHS, temperature of reaction is 15 ℃, reacted 1.5 hours, EDC concentration is 30mg/mL, and the mol ratio of EDC and NHS is 5/2, phosphate buffer soln pH is 7.4, then place PBS (pH the is 7.4) solution of the BSA of 2mg/mL, reacted 1 hour, temperature of reaction is 20 ℃, reaction is at room temperature cleaned with deionized water after finishing, and namely makes the BSA pattern.
Embodiment 5
SBS is dissolved in the homogeneous SBS solution that dithiocarbonic anhydride makes 0.5mg/mL, the molar content of styrene units is 35% among the SBS, SBS solution with syringe extraction 0.1mL under the room temperature spreads on the ice face equably, be placed on rapidly the environment of relative humidity 85%, take out after solvent evaporates and make through SBS honeycomb-patterned ordered porous-film, its aperture size is 9 μ m; Gold plaque is placed piranha solution (30%H
2O
2/ 98% H
2SO
4, mass percent, v/v=1: soaked 10 minutes 3), after taking out with a large amount of washed with de-ionized water, after nitrogen dries up at the through SBS honeycomb-patterned ordered of gold plaque surface coverage porous-film as template, and thermal treatment 1.5 minutes in 80 ℃ of baking ovens; The above-mentioned gold plaque that makes is immersed in the ethanolic soln of the MPA of 10mM, temperature of reaction is 20 ℃, and 5 hours reaction times is then successively with ethanol, washed with de-ionized water gold plaque; The above-mentioned gold plaque that makes is soaked in the dithiocarbonic anhydride 4 hours, successively with ethanol, washed with de-ionized water gold plaque surface, removes template afterwards; The above-mentioned gold plaque that makes is immersed in the phosphate buffer soln of EDC and NHS, temperature of reaction is 25 ℃, reacted 1 hour, EDC concentration is 20mg/mL, and the mol ratio of EDC and NHS is 5/2, phosphate buffer soln pH is 7.4, then place PBS (pH the is 7.4) solution of the FN of 0.5mg/mL, reacted 2 hours, temperature of reaction is 20 ℃, reaction is at room temperature cleaned with phosphate buffer soln after finishing, and namely makes the FN pattern.
Embodiment 6
The PS-b-PDMAEMA/SIS mixture is dissolved in chloroform makes homogeneous PS-b-PDMAEMA/SIS mixture solution, the concentration of PS-b-PDMAEMA is 1mg/mL, the concentration of SIS is 0.5mg/mL, the molar content of styrene units is 65% among the PS-b-PDMAEMA/SIS, PS-b-PDMAEMA/SIS solution with syringe extraction 0.1mL under the room temperature spreads on the ice face equably, be placed on rapidly the environment of relative humidity 80%, take out after solvent evaporates and make through PS-b-PDMAEMA/SIS honeycomb-patterned ordered porous-film, its aperture size is 5 μ m; Gold plaque is placed piranha solution (30%H
2O
2/ 98% H
2SO
4Mass percent, v/v=1: soaked 10 minutes 3), take out rear with a large amount of washed with de-ionized water, after nitrogen dries up at the through PS-b-PDMAEMA/SIS honeycomb-patterned ordered of gold plaque surface coverage porous-film as template, and thermal treatment 1 minute in 80 ℃ of baking ovens; The above-mentioned gold plaque that makes is immersed in the ethanolic soln of the MUA of 5mM, temperature of reaction is 25 ℃, and 10 hours reaction times is then successively with ethanol, washed with de-ionized water gold plaque; The above-mentioned gold plaque that makes is soaked in the chloroform 5 hours, successively with ethanol, washed with de-ionized water gold plaque surface, removes template afterwards; The above-mentioned gold plaque that makes is immersed in the phosphate buffer soln of EDC and NHS, temperature of reaction is 30 ℃, reacted 0.5 hour, EDC concentration is 45mg/mL, and the mol ratio of EDC and NHS is 5/2, phosphate buffer soln pH is 7.4, then place the poly-lysine aqueous solution of 50mg/mL, reacted 0.5 hour, temperature of reaction is 30 ℃, reaction is at room temperature cleaned with deionized water after finishing, and namely makes the poly-lysine pattern.
Embodiment 7
PS-b-PAA is dissolved in the homogeneous PS-b-PAA solution that tetrahydrofuran (THF) makes 1.5mg/mL, the molar content of styrene units is 60% among the PS-b-PAA, PS-b-PAA solution with syringe extraction 0.1mL under the room temperature spreads on the ice face equably, be placed on rapidly the environment of relative humidity 75%, take out after solvent evaporates and make through PS-b-PAA honeycomb-patterned ordered porous-film, its aperture size is 4 μ m; Gold plaque is placed piranha solution (30%H
2O
2/ 98% H
2SO
4, mass percent, v/v=1: soaked 10 minutes 3), after taking out with a large amount of washed with de-ionized water, after nitrogen dries up at the through PS-b-PAA honeycomb-patterned ordered of gold plaque surface coverage porous-film as template, and thermal treatment 3 minutes in 80 ℃ of baking ovens; The above-mentioned gold plaque that makes is immersed in the ethanolic soln of the MPA of 5mM, temperature of reaction is 25 ℃, and 8 hours reaction times is then successively with ethanol, washed with de-ionized water gold plaque; The above-mentioned gold plaque that makes is soaked in the tetrahydrofuran (THF) 2.5 hours, successively with ethanol, washed with de-ionized water gold plaque surface, removes template afterwards; The above-mentioned gold plaque that makes is immersed in the phosphate buffer soln of EDC and NHS, temperature of reaction is 20 ℃, reacted 1.5 hours, EDC concentration is 15mg/mL, and the mol ratio of EDC and NHS is 5/2, phosphate buffer soln pH is 7.4, then place the GSH aqueous solution of 5mg/mL, reacted 1 hour, temperature of reaction is 20 ℃, reaction is at room temperature cleaned with phosphate buffer soln after finishing, and namely makes the GSH pattern.
Embodiment 8
PS-b-PAA is dissolved in the homogeneous PS-b-PAA solution that tetrahydrofuran (THF) makes 1.5mg/mL, the molar content of styrene units is 60% among the PS-b-PAA, PS-b-PAA solution with syringe extraction 0.1mL under the room temperature spreads on the ice face equably, be placed on rapidly the environment of relative humidity 75%, take out after solvent evaporates and make through PS-b-PAA honeycomb-patterned ordered porous-film, its aperture size is 4 μ m; Gold plaque is placed piranha solution (30%H
2O
2/ 98% H
2SO
4, mass percent, v/v=1: soaked 10 minutes 3), after taking out with a large amount of washed with de-ionized water, after nitrogen dries up at the through PS-b-PAA honeycomb-patterned ordered of gold plaque surface coverage porous-film as template, and thermal treatment 3 minutes in 80 ℃ of baking ovens; The above-mentioned gold plaque that makes is immersed in the ethanolic soln of the MPA of 5mM, temperature of reaction is 25 ℃, and 8 hours reaction times is then successively with ethanol, washed with de-ionized water gold plaque; The above-mentioned gold plaque that makes is soaked in the tetrahydrofuran (THF) 2.5 hours, successively with ethanol, washed with de-ionized water gold plaque surface, removes template afterwards; The above-mentioned gold plaque that makes is immersed in the phosphate buffer soln of EDC and NHS, temperature of reaction is 15 ℃, reacted 2 hours, EDC concentration is 30mg/mL, and the mol ratio of EDC and NHS is 5/2, phosphate buffer soln pH is 7.4, then place PBS (pH the is 7.4) solution of the gelatin of 15mg/mL, reacted 2 hours, temperature of reaction is 20 ℃, reaction is at room temperature cleaned with deionized water after finishing, and namely makes the gelatin pattern.
Embodiment 9
PS-b-PDMAEMA is dissolved in the homogeneous PS-b-PDMAEMA solution that benzene makes 1mg/mL, the molar content of styrene units is 80% among the PS-b-PDMAEMA, PS-b-PDMAEMA solution with syringe extraction 0.1mL under the room temperature spreads on the ice face equably, be placed on rapidly the environment of relative humidity 80%, take out after solvent evaporates and make through PS-b-PDMAEMA honeycomb-patterned ordered porous-film, its aperture size is 2 μ m; Gold plaque is placed piranha solution (30%H
2O
2/ 98% H
2SO
4, mass percent, v/v=1: soaked 10 minutes 3), after taking out with a large amount of washed with de-ionized water, after nitrogen dries up at the through PS-b-PDMAEMA honeycomb-patterned ordered of gold plaque surface coverage porous-film as template, and thermal treatment 1 minute in 80 ℃ of baking ovens; The above-mentioned gold plaque that makes is immersed in the ethanolic soln of the MUA of 15mM, temperature of reaction is 20 ℃, and 4 hours reaction times is then successively with ethanol, washed with de-ionized water gold plaque; The above-mentioned gold plaque that makes is soaked in the benzene 3 hours, successively with ethanol, washed with de-ionized water gold plaque surface, removes template afterwards; The above-mentioned gold plaque that makes is immersed in the phosphate buffer soln of EDC and NHS, temperature of reaction is 20 ℃, reacted 2 hours, EDC concentration is 25mg/mL, and the mol ratio of EDC and NHS is 5/2, phosphate buffer soln pH is 7.4, then place the acetic acid aqueous solution (mass percentage concentration of acetic acid aqueous solution acetic acid is 0.3%) of the collagen of 4mg/mL, reacted 2 hours, temperature of reaction is 30 ℃, reaction is at room temperature cleaned with phosphate buffer soln after finishing, and namely makes the collagen pattern.
Embodiment 10
PS-b-PDMAEMA is dissolved in the homogeneous PS-b-PDMAEMA solution that toluene makes 1.5mg/mL, the molar content of styrene units is 70% among the PS-b-PDMAEMA, PS-b-PDMAEMA solution with syringe extraction 0.1mL under the room temperature spreads on the ice face equably, be placed on rapidly the environment of relative humidity 65%, take out after solvent evaporates and make through PS-b-PDMAEMA honeycomb-patterned ordered porous-film, its aperture size is 2.5 μ m; Gold plaque is placed piranha solution (30%H
2O
2/ 98% H
2SO
4, mass percent, v/v=1: soaked 10 minutes 3), after taking out with a large amount of washed with de-ionized water, after nitrogen dries up at the through PS-b-PDMAEMA honeycomb-patterned ordered of gold plaque surface coverage porous-film as template, and thermal treatment 1.5 minutes in 80 ℃ of baking ovens; The above-mentioned gold plaque that makes is immersed in the ethanolic soln of the MPA of 20mM, temperature of reaction is 15 ℃, and 3 hours reaction times is then successively with ethanol, washed with de-ionized water gold plaque; The above-mentioned gold plaque that makes is soaked in the toluene 2.5 hours, successively with ethanol, washed with de-ionized water gold plaque surface, removes template afterwards; The above-mentioned gold plaque that makes is immersed in the phosphate buffer soln of EDC and NHS, temperature of reaction is 30 ℃, reacted 1 hour, EDC concentration is 10mg/mL, and the mol ratio of EDC and NHS is 5/2, phosphate buffer soln pH is 7.4, then place PBS (pH the is 7.4) solution of the HRP of 2mg/mL, reacted 2 hours, temperature of reaction is 20 ℃, reaction is at room temperature cleaned with deionized water after finishing, and namely makes the HRP pattern.
Claims (10)
1. a method for preparing the biomolecules pattern on the gold plaque surface is characterized in that, may further comprise the steps:
(1) polymer dissolution is made the polymers soln of homogeneous in solvent, under 15 ℃～30 ℃ with the polymers soln uniform spreading on the ice face, place rapidly the environment with humidity, after solvent evaporates, take out and make through polymkeric substance honeycomb-patterned ordered porous-film; Described polymkeric substance is styrol copolymer;
(2) gold plaque is placed piranha solution soaked 10 minutes～15 minutes, use washed with de-ionized water after taking out, the through polymkeric substance honeycomb-patterned ordered porous-film that makes in gold plaque surface coverage step (1) after nitrogen dries up is as template, and at 80 ℃～85 ℃ thermal treatments 1 minute～5 minutes, the gold plaque after obtaining processing;
(3) gold plaque after processing in the step (2) is immersed in the polyfunctional group organic solution with sulfydryl, 15 ℃～30 ℃ reactions 0.5 hour～12 hours, then use successively ethanol, washed with de-ionized water gold plaque, obtain the gold plaque that there is self assembled monolayer on the surface;
(4) surface in the step (3) there is the gold plaque of self assembled monolayer be soaked in the releasing agent 0.2 hour～5 hours, use successively afterwards ethanol, washed with de-ionized water gold plaque surface, remove template, obtain the gold plaque that there is the patterning self assembled monolayer on the surface;
(5) surface in the step (4) there is the gold plaque of patterning self assembled monolayer activate, then place biomolecule solution, 15 ℃～30 ℃ reactions 0.2 hour～2 hours, after finishing, reaction with buffered soln or washed with de-ionized water, makes the biomolecules pattern.
2. according to claim 1 in the surperficial method for preparing the biomolecules pattern of gold plaque, it is characterized in that, in the step (1), described styrol copolymer is one or more in styrene/methacrylic acid hydroxy methacrylate segmented copolymer, styrene/methacrylic acid dimethylaminoethyl segmented copolymer, styrene/acrylic segmented copolymer, styrene/isoprene/styrene segmented copolymer, the styrene/butadiene/styrene block copolymers;
Described solvent is dithiocarbonic anhydride, methylene dichloride, chloroform, tetrahydrofuran (THF), benzene or toluene.
3. the method for preparing the biomolecules pattern on the gold plaque surface according to claim 1 and 2 is characterized in that, in the step (1), the molar content of styrene units is 30%～99% in the described styrol copolymer.
4. the method for preparing the biomolecules pattern on the gold plaque surface according to claim 1 is characterized in that, in the step (1), the polymer concentration in the described polymers soln is 0.1mg/mL～5.0mg/mL.
5. the method for preparing the biomolecules pattern on the gold plaque surface according to claim 1 is characterized in that, in the step (1), described humidity is relative humidity 60%～90%.
6. according to claim 1 in the surperficial method for preparing the biomolecules pattern of gold plaque, it is characterized in that, in the step (3), described polyfunctional group organic solution with sulfydryl is that 3-thiohydracrylic acid concentration is that ethanolic soln or the 11-sulfydryl undecanoic acid concentration of the 3-thiohydracrylic acid of 0.1mmol/L～20mmol/L is the ethanolic soln of the 11-sulfydryl undecanoic acid of 0.1mmol/L～20mmol/L.
7. the method for preparing the biomolecules pattern on the gold plaque surface according to claim 1 is characterized in that, in the step (4), described releasing agent is dithiocarbonic anhydride, methylene dichloride, chloroform, tetrahydrofuran (THF), benzene or toluene.
8. according to claim 1 in the surperficial method for preparing the biomolecules pattern of gold plaque, it is characterized in that, in the step (5), described biomolecules is a kind of in horseradish peroxidase, glucose oxidase, catalase, albumin, fibronectin, poly-lysine, gsh, gelatin, the collagen.
9. the method for preparing the biomolecules pattern on the gold plaque surface according to claim 1 is characterized in that, in the step (5), the concentration of biomolecules is 0.001mg/mL～50mg/mL in the described biomolecule solution.
10. according to claim 1 in the surperficial method for preparing the biomolecules pattern of gold plaque, it is characterized in that, in the step (5), the step of described activation comprises: have the gold plaque of patterning self assembled monolayer to be immersed in the phosphate buffer soln of 1-ethyl-3-(dimethyl aminopropyl) phosphinylidyne diimine and N-maloyl imines on surface in the step (4), 15 ℃～30 ℃ reactions 0.2 hour～2 hours; Wherein the concentration of 1-ethyl-3-(dimethyl aminopropyl) phosphinylidyne diimine is 2mg/mL～50mg/mL, and the mol ratio of 1-ethyl-3-(dimethyl aminopropyl) phosphinylidyne diimine and N-maloyl imines is 5/2, and phosphate buffer soln pH is 7.4.
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