CN102912343A - Method for preparing biological molecule pattern on gold sheet - Google Patents

Abstract

The invention discloses a method for preparing a biomolecular pattern on the surface of a gold sheet, comprising: (1) preparing a through-type polymer honeycomb ordered porous membrane; (2) covering the surface of the gold sheet with a through-type polymer honeycomb organic (3) adding a multifunctional organic solution with a mercapto group to the surface of the above-mentioned gold flakes, selectively introducing functional groups on the surface of the gold flakes in the membrane pores to form a patterned monolayer surface; (4) adding The above gold sheet is soaked in a release agent to remove the through-type polymer honeycomb ordered porous film; (5) a biomolecular solution is dropped on the surface of the gold sheet on which the mercapto compound has been patterned and fixed, and the biomolecular pattern is obtained after cleaning. The method of the present invention does not rely on the existing patterning technologies such as photolithography and electron beam etching, and is an effective method for preparing biomolecular patterns with low cost, simple and convenient, and has good applications in the fields of biosensing and bioanalysis. Application prospect.

Description

A method for preparing biomolecular patterns on the surface of gold flakes

technical field

The invention relates to the field of material chemistry, in particular to a method for preparing biomolecular patterns on the surface of a gold sheet.

Background technique

Patterning of biomolecules is a microarray technology that immobilizes biomolecules at specified positions on the solid surface and avoids non-specific adsorption in other areas. This technology provides a powerful tool for the study of the interaction between proteins, enzymes, sugars, nucleic acids and other biomolecules and their receptors, and has been widely used in the fields of biosensors, biomedical instruments, microfluidic chips and tissue engineering. Gold flakes are one of the commonly used solid phase substrates.

The currently widely used methods for preparing biomolecular patterns mainly include photolithography, mechanical micro-spotting and soft lithography. Among them, the photolithography and mechanical micro-spotting methods apply photoelectron etching technology, which requires photomasks, electron beam emitters and skilled operators, and consumes high manpower, material and financial resources. The biomolecules obtained after the template is determined The size of the array is relatively fixed, and it is not easy to realize dynamic adjustment. Soft lithography usually uses polydimethylsiloxane (PDMS) as a stamp, which is used as a template to construct biomolecular arrays on the substrate material, which is more convenient than photolithography. Chinese invention patent application CN1425920A discloses a method for co-patterning immobilized biomacromolecules on the surface of inorganic silicon materials. PDMS stamp micro-contact printing biomacromolecules to form micropatterns, drop another biomacromolecule solution on the surface of inorganic silicon material that has been patterned and immobilized with one biomacromolecule, so that the latter can be fixed on the area not covered by the former After washing and ultrasonic cleaning, the co-patterned immobilization of two biomacromolecules on the same surface is obtained. Chinese invention patent application CN02160335.9 discloses a method of patterning and immobilizing biomacromolecules on the active surface of polymers using micro-transfer technology, which is to selectively "load" biomacromolecules into polydimethylsiloxane stamps In the micro-well, a layer of condensed water is formed on the surface, and the original water in the micro-well and the water diffused laterally in the contact area are used to redissolve the biomacromolecules after the seal and the modified polymer film are pressed tightly, so that the biomacromolecules can be redissolved in the form of a solution. The surface groups undergo chemical reactions to achieve patterned immobilization of biomacromolecules on the active surface of the polymer; this method is simple, flexible, and reproducible. The well depth requirement is low, and it is a simple and cheap effective method for patterning and immobilizing biomacromolecules, especially for biomolecules/active surface systems with low reactivity, in the construction of cell-based biological devices, tissue engineering and cell It has good application prospects in fields such as basic biological research. However, the process of preparing PDMS stamps also relies on techniques such as photolithography or electron beam etching, which hinders the promotion of soft etching. It can be seen that the existing technologies for preparing biomolecular patterns almost all rely on photolithography or electron beam etching, which require large-scale equipment and complicated operations, and cannot fully meet the needs of actual preparation processes.

Contents of the invention

The purpose of the present invention is to provide a method for preparing biomolecular patterns on the surface of a gold sheet with very simple operation process, mild reaction conditions and low cost. The method does not depend on technologies such as photoetching or electron beam etching.

The basic principle of the method of the present invention is to use the through-type polymer honeycomb ordered porous membrane prepared by the water droplet template method as a template, cover the template on the surface of the cleaned gold sheet and assemble a monomolecular layer, and then selectively inject biomolecules The biomolecular pattern is fixed on the membrane hole area of the perforated polymer honeycomb ordered porous membrane.

A method for preparing a biomolecular pattern on the surface of a gold sheet, comprising the following steps:

(1) Dissolve the polymer in a solvent to prepare a uniform polymer solution, spread the polymer solution evenly on the ice surface at 15°C to 30°C, quickly place it in a humid environment, and take it out after the solvent evaporates. A perforated polymer honeycomb ordered porous membrane; the polymer is a styrene copolymer;

(2) Soak the gold sheet in piranha solution for 10 to 15 minutes, take it out, wash it with deionized water, dry it with nitrogen, and cover the surface of the gold sheet with the through-type polymer honeycomb ordered in step (1). The porous membrane is used as a template, and heat-treated at 80°C to 85°C for 1 minute to 5 minutes to obtain the treated gold sheet;

(3) Submerge the gold flakes treated in step (2) in a solution of multifunctional organic matter with mercapto groups, react at ambient temperature (preferably 15°C to 30°C) for 0.5 hours to 12 hours, and then use ethanol, and remove Wash the gold flakes with ionized water to obtain gold flakes with a self-assembled monolayer on the surface;

(4) Soak the gold sheet with self-assembled monolayer on the surface in step (3) in the release agent for 0.2 hours to 5 hours, then wash the surface of the gold sheet with ethanol and deionized water successively, remove the template, and obtain the surface with Au flakes with patterned self-assembled monolayers;

(5) Activate the gold sheet with a patterned self-assembled monolayer on the surface in step (4), then place it in a biomolecular solution, and react at 15°C to 30°C for 0.2 to 2 hours. After the reaction, use a buffer solution or Wash with deionized water to make biomolecular patterns.

In step (1), the polymer is selected from styrene copolymer, which has appropriate styrene units, and can obtain a through-type polymer honeycomb ordered porous membrane with uniform pore size.

Described styrene copolymer is preferably styrene/hydroxyethyl methacrylate block copolymer (PS-b-PHEMA), styrene/dimethylaminoethyl methacrylate block copolymer (PS-b-PDMAEMA ), styrene/acrylic acid block copolymer (PS-b-PAA), styrene/isoprene/styrene block copolymer (SIS), styrene/butadiene/styrene block copolymer ( One or more of SBS). The mole percentage of styrene units in the styrene copolymer is preferably 30%-99%.

The solvent is selected according to the polymer, and the principle is to select a solvent with a high saturated vapor pressure, preferably carbon disulfide, methylene chloride, chloroform, tetrahydrofuran, benzene or toluene.

The polymer concentration in the polymer solution is preferably 0.1 mg/mL˜5.0 mg/mL.

The humidity is selected according to the polymer, preferably relative humidity of 60% to 90%.

The amount of the polymer solution is selected according to the membrane area of the through-type polymer honeycomb ordered porous membrane to be prepared, the more the polymer solution is used, the larger the membrane area is, and the present invention forms about 2 square centimeters area of the film as an example, the amount of polymer solution is 0.1mL.

In step (2), the piranha solution can be commercially available or prepared by existing methods, generally 30% (mass percentage) H ₂ O ₂ and 98% (mass percentage) H ₂ SO ₄ by volume Mixed at a ratio of 1:3.

In step (2), the template only allows the surface of the gold sheet in the membrane hole area to be in contact with other substances, while covering the surface of the gold sheet in the non-membrane hole area.

In step (3), the multifunctional organic compound with mercapto groups forms a self-assembled monomolecular layer on the surface of the gold sheet.

The described polyfunctional organic matter solution with mercapto group is 3-mercaptopropionic acid (MPA) concentration is 0.1mmol/L～20mmol/L ethanol solution of 3-mercaptopropionic acid or 11-mercaptoundecanoic acid (MUA) Ethanol solution of 11-mercaptoundecanoic acid with a concentration of 0.1mmol/L to 20mmol/L.

In step (4), the release agent is preferably carbon disulfide, methylene chloride, chloroform, tetrahydrofuran, benzene or toluene.

In step (5), the biomolecules are polypeptides, enzymes, protein biomolecules and derivatives thereof, horseradish peroxidase (HR), glucose oxidase (GOX), catalase (CAT ), albumin (BSA), fibronectin (FN), polylysine, glutathione (GSH), gelatin, collagen, etc. The solvent in the described biomolecule solution is selected from the buffer solution or aqueous solution of the corresponding pH value of the above-mentioned biomolecule, such as the phosphate buffer saline (PBS) solution of BSA, the PBS solution of gelatin, the acetic acid aqueous solution of collagen (acetic acid in the acetic acid aqueous solution). The mass percent concentration is 0.3%), etc.

The concentration of biomolecules in the biomolecules solution is preferably 0.001 mg/mL-50 mg/mL, which can be adjusted according to different types of biomolecules.

The step of described activation comprises: immerse the gold sheet that patterned self-assembled monolayer is arranged on the surface in step (4) in 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC ) and N-hydroxysuccinimide (NHS) in a phosphate buffer solution, react at ambient temperature (preferably 15°C to 30°C) for 0.2 hours to 2 hours; in the phosphate buffer solution of EDC and NHS, the EDC The concentration is 2mg/mL～50mg/mL, the molar ratio of EDC to NHS is 5/2, and the pH of the phosphate buffer solution is 7.4.

Compared with prior art, the present invention has following advantage:

The method of the present invention comprises the following steps: (1) preparation of a through-type polymer honeycomb ordered porous membrane; (2) covering the through-type polymer honeycomb ordered porous membrane on the surface of the gold sheet; Add a multifunctional organic solution with mercapto groups to the surface, and selectively introduce functional groups on the surface of the gold flakes in the membrane pores to form a patterned monolayer surface; (4) soak the above gold flakes in an appropriate solvent to remove Through-type polymer honeycomb ordered porous membrane; (5) drop a biomolecular solution on the surface of the gold sheet on which the mercapto compound has been patterned and fixed, so that the biomolecule is specifically fixed on the area grafted with the mercapto compound. After cleaning, That is, the patterned immobilization of biomolecules on the surface of the gold sheet is obtained. In the present invention, the biomolecular pattern can control the size of the through-type polymer honeycomb ordered porous membrane through the polymer type, polymer concentration, relative humidity, polymer solvent, etc., or adjust the reaction time, reaction temperature and mercapto polyfunctional organic matter and biomolecule solution concentration to control.

The most prominent advantage of the present invention is that it does not rely on existing patterning technologies such as photolithography and electron beam etching, and avoids the use of large-scale and expensive instruments and equipment. Therefore, the method has simple operation process, mild reaction conditions, good repeatability, and low cost. It is cheap, immobilized biomolecules with high firmness and can realize dynamic controllable adjustment of patterned size. The prepared biomolecular patterned gold sheet surface has good application prospects in the fields of biosensing and bioanalysis based on quartz crystal microbalance and surface plasmon resonance.

Detailed ways

The present invention is described in more detail through the following examples, but the examples do not constitute a limitation to the present invention.

Example 1

Dissolve PS-b-PHEMA in carbon disulfide to prepare a 1 mg/mL homogeneous PS-b-PHEMA solution. The molar percentage of styrene units in PS-b-PHEMA is 99%. Draw 0.1 mL of PS with a syringe at room temperature - The b-PHEMA solution is evenly spread on the ice (the amount of solution can be changed, and the film-forming area of different solution amounts is different), and it is quickly placed in an environment with a relative humidity of 75%, and the through-type PS is obtained after the solvent is volatilized. -b-PHEMA honeycomb ordered porous membrane with a pore size of 2 μm; gold flakes are placed in piranha solution (30% H ₂ O ₂ /98% H ₂ SO ₄ , mass percent, v/v=1:3) Immerse in the medium for 10 minutes, take it out, wash it with a large amount of deionized water, blow it dry with nitrogen, cover the surface of the gold sheet with a through-type PS-b-PHEMA honeycomb ordered porous membrane as a template, and heat-treat it in an oven at 80°C for 5 minutes; The gold flakes after the above heat treatment are immersed in the ethanol solution of MPA whose MPA concentration is 20mmol/L (mM), the reaction temperature is 15°C, and the reaction time is 0.5 hour, then the gold flakes are cleaned with ethanol and deionized water successively; The final gold flakes were immersed in carbon disulfide for 0.2 hours, and then the surface of the gold flakes was washed with ethanol and deionized water successively to remove the template; the gold flakes prepared above were immersed in the phosphate buffer solution of EDC and NHS, and the reaction temperature was 30 ℃, reacted for 0.2 hours, wherein the concentration of EDC was 2 mg/mL, the molar ratio of EDC to NHS was 5/2, the pH of the phosphate buffer solution was 7.4, and then placed in 0.001 mg/mL of HRP in PBS (pH 7.4) The solution was reacted for 0.2 hours at a reaction temperature of 15° C., and after the reaction was completed, it was washed with a phosphate buffer solution at room temperature to obtain an HRP pattern.

Example 2

Dissolve PS-b-PDMAEMA in carbon disulfide to prepare a 1 mg/mL homogeneous PS-b-PDMAEMA solution. The molar percentage of styrene units in PS-b-PDMAEMA is 90%. Draw 0.1 mL of PS with a syringe at room temperature - The b-PDMAEMA solution is evenly spread on the ice surface, quickly placed in an environment with a relative humidity of 80%, and taken out after the solvent volatilizes to obtain a through-type PS-b-PDMAEMA honeycomb-shaped ordered porous membrane with a pore size of 2.5 μm; soak the gold sheet in piranha solution (30% H ₂ O ₂ /98% H ₂ SO ₄ , mass percentage, v/v=1:3) for 10 minutes, take it out and wash it with a large amount of deionized water, After drying with nitrogen, cover the through-type PS-b-PDMAEMA honeycomb ordered porous film on the surface of the gold sheet as a template, and heat-treat it in an oven at 80°C for 1 minute; immerse the above-prepared gold sheet in 0.1mM MPA ethanol solution, the reaction temperature is 30°C, the reaction time is 12 hours, and then the gold flakes are washed with ethanol and deionized water successively; the gold flakes prepared above are soaked in carbon disulfide for 5 hours, and then the gold flakes are washed with ethanol and deionized water in turn surface, remove the template; immerse the gold flakes prepared above in the phosphate buffer solution of EDC and NHS, the reaction temperature is 15°C, react for 2 hours, the concentration of EDC is 50mg/mL, and the molar ratio of EDC to NHS is 5/ 2. The pH of the phosphate buffer solution is 7.4, and then placed in 10 mg/mL GOX in PBS (pH 7.4) solution, reacted for 2 hours, and the reaction temperature is 30 ° C. After the reaction, wash with deionized water at room temperature, that is A GOX pattern is produced.

Example 3

Dissolve PS-b-PAA in dichloromethane to prepare a 5mg/mL homogeneous PS-b-PAA solution, the molar percentage of styrene units in PS-b-PAA is 95%, draw 0.1mL with a syringe at room temperature The PS-b-PAA solution was evenly spread on the ice surface, quickly placed in an environment with a relative humidity of 90%, and taken out after the solvent volatilized to obtain a through-type PS-b-PAA honeycomb-shaped ordered porous membrane. The size is 3 μm; soak the gold sheet in piranha solution (30% H ₂ O ₂ /98% H ₂ SO ₄ , mass percentage, v/v=1:3) for 10 minutes, take it out and wash it with a large amount of deionized water , after drying with nitrogen, cover the through-type PS-b-PAA honeycomb ordered porous film on the surface of the gold sheet as a template, and heat-treat it in an oven at 80°C for 5 minutes; Ethanol solution, the reaction temperature is 30°C, the reaction time is 12 hours, and then the gold flakes are washed with ethanol and deionized water successively; the gold flakes prepared above are soaked in dichloromethane for 3 hours, and then washed with ethanol and deionized water successively Clean the surface of the gold flakes and remove the template; immerse the gold flakes prepared above in the phosphate buffer solution of EDC and NHS, the reaction temperature is 20°C, react for 1 hour, the concentration of EDC is 10mg/mL, the molar ratio of EDC to NHS 5/2, the pH of the phosphate buffer solution is 7.4, and then placed in 5mg/mL of CAT in PBS (pH is 7.4) solution, reacted for 1 hour, the reaction temperature is 20 ° C, after the reaction, use the phosphate buffer solution at room temperature After cleaning, the CAT pattern is obtained.

Example 4

Dissolve SIS in carbon disulfide to prepare a uniform SIS solution of 0.1mg/mL. The molar percentage of styrene units in SIS is 30%. Draw 0.1mL of SIS solution with a syringe at room temperature and spread it evenly on the ice surface. It is placed in an environment with a relative humidity of 90%, and after the solvent is volatilized, it is taken out to obtain a through-type SIS honeycomb ordered porous membrane with a pore size of 8 μm; the gold sheet is placed in a piranha solution (30% H ₂ O ₂ /98% Soak in H ₂ SO ₄ (mass percentage, v/v=1:3) for 10 minutes, take it out, wash it with a large amount of deionized water, dry it with nitrogen, and cover the surface of the gold sheet with a through-type SIS honeycomb ordered porous membrane as a template , and heat treatment in an oven at 80° C. for 1 minute; immerse the gold flakes prepared above in a 20 mM ethanol solution of MUA, the reaction temperature is 15° C., and the reaction time is 2 hours, and then wash the gold flakes with ethanol and deionized water successively; Soak the above-mentioned gold flakes in carbon disulfide for 5 hours, then wash the surface of the gold flakes with ethanol and deionized water successively to remove the template; immerse the above-mentioned gold flakes in the phosphate buffer solution of EDC and NHS, react The temperature is 15°C, the reaction is 1.5 hours, the EDC concentration is 30mg/mL, the molar ratio of EDC to NHS is 5/2, the pH of the phosphate buffer solution is 7.4, and then placed in 2mg/mL of BSA in PBS (pH 7.4) The solution was reacted for 1 hour at a reaction temperature of 20° C., and after the reaction was completed, it was washed with deionized water at room temperature to obtain a BSA pattern.

Example 5

Dissolve SBS in carbon disulfide to prepare a 0.5mg/mL uniform SBS solution. The molar percentage of styrene units in SBS is 35%. Use a syringe to extract 0.1mL of SBS solution at room temperature and spread it evenly on the ice. It is placed in an environment with a relative humidity of 85%, and after the solvent is volatilized, it is taken out to obtain a through-type SBS honeycomb ordered porous membrane with a pore size of 9 μm; the gold sheet is placed in a piranha solution (30% H ₂ O ₂ /98% Soak in H ₂ SO ₄ (mass percentage, v/v=1:3) for 10 minutes, take it out, wash it with a large amount of deionized water, dry it with nitrogen, and cover the surface of the gold sheet with a through-type SBS honeycomb ordered porous membrane as a template , and heat treatment in an oven at 80°C for 1.5 minutes; the gold flakes prepared above were immersed in an ethanol solution of 10mM MPA, the reaction temperature was 20°C, and the reaction time was 5 hours, and then the gold flakes were washed with ethanol and deionized water successively; Soak the above-mentioned gold flakes in carbon disulfide for 4 hours, then wash the surface of the gold flakes with ethanol and deionized water successively to remove the template; immerse the above-mentioned gold flakes in the phosphate buffer solution of EDC and NHS, react The temperature was 25°C, reacted for 1 hour, the EDC concentration was 20mg/mL, the molar ratio of EDC to NHS was 5/2, the pH of the phosphate buffer solution was 7.4, and then placed in 0.5mg/mL of FN in PBS (pH 7.4 ) solution, reacted for 2 hours, and the reaction temperature was 20°C. After the reaction was completed, it was washed with a phosphate buffer solution at room temperature to obtain an FN pattern.

Example 6

The PS-b-PDMAEMA/SIS mixture was dissolved in chloroform to obtain a homogeneous PS-b-PDMAEMA/SIS mixture solution, the concentration of PS-b-PDMAEMA was 1mg/mL, the concentration of SIS was 0.5mg/mL, PS-b- The molar percentage of styrene units in PDMAEMA/SIS is 65%. At room temperature, draw 0.1mL of PS-b-PDMAEMA/SIS solution with a syringe and spread it evenly on the ice surface, and quickly place it at a relative humidity of 80%. After the solvent volatilized, it was taken out to obtain a through-type PS-b-PDMAEMA/SIS honeycomb ordered porous membrane with a pore size of 5 μm; the gold sheet was placed in a piranha solution (30% H ₂ O ₂ /98% H ₂ SO ₄ , mass percent, v/v=1:3) for 10 minutes, took it out, washed it with a large amount of deionized water, dried it with nitrogen, and covered the through-type PS-b-PDMAEMA/SIS honeycomb layer on the surface of the gold sheet. The sequenced porous membrane was used as a template, and heat-treated in an oven at 80°C for 1 minute; the gold flakes prepared above were immersed in a 5 mM ethanol solution of MUA, the reaction temperature was 25°C, and the reaction time was 10 hours, followed by ethanol, deionized Wash the gold flakes with water; soak the gold flakes prepared above in chloroform for 5 hours, then wash the surface of the gold flakes with ethanol and deionized water in turn to remove the template; immerse the gold flakes prepared above in the phosphate salt of EDC and NHS In the buffer solution, the reaction temperature is 30°C, the reaction is 0.5 hours, the EDC concentration is 45mg/mL, the molar ratio of EDC to NHS is 5/2, the pH of the phosphate buffer solution is 7.4, and then placed in 50mg/mL polylysine Aqueous acid solution was reacted for 0.5 hours, and the reaction temperature was 30°C. After the reaction was completed, it was washed with deionized water at room temperature to prepare poly-lysine patterns.

Example 7

Dissolve PS-b-PAA in tetrahydrofuran to prepare a 1.5mg/mL homogeneous PS-b-PAA solution, the molar percentage of styrene units in PS-b-PAA is 60%, draw 0.1mL of it with a syringe at room temperature Spread the PS-b-PAA solution evenly on the ice, quickly place it in an environment with a relative humidity of 75%, and take it out after the solvent evaporates to obtain a through-type PS-b-PAA honeycomb-shaped ordered porous membrane. 4 μm; soak the gold sheet in piranha solution (30% H ₂ O ₂ /98% H ₂ SO ₄ , mass percent, v/v=1:3) for 10 minutes, take it out and wash it with a large amount of deionized water, After drying with nitrogen, cover the through-type PS-b-PAA honeycomb ordered porous membrane on the surface of the gold sheet as a template, and heat-treat it in an oven at 80°C for 3 minutes; immerse the above-prepared gold sheet in a 5mM ethanol solution of MPA , the reaction temperature is 25°C, the reaction time is 8 hours, and then the gold flakes are washed with ethanol and deionized water in turn; the gold flakes prepared above are soaked in tetrahydrofuran for 2.5 hours, and then the surface of the gold flakes is washed with ethanol and deionized water in turn , remove the template; immerse the gold flakes prepared above in the phosphate buffer solution of EDC and NHS, the reaction temperature is 20°C, react for 1.5 hours, the concentration of EDC is 15mg/mL, and the molar ratio of EDC to NHS is 5/2 , pH of phosphate buffer solution is 7.4, then put in 5mg/mL GSH aqueous solution, react for 1 hour, the reaction temperature is 20°C, wash with phosphate buffer solution at room temperature after the reaction, and the GSH pattern is obtained.

Example 8

Dissolve PS-b-PAA in tetrahydrofuran to prepare a 1.5mg/mL homogeneous PS-b-PAA solution, the molar percentage of styrene units in PS-b-PAA is 60%, draw 0.1mL of it with a syringe at room temperature Spread the PS-b-PAA solution evenly on the ice, quickly place it in an environment with a relative humidity of 75%, and take it out after the solvent evaporates to obtain a through-type PS-b-PAA honeycomb-shaped ordered porous membrane. 4 μm; soak the gold sheet in piranha solution (30% H ₂ O ₂ /98% H ₂ SO ₄ , mass percent, v/v=1:3) for 10 minutes, take it out and wash it with a large amount of deionized water, After drying with nitrogen, cover the through-type PS-b-PAA honeycomb ordered porous membrane on the surface of the gold sheet as a template, and heat-treat it in an oven at 80°C for 3 minutes; immerse the above-prepared gold sheet in a 5mM ethanol solution of MPA , the reaction temperature is 25°C, the reaction time is 8 hours, and then the gold flakes are washed with ethanol and deionized water in turn; the gold flakes prepared above are soaked in tetrahydrofuran for 2.5 hours, and then the surface of the gold flakes is washed with ethanol and deionized water in turn , remove the template; immerse the gold flakes prepared above in the phosphate buffer solution of EDC and NHS, the reaction temperature is 15°C, react for 2 hours, the concentration of EDC is 30mg/mL, and the molar ratio of EDC to NHS is 5/2 , the pH of the phosphate buffer solution is 7.4, and then placed in 15 mg/mL of gelatin in PBS (pH is 7.4) solution, reacted for 2 hours, the reaction temperature is 20 ° C, after the reaction, wash with deionized water at room temperature, that is, the preparation Gelatin pattern.

Example 9

Dissolve PS-b-PDMAEMA in benzene to prepare a 1 mg/mL homogeneous PS-b-PDMAEMA solution. The molar percentage of styrene units in PS-b-PDMAEMA is 80%. Draw 0.1 mL of PS with a syringe at room temperature - The b-PDMAEMA solution is evenly spread on the ice surface, quickly placed in an environment with a relative humidity of 80%, and taken out after the solvent volatilizes to obtain a through-type PS-b-PDMAEMA honeycomb-shaped ordered porous membrane with a pore size of 2 μm; soak the gold sheet in piranha solution (30% H ₂ O ₂ /98% H ₂ SO ₄ , mass percent, v/v=1:3) for 10 minutes, take it out and wash it with a large amount of deionized water, nitrogen After blowing dry, cover the through-type PS-b-PDMAEMA honeycomb ordered porous membrane on the surface of the gold sheet as a template, and heat-treat it in an oven at 80°C for 1 minute; immerse the above-mentioned gold sheet in a 15mM ethanol solution of MUA, The reaction temperature is 20°C, the reaction time is 4 hours, and then the gold flakes are washed successively with ethanol and deionized water; the gold flakes prepared above are soaked in benzene for 3 hours, and then the surface of the gold flakes is washed with ethanol and deionized water successively, Remove the template; immerse the above-prepared gold flakes in a phosphate buffer solution of EDC and NHS, the reaction temperature is 20°C, react for 2 hours, the EDC concentration is 25mg/mL, and the molar ratio of EDC to NHS is 5/2, The pH of the phosphate buffer solution is 7.4, and then placed in 4 mg/mL collagen acetic acid aqueous solution (the mass percent concentration of acetic acid in the acetic acid aqueous solution is 0.3%), reacted for 2 hours, and the reaction temperature was 30 ° C. The buffer solution was washed at room temperature, and the collagen pattern was prepared.

Example 10

Dissolve PS-b-PDMAEMA in toluene to obtain a 1.5 mg/mL homogeneous PS-b-PDMAEMA solution. The molar percentage of styrene units in PS-b-PDMAEMA is 70%, and draw 0.1 mL of it with a syringe at room temperature. Spread the PS-b-PDMAEMA solution evenly on the ice surface, quickly place it in an environment with a relative humidity of 65%, and take it out after the solvent volatilizes to obtain a through-type PS-b-PDMAEMA honeycomb-shaped ordered porous membrane. The pore size 2.5 μm; soak the gold sheet in piranha solution (30% H ₂ O ₂ /98% H ₂ SO ₄ , mass percentage, v/v=1:3) for 10 minutes, take it out and wash it with a large amount of deionized water , after drying with nitrogen, cover the through-type PS-b-PDMAEMA honeycomb ordered porous film on the surface of the gold sheet as a template, and heat-treat it in an oven at 80°C for 1.5 minutes; immerse the above-mentioned gold sheet in 20mM MPA ethanol Solution, the reaction temperature is 15°C, the reaction time is 3 hours, and then the gold flakes are washed with ethanol and deionized water in sequence; the gold flakes prepared above are soaked in toluene for 2.5 hours, and then the gold flakes are washed with ethanol and deionized water in turn surface, remove the template; immerse the above-prepared gold flakes in a phosphate buffer solution of EDC and NHS, the reaction temperature is 30°C, react for 1 hour, the concentration of EDC is 10mg/mL, and the molar ratio of EDC to NHS is 5/ 2. The pH of the phosphate buffer solution is 7.4, and then placed in 2 mg/mL of HRP in PBS (pH 7.4) solution, reacted for 2 hours, the reaction temperature is 20 ° C, after the reaction, wash with deionized water at room temperature, that is An HRP pattern is made.

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.