CN105837730B - Method for constructing bioactive surface by combining layer-by-layer assembly technology and host-guest interaction - Google Patents

Abstract

The invention relates to the fields of biomedical engineering and polymer materials, and provides a method for constructing a bioactive surface in combination with layer-by-layer assembly technology and host-guest interaction. The present invention introduces the guest molecule adamantane (Ada) group into polyacrylic acid (PAA) through a simple copolymerization technique to obtain a copolymer of acrylic acid monomer and adamantyl methyl acrylate monomer (P(AA‑Ada)); Polyelectrolyte multilayer films of P(AA‑Ada) and polyallylamine (PAH) were then deposited on the surface of various aminated substrates using layer-by-layer (LbL) technology. A large number of adamantane groups contained in the polyelectrolyte multilayer film can be used to immobilize β-cyclodextrin (β-CD) derivatives modified with different biomolecules at high density, so as to obtain a bioactive surface with corresponding functions. The outstanding features of the present invention are: the method is universal and can be applied to substrates with different chemical properties and shapes; and the properties of the constructed bioactive surface can be adjusted, which is applicable to a wide range of biomedical fields.

Description

A combination of layer-by-layer assembly technology and host-guest interaction to construct bioactive surfaces Methods

technical field

The invention relates to the field of biomedical functional polymer materials, in particular to a preparation method for constructing bioactive surfaces on various substrates in combination with layer-by-layer assembly technology and host-guest interaction.

Background technique

Bioactive surface refers to the immobilization of biological functional molecules such as proteins, polypeptides, nucleic acids, polysaccharides, etc. on the surface of synthetic materials by physical or chemical methods, and then endows the surface with specific bioactive functions (such as biomolecular recognition performance, bactericidal performance, anticoagulant performance) , anti-fouling properties, etc.), which have been widely used in many fields of biomedical engineering and biotechnology. Therefore, how to modify the surface of biological materials in a simple, efficient and universal way to construct bioactive surfaces is of great significance. Usually, the bioactive surface is constructed by first forming a layer of matrix on the surface of the material, and then immobilizing or loading biofunctional molecules in the matrix. Polymers are widely used as substrates due to their good biocompatibility, multiple active sites, strong functionality, and easy surface modification. The commonly used polymer matrix preparation methods mainly include self-assembly, Langmuir-Blodgett membrane, polymer coating, surface-initiated polymerization, layer-by-layer assembly technology, etc. Among them, the layer-by-layer assembly technology is a modification method that adsorbs alternate layers of polymers with different charges on the surface of the substrate based on electrostatic interactions. Compared with other methods, layer-by-layer assembly technology has many advantages such as simple operation, convenient control of polymer film parameters, and almost any substrate with any shape and physical properties. In particular, the polymer film formed by the layer-by-layer assembly technique has a three-dimensional structure, which is very suitable for loading and immobilizing bioactive molecules, and the number of immobilized biomolecules can be effectively controlled by simply changing the thickness of the multilayer film.

A common method for immobilizing biomolecules on the surface of materials is to chemically bind the biomolecules to the substrate through covalent bonds. Although this method has a strong binding force, it usually requires complicated operations and the use of organic solvents. In recent years, the use of affinity immobilization based on weak interactions has received extensive attention. Host-guest interaction is a common supramolecular chemical interaction. After the host molecule (or guest molecule) is post-modified by biomolecules, the immobilization of biomolecules can be easily realized under mild conditions through host-guest interaction. β-cyclodextrin (β-CD) is a common host molecule, and its hydrophobic cavity facilitates the insertion of various guest small molecules. β-CD itself has multiple conveniently activated hydroxyl groups, which can realize the binding of multiple biological small molecule ligands on one molecule, thereby increasing the local ligand density and making the post-modified β-CD have higher biological activity. In addition, from a chemical point of view, we can post-modify β-CD to obtain β-CD derivatives with different functions. Applying host-guest interactions, it is possible to achieve bifunctional or even multifunctional endowment on the same surface by simply mixing two or even multiple biomolecular post-modified β-CD derivatives.

The present invention selects polyanion/polycation pair polyacrylic acid/polyacrylamine (PAA/PAH) commonly used in layer-by-layer assembly and typical host-guest pair β-cyclodextrin/adamantane (β-CD/Ada) as models. First, the Ada group was introduced into the polyanion PAA by a simple copolymerization technique to obtain the copolymer P(AA-Ada), and then the P(AA-Ada)/PAH multilayer was deposited on the surface of various substrates by using the layer-by-layer assembly technique Finally, the β-CD modified with bioactive molecules was introduced into the polyelectrolyte multilayer film through host-guest interaction to obtain a bioactive surface with corresponding functions.

Contents of the invention

The technical problem to be solved by the present invention is to provide a simple and universal bioactive surface preparation method for the problems of complex reaction process, limited substrate, limited types of immobilized bioactive molecules, and single function. , a surface modification method that can immobilize a variety of bioactive molecules and endow the surface with a variety of biological functions.

The present invention solves its technical problem and adopts the following technical solutions:

A preparation method for constructing materials with corresponding biological functions provided by the present invention is characterized in that it comprises the following steps:

(1) Preparation of modified surface of polyelectrolyte multilayer film:

The aminated substrate was soaked in a solution of a copolymer of acrylic acid monomer and adamantylmethanol 1-acrylate monomer (P(AA-Ada)) for a period of time and then in a solution of polyallylamine (PAH) For a period of time, one layer of P(AA-Ada)/PAH bimolecular layer is obtained on the surface of the aminated substrate, and the polyelectrolyte with several layers of P(AA-Ada)/PAH bimolecular layer is obtained after repeated soaking several times. Multilayer film modified surface;

(2) Polyelectrolyte multilayer membrane modified surface immobilized bioactive molecules:

The obtained polyelectrolyte multilayer modified surface is placed in a solution containing the β-CD derivative modified with bioactive molecules for reaction, and the polyelectrolyte multilayer modified surface with bioactive molecules immobilized is obtained.

Another preparation method for constructing materials with corresponding biological functions provided by the present invention is characterized in that it includes the following steps:

(1) Preparation of polyanion P(AA-Ada) containing adamantane groups:

According to a certain molar ratio, acrylic acid monomer, 1-adamantyl acrylate monomer, chain transfer agent and initiator are added to the reaction solution for polymerization reaction. After the reaction, the method of sedimentation separation and purification is used to obtain the The polyanion P(AA-Ada);

(2) Preparation of modified surface of polyelectrolyte multilayer film:

The aminated substrate was soaked in a solution of a copolymer of acrylic acid monomer and adamantylmethanol 1-acrylate monomer (P(AA-Ada)) for a period of time and then in a solution of polyallylamine (PAH) For a period of time, one layer of P(AA-Ada)/PAH bimolecular layer is obtained on the surface of the aminated substrate, and the polyelectrolyte with several layers of P(AA-Ada)/PAH bimolecular layer is obtained after repeated soaking several times. Multilayer film modified surface;

(3) Polyelectrolyte multilayer film modified surface to immobilize bioactive molecules:

The obtained polyelectrolyte multilayer modified surface is placed in a solution containing the β-CD derivative modified with bioactive molecules for reaction, and the polyelectrolyte multilayer modified surface with bioactive molecules immobilized is obtained.

Further, the bioactive molecule is biotin, mannose or arginine-glutamic acid-aspartic acid-valine polypeptide.

Further, the preparation process of the β-CD derivative modified with the biotin molecule is as follows: the β-CD (β-CD-(N ₃ ) ₇ ) modified by propargyl biotin and 7 azido groups Click reaction to prepare biotin-modified β-CD.

Further, the preparation process of the β-CD derivative modified with the mannose molecule is as follows: β-CD (β-CD-(N ₃ ) ₇ ) modified with propargyl mannose and 7 azide groups Click reaction to prepare mannose-modified β-CD.

Further, the preparation process of the β-CD derivative modified with the arginine-glutamic acid-aspartic acid-valine polypeptide molecule is as follows: propargyl-arginine-glutamic acid-aspartate Arginine-Glutamate-Valine Polypeptide and β-CD Modified with 7 Azide Groups (β-CD-(N ₃ ) ₇ ) Click Reaction to Prepare Arginine-Glutamate-Aspartate-Valine Amino acid peptide modified β-CD.

Further, the aminated base material is single crystal silicon or glass, and the preparation process of the aminated single crystal silicon sheet or glass sheet is as follows: the single crystal silicon sheet or glass sheet is placed in 3-aminopropyl-three Ethoxysilane (APTES) in toluene solution to react for a period of time to obtain surface aminated single crystal silicon or glass substrate.

Further, the aminated substrate is a gold-plated single crystal silicon wafer, and the preparation process of the aminated gold-plated single crystal silicon wafer is as follows: placing the gold-plated single crystal silicon wafer in an aqueous solution of mercaptoethylamine hydrochloride for a period of reaction time, a gold-plated single crystal silicon substrate with surface amination was obtained.

Further, the solution of the β-CD derivative modified with the bioactive molecule is an aqueous solution, a phosphate buffer solution or other solutions that can dissolve the corresponding bioactive molecule.

Further, the polymerization is reversible addition-fragmentation chain transfer polymerization, and the chain transfer agent is 2-(phenylthioformylthio)propionic acid; the initiator is azobisisobutyronitrile, azo Diisoheptanonitrile, dibenzoyl peroxide, diethylhexyl peroxydicarbonate, cumene hydroperoxide, potassium persulfate-sulfite system or hydrogen peroxide-ferrite system.

Further, the solution containing the chain transfer agent is N,N-dimethylformamide solution, and the solution containing the initiator is N,N-dimethylformamide solution.

Further, the preparation method of β-CD modified with 7 azide groups (β-CD-(N ₃ ) ₇ ) is as follows: firstly, the hydroxyl group at the sixth position of β-CD is substituted with bromine (β-CD-(Br) ₇ ) or iodine substitution (β-CD-(I) ₇ ), followed by azidation with sodium azide.

The present invention provides a preparation method for constructing materials with corresponding biological functions, which includes the following detailed steps:

(1) Preparation of polyanion P(AA-Ada) containing adamantane groups:

In the reaction device, acrylic acid monomer, 1-adamantyl acrylate monomer, chain transfer agent and initiator are added to the reaction solution according to a certain molar ratio, and the reaction is stirred under the protection of nitrogen. The reaction temperature is 65°C. The time is 24 hours; after the reaction is completed, it is settled with ether, and after separation and purification, the polyanion P(AA-Ada) containing adamantyl group can be obtained; the P(AA-Ada) is 1-adamantyl methyl acrylate The English abbreviation for the copolymer of monomer and acrylic acid monomer;

The chain transfer agent is 2-(phenylthioformylthio)propionic acid; the initiator is azobisisobutyronitrile, etc.; the reaction solution is DMF solution.

(2) Preparation of modified surface of polyelectrolyte multilayer film:

Prepare polyelectrolyte solutions, including polyanion P(AA-Ada) solution and polycation PAH solution; soak the aminated substrate in P(AA-Ada) solution, wash to remove weakly bound polymers, and then soak In the PAH solution, after cleaning, a P(AA-Ada)/PAH bimolecular layer can be obtained on the surface. This process is repeated several times to finally reach the required number of layers, and the modified surface of the polyelectrolyte multilayer film is obtained;

The polyelectrolyte solution is an acetic acid/sodium acetate buffer solution with a pH value of 5.0; the surface cleaning solution taken out from the polyelectrolyte solution is an acetic acid/sodium acetate buffer solution with a pH value of 5.0; the aminated substrate Monocrystalline silicon, glass or gold-plated monocrystalline silicon.

The preparation process of the base material of described amination comprises the following steps:

(a) Pre-treat the cleaned single crystal silicon wafer or glass wafer with "piranha" solution (sulfuric acid:hydrogen peroxide=7:3, volume ratio), and then place it in 3-aminopropyl-triethoxysilane (APTES) in toluene solution to react overnight to obtain surface aminated single crystal silicon or glass substrate;

(b) Pretreat the gold-plated monocrystalline silicon wafers cleaned by the UV-ozone meter with a mixed solution of ammonia, hydrogen peroxide and deionized water (ammonia: hydrogen peroxide: deionized water = 1:1:5, volume ratio), and then Placed in an aqueous solution of mercaptoethylamine hydrochloride to react overnight to obtain a gold-plated single crystal silicon substrate with an aminated surface;

(3) Polyelectrolyte multilayer film modified surface to immobilize bioactive molecules:

Put the modified surface of the obtained polyelectrolyte multilayer film in a solution containing β-CD derivatives modified with biomolecules for reaction, the reaction temperature is room temperature, and the reaction time is 12 to 24 hours. After cleaning the surface, the modified surface of polyelectrolyte multilayer film immobilized with bioactive molecules can be obtained;

The biologically active molecules are biotin, mannose and REDV polypeptide; the solution of the β-CD derivative modified with the biologically active molecules is aqueous solution, phosphate buffer solution or other solutions that can dissolve the corresponding biologically active molecules.

The preparation process of the β-CD derivative modified with biologically active molecules comprises the following steps:

(a) Preparation of β-CD modified with 7 azide groups (β-CD-(N ₃ ) ₇ );

(b) Preparation of biotin-modified β-CD:

In the reaction device, first add β-CD-(N ₃ ) ₇ and biotin-PEG ₄ -alkyne, then add the catalyst/ligand solution under the protection of nitrogen, stir the reaction, the reaction temperature is 50°C, and the reaction time is 36 hours After the reaction, the mixed solution was dialyzed with a dialysis bag of MWCO 1000, and the biotin-modified β-CD could be obtained after freeze-drying.

(c) Preparation of mannose-modified β-CD:

Under the protection of nitrogen, add 1-(2'-propargyl)-α-D-mannose and β-CD-(N ₃ ) ₇ into the catalyst system solution for click reaction, the reaction temperature is 50°C, and the reaction time is After 24 hours, the solvent was removed after the reaction, and the precipitate was collected by centrifugation with anhydrous methanol, and the mannose-modified β-CD could be obtained after purification.

(d) Preparation of β-CD modified by REDV polypeptide:

Under the protection of nitrogen, add propargyl-REDV and β-CD-(N ₃ ) ₇ into the catalyst system solution for click reaction, the reaction temperature is 50°C, the reaction time is 24 hours, and the post-reaction treatment is to use MWCO 500 The β-CD modified by REDV polypeptide can be obtained by dialyzing the dialysis bag and freeze-drying.

Preferably, the preparation method described in step (a) is one or both of the following:

Firstly, the hydroxyl group at the sixth position of β-CD is replaced by bromine (β-CD-(Br) ₇ ), and then azidated with sodium azide;

The hydroxyl group at the sixth position of β-CD is replaced with iodine (β-CD-(I) ₇ ), and then azide is carried out with sodium azide.

Preferably, the catalyst/ligand solution described in step (b) is a dimethyl sulfoxide solution, and the catalyst/ligand system is 2,2'-bipyridyl/copper bromide and the like.

Preferably, the catalyst solution described in step (c) is a dimethyl sulfoxide solution, and the catalyst/ligand system is 2,2'-bipyridine/copper bromide and the like.

Preferably, the catalyst system solution described in step (d) is a mixed solution of dimethyl sulfoxide/water, and the catalyst system is copper sulfate/sodium ascorbate and the like.

After the above steps, a bioactive surface with high-density immobilized biofunctional molecules with corresponding biological functions is finally obtained.

Compared with the prior art, the present invention has the following main advantages:

The method provided by the invention is a method for preparing a bioactive surface by combining layer-by-layer assembly technology and host-guest interaction. Compared with the prior art, the present invention has the following prominent features:

1. Simple and easy to operate: layer-by-layer assembly and host-guest reaction conditions are mild, and can be completed at room temperature and in an aqueous solution environment.

2. Wide applicability: The method of constructing polyelectrolyte multilayer films by layer-by-layer assembly technology can be applied to substrates of different chemical properties and shapes, and can be realized on the surface of any substrate containing amino groups. It is suitable for biological detection, tissue Engineering, protein separation and purification and many other biomedical fields.

3. Adjustable surface properties: On the one hand, the capacity of loaded biomolecules can be controlled simply by changing the number of layers assembled, and on the other hand, β-CD modified with different biofunctional molecules can be designed to endow the surface with different biological functions. For example, after immobilizing biotin-modified β-CD on the surface of the polyelectrolyte multilayer film obtained by this method, avidin can be selectively bound (see Figure 1); after immobilizing mannose-modified β-CD on the surface, it can Specific adsorption of concanavalin (ConA) (see Figure 2); after immobilizing REDV polypeptide-modified β-CD on the surface, it can promote the adhesion of surface endothelial cells (see Figure 3).

Description of drawings

Figure 1 shows the adsorption of fluorescence-modified avidin on the surface of a polyelectrolyte multilayer film on a single crystal silicon wafer before and after immobilization of biotin-modified β-CD;

Figure 2 shows the adsorption of fluorescence-modified concanavalin on the surface of the gold-plated single-crystal silicon wafer polyelectrolyte multilayer film before and after immobilization of mannose-modified β-CD.

Figure 3 shows the adhesion to endothelial cells before and after the REDV polypeptide-modified β-CD is immobilized on the surface of the polyelectrolyte multilayer film on the gold-plated single crystal silicon substrate.

detailed description

The method provided by the present invention for constructing a biologically active surface in combination with layer-by-layer assembly technology and host-guest interaction is to firstly carry out amination pretreatment on the surface of the substrate, and then use polyelectrolyte P (AA-Ada) and PAH through layer-by-layer assembly technology A polyelectrolyte multilayer film containing Ada groups was deposited on the surface. Finally, by using the host-guest interaction between Ada and β-CD, β-CD modified with different bioactive molecules was immobilized on the surface of the polyelectrolyte multilayer membrane, endowing the surface with corresponding biological functions.

Below by embodiment, the present invention is described further, but does not limit the present invention.

Example 1

Combining layer-by-layer assembly technology and host-guest interaction to construct a surface with biomolecular recognition function on the surface of a single crystal silicon wafer

The monocrystalline silicon wafers cleaned ultrasonically with acetone solution were pretreated with "piranha" solution (sulfuric acid:hydrogen peroxide=7:3, volume ratio), and then placed in 3-aminopropyl-triethoxysilane (APTES) react in a toluene solution for 18 hours to obtain a monocrystalline silicon wafer with surface amination. A polyanion P(AA-Ada) solution and a polycation PAH solution were respectively prepared, and the solvent was an acetic acid/sodium acetate buffer solution with a pH value of 5.0 and a concentration of 0.05 mol/L. Soak the aminated monocrystalline silicon wafer in the P(AA-Ada) solution, wash it with acetic acid/sodium acetate buffer solution to remove the weakly bonded polymer, and then soak it in the PAH solution. After cleaning, a P (AA-Ada)/PAH bimolecular layer, this process is repeated several times until the required number of layers is reached, and a polyelectrolyte multilayer film modified single crystal silicon wafer can be obtained.

Firstly, the hydroxyl group at the sixth position of β-CD is replaced by bromine (β-CD-(Br) ₇ ), and then azide is used to prepare β-CD modified with 7 azido groups (β-CD -(N ₃ ) ₇ ); Finally, click reaction was performed on β-CD-(N ₃ ) ₇ and propargyl biotin to obtain biotin-modified β-CD (β-CD-(biotin) ₇ ).

The above polyelectrolyte multilayer modified single crystal silicon wafer was placed in 1 millimole per liter of β-CD-(biotin) ₇ aqueous solution, and reacted overnight at room temperature. After the reaction, the surface is washed with water to obtain a biotin-immobilized surface with a biomolecular recognition function.

Example 2

Combining layer-by-layer assembly technology and host-guest interaction to construct a surface with specific adsorption function of concanavalin on the surface of gold-plated single crystal silicon wafer

The gold-plated monocrystalline silicon wafers cleaned by the UV-ozone meter were pretreated with a mixed solution of ammonia, hydrogen peroxide and deionized water (ammonia: hydrogen peroxide: deionized water = 1:1:5, volume ratio), and then placed in React overnight in an aqueous solution of mercaptoethylamine hydrochloride to obtain a gold-plated single-crystal silicon chip whose surface is aminated. A polyanion P(AA-Ada) solution and a polycation PAH solution were respectively prepared, and the solvent was an acetic acid/sodium acetate buffer solution with a pH value of 5.0 and a concentration of 0.05 mol/L. Soak the aminated gold-plated single crystal silicon wafer in the P(AA-Ada) solution, wash it with acetic acid/sodium acetate buffer solution to remove the weakly bonded polymer, and then soak it in the PAH solution. After cleaning, a A bimolecular layer of P(AA-Ada)/PAH, this process is repeated several times to finally reach the required number of layers, and a gold-plated single crystal silicon wafer modified by a polyelectrolyte multilayer film can be obtained.

Firstly, the hydroxyl group at the sixth position of β-CD is replaced by bromine (β-CD-(Br) ₇ ), and then azide is used to prepare β-CD modified with 7 azido groups (β-CD -(N ₃ ) ₇ ); Finally, click reaction was performed on β-CD-(N ₃ ) ₇ and 1-(2'-propargyl)-α-D-mannose to obtain mannose-modified β-CD ( β-CD-(mannose) ₇ ).

The above-mentioned gold-plated single-crystal silicon wafer modified by the polyelectrolyte multilayer film was placed in 1 millimole per liter of β-CD-(mannose) ₇ aqueous solution, and reacted overnight at room temperature. After the reaction, the surface is washed with water to obtain a surface with the function of specifically adsorbing concanavalin (Con A) immobilized with mannose.

Example 3

Combining layer-by-layer assembly technology and host-guest interaction to construct a surface with the function of promoting endothelial cell adhesion on the surface of gold-plated single crystal silicon wafer

The gold-plated monocrystalline silicon wafers cleaned by the ultraviolet ozone instrument are pretreated with a mixed solution of ammonia, hydrogen peroxide and deionized water (ammonia: hydrogen peroxide: deionized water = 1:1:5, volume ratio), and then placed in React overnight in an aqueous solution of mercaptoethylamine hydrochloride to obtain a gold-plated single-crystal silicon chip whose surface is aminated. A polyanion P(AA-Ada) solution and a polycation PAH solution were respectively prepared, and the solvent was an acetic acid/sodium acetate buffer solution with a pH value of 5.0 and a concentration of 0.05 mol/L. Soak the aminated gold-plated single crystal silicon wafer in the P(AA-Ada) solution, wash it with acetic acid/sodium acetate buffer solution to remove the weakly bonded polymer, and then soak it in the PAH solution. After cleaning, a A bimolecular layer of P(AA-Ada)/PAH, this process is repeated several times to finally reach the required number of layers, and a gold-plated single crystal silicon wafer modified by a polyelectrolyte multilayer film can be obtained.

Firstly, the hydroxyl group at the sixth position of β-CD is replaced by bromine (β-CD-(Br) ₇ ), and then azide is used to prepare β-CD modified with 7 azido groups (β-CD -(N ₃ ) ₇ ); Finally, click reaction was performed on β-CD-(N ₃ ) ₇ and propargyl-REDV polypeptide to obtain β-CD modified by REDV polypeptide (β-CD-(REDV) ₇ ).

The above-mentioned gold-plated single crystal silicon wafer modified by polyelectrolyte multilayer film was placed in 1 millimole per liter of β-CD-(REDV) ₇ aqueous solution, and reacted overnight at room temperature. After the reaction, the surface is washed with water to obtain a surface with the function of promoting endothelial cell adhesion immobilized with the REDV polypeptide.

The method has potential application prospects in many fields of biomedicine and biotechnology. It should be pointed out that the methodology is not limited to PAA/PAH and β-CD/Ada used in our work, but can be extended to other polymers and other host-guest pairs. For fields that require bifunctional or even multifunctional surfaces, it can be achieved simply by introducing two or more cyclodextrin molecules with different ligands by hybrid co-introduction.

The above is inspired by the ideal embodiment of the present invention. Through the above description, relevant personnel can make various changes and modifications within the scope of not departing from the technical idea of the present invention. The technical scope of the present invention is not limited to the content in the specification, and must be determined according to the scope of the claims.

Claims (10)

1. a kind of method that combination laminated assembling technology and host-guest interaction build bioactivity surface, it is characterised in that Comprise the following steps：

（1）The preparation of polyelectrolyte multilayer film modified surface：

Amidized base material is immersed in the copolymer p (AA-Ada) of acrylic monomers and 1- acrylic acid adamantane methanol ester monomers Solution in a period of time, a period of time is then immersed in polypropylene amine PAH solution again, i.e., is obtained in amidized substrate surface To one layer of P (AA-Ada)/PAH bilayer, immersion is repeated several times, if being had double points of dried layer P (AA-Ada)/PAH The polyelectrolyte multilayer film modified surface of sublayer；

（2）Polyelectrolyte multilayer film modified surface immobilizing biologically active molecule：

Gained is placed in polyelectrolyte multilayer film modified surface and spread out containing the beta-schardinger dextrin for having modified bioactive molecule Reacted in biological solution, that is, obtain being fixed with the polyelectrolyte multilayer film modified surface of bioactive molecule.

2. a kind of method that combination laminated assembling technology and host-guest interaction build bioactivity surface, it is characterised in that Comprise the following steps：

（1）The preparation of polyanion P (AA-Ada) containing adamantane group：

Acrylic monomers, 1- acrylic acid adamantane methanol ester monomer, chain-transferring agent and initiator are added by certain mol ratio Polymerisation is carried out into reaction solution, the method purified after reaction using sedimentation separation produces poly- the moon containing adamantane group Ion P (AA-Ada)；

（2）The preparation of polyelectrolyte multilayer film modified surface：

Amidized base material is immersed in the copolymer p (AA-Ada) of acrylic monomers and 1- acrylic acid adamantane methanol ester monomers Solution in a period of time, a period of time is then immersed in polypropylene amine PAH solution again, i.e., is obtained in amidized substrate surface To one layer of P (AA-Ada)/PAH bilayer, immersion is repeated several times, if being had double points of dried layer P (AA-Ada)/PAH The polyelectrolyte multilayer film modified surface of sublayer；

（3）Polyelectrolyte multilayer film modified surface immobilizing biologically active molecule：

Gained is placed in polyelectrolyte multilayer film modified surface and spread out containing the beta-schardinger dextrin for having modified bioactive molecule Reacted in biological solution, that is, obtain being fixed with the polyelectrolyte multilayer film modified surface of bioactive molecule.

3. method according to claim 1 or 2, it is characterised in that：Described bioactive molecule is biotin, mannose Or arginine-glutamic acid-aspartic acid-valine polypeptide.

4. method according to claim 3, it is characterised in that：It is modified with the system of the β-cdderivatives of the biotin molecule It is for process：β-CD by propargyl biotin and the modification of 7 azido groups are：β-CD-(N₃)₇, click-reaction is carried out, is made β-the CD of biotin modification.

5. method according to claim 3, it is characterised in that：It is modified with the system of the β-cdderivatives of the mannose molecules It is for process：β-CD by propargyl mannose and the modification of 7 azido groups are：β-CD-(N₃)₇, click-reaction is carried out, is made β-the CD of mannose-modified.

6. method according to claim 3, it is characterised in that：It is modified with the arginine-glutamic acid-aspartic acid-figured silk fabrics The preparation process of the β-cdderivatives of propylhomoserin peptide molecule is：Propargyl-arginine-glutamic acid-aspartic acid-valine is more Peptide and the β-CD of 7 azido group modifications are：β-CD-(N₃)₇, click-reaction is carried out, arginine-glutamic acid-asparagus fern ammonia is made Peptide modified β-the CD of acid-valine.

7. method according to claim 1 or 2, it is characterised in that：The amidized base material is monocrystalline silicon or glass, institute The preparation process for stating amidized monocrystalline silicon piece or sheet glass is：Monocrystalline silicon piece or sheet glass are placed in the second of 3- aminopropyls-three TMOS（APTES）Toluene solution in react a period of time, obtain the monocrystalline silicon or glass baseplate of surface amination.

8. method according to claim 1 or 2, it is characterised in that：The amidized base material is gold-plated monocrystalline silicon piece, institute The preparation process for stating amidized gold-plated monocrystalline silicon piece is：Gold-plated monocrystalline silicon piece is placed in the aqueous solution of mercaptoethylamine hydrochloride Reaction a period of time, obtain the gold-plated single crystal silicon substrate of surface amination.

9. method according to claim 1 or 2, it is characterised in that：The β-cdderivatives of bioactive molecule are modified Solution be the aqueous solution, phosphate buffer solution or other can dissolve the solution of corresponding bioactive molecule.

10. method according to claim 2, it is characterised in that：Described is polymerized to Invertible ideal, The chain-transferring agent is 2-（Phenyl formyl sulfenyl）Propionic acid；Described initiator is azodiisobutyronitrile, azo two different heptan Nitrile, dibenzoyl peroxide, dicetyl peroxydicarbonate ethylhexyl, isopropyl benzene hydroperoxide, potassium peroxydisulfate-sulphite body System or hydrogen peroxide-perferrite system.