CN101810888B - Preparation method for material with high density fixed biologically functional molecule - Google Patents

Abstract

The invention relates to a method for preparing a surface with biologically active functions. The method is: graft polymethacrylate oligoethylene glycol ester with excellent non-specific protein adsorption ability on the material surface by atom transfer radical polymerization (ATRP) technology (POEGMA); then use the secondary initiation of ATRP to graft polymethacryloxysuccinimide (PNHSMA) at the end of POEGMA, and use the large amount of active ester groups contained in the side chain of PNHSMA to fix the amino group with high density. Biomolecules. The bioactive functional surface provided by the present invention can significantly increase the graft density of biomolecules on the surface, reduce the adverse effects caused by non-specific protein adsorption, and effectively maintain the activity of immobilized biomolecules; it can immobilize various biomolecules, Applicable to a wide range of biomedical fields.

Description

A preparation method of a material for high-density immobilization of biofunctional molecules

technical field

The invention relates to the fields of biological detection, tissue engineering and polymer materials, in particular to a method for preparing a biological functional surface capable of high-density immobilization of biological molecules while repelling non-specific protein adsorption.

Background technique

Using biological principles to immobilize biologically active substances such as proteins, cell growth factors, enzymes, and polypeptides on the surface of the material can effectively improve various biological functions of the material (such as anticoagulant performance, activity of binding to antibodies, and ability to bind to cells) Adhesion, etc.), which has important application value in various biomedical fields such as biological detection, tissue engineering, protein separation and purification, and artificial implant materials (Pascal J.et al.Angew.Chem.Int.Ed., 2008 , 47: 9618-9647; Chen H. et al. Prog. Polym. Sci., 2008, 33: 1059-1087). For example: immobilized heparin can endow the surface with anticoagulant function to improve its blood compatibility; immobilized extracellular matrix proteins such as fibronectin and collagen can effectively promote the adhesion and reproduction of surface cells. However, while immobilizing as many bioactive molecules as possible on the surface of the material, the surface must also have the ability to repel the adsorption of non-specific proteins. This is because in the field of biological detection such as biosensors and biochips, the background noise caused by non-specific proteins is the main factor hindering the accuracy of detection; and in the fields of artificial implant materials and tissue engineering scaffolds, it is also necessary to reduce non-specific Adverse reactions such as thrombosis, bacterial adhesion, and inflammation caused by heterosexual protein adsorption.

The current preparation strategies for such biofunctional surfaces generally combine bioinert surfaces with immobilization of bioactive molecules. For example, Xu et al. (Xu et al. Biomacromolecules, 2009, 10: 1665-1674) grafted POEGMA with excellent non-specific protein adsorption function on the surface, and activated the hydroxyl group at the end of its side chain, thereby immobilizing the immune system. Globulin (IgG) is used to prepare biosensors with high signal-to-noise ratio. Tugulu et al. (Tugulu et al. Biomaterials, 2007, 28: 2536-2546) grafted POEGMA on the surface, immobilized cell adhesion polypeptides after hydroxyl activation, and effectively promoted the adhesion and spreading of cells on the surface. However, these preparation methods all need to go through the process of activating functional groups, and cannot directly immobilize biomolecules. learning function.

Contents of the invention

The technical problem to be solved by the present invention is: aiming at the problems of complex reaction process and low density of immobilized biomolecules existing in the current bioactive surface preparation method, to provide a method that is easy to operate, can immobilize biomolecules at high density and repel non-specific proteins at the same time. Surface Modification Methods by Adsorption.

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

The preparation method of the high-density immobilized biofunctional molecular material provided by the present invention comprises the following steps:

(1) Preparation of POEGMA modified surface:

Place the surface of the material immobilized with the initiator in a solution containing the catalyst/ligand system and OEGMA monomer under the protection of nitrogen for ATRP reaction, the reaction temperature is 0-45°C, and the reaction time is 0.5-6 hours to obtain POEGMA Modified surface; POEGMA is the English abbreviation of oligoethylene glycol methacrylate, OEGMA is the English abbreviation of oligoethylene glycol methacrylate, and ATRP is the English abbreviation of atom transfer radical polymerization.

The material on which the initiator is fixed can be single crystal silicon or glass; the initiator is α-bromobutyryl bromide, α-bromoisobutyryl bromide, or α-bromopropionyl bromide.

The catalyst/ligand system can be copper bromide/pentamethyldiethylenetriamine, or copper bromide/ascorbic acid.

The OEGMA monomer solution is an aqueous solution, a methanol solution, or a mixed solution of water and methanol.

(2) Preparation of NHSMA:

In the reaction device, slowly drop methacryloyl chloride into the NHS solution at a molar ratio of 1:1 to 1.2:1, stir and react in the presence of triethylamine, and the reaction temperature is 0 to 35°C. The reaction time is 1 to 12 hours, and the monomer solution of NHSMA is obtained; then the monomer solution is recrystallized to obtain white crystal NHSMA; the NHSMA is the English abbreviation of methacryloyloxysuccinimide, and the NHS It is the abbreviation of N-hydroxysuccinimide.

The NHS solution is a chloroform solution of N-hydroxysuccinimide, and the amount of triethylamine is 10-25% of the solution in terms of W/V.

(3) Preparation of POEGMA-PNHSMA modified surface:

The obtained POEGMA modified surface is placed in the monomer solution containing the catalyst/ligand system and the obtained NHSMA to carry out a second ATRP reaction, the reaction temperature is 70-100 ° C, and the reaction time is 2-6 hours to obtain POEGMA-PNHSMA Modified surface.

The catalyst/ligand system can use copper bromide/pentamethyldiethylenetriamine, or copper bromide/pentamethyldiethylenetriamine.

The NHSMA monomer solution is an anisole solution or a dimethyl sulfoxide solution.

(4) POEGMA-PNHSMA modified surface immobilization bioactive molecules:

The obtained POEGMA-PNHSMA modified surface is placed in a solution containing biologically active molecules for reaction, the temperature is controlled at 0-40°C, and the reaction time is 2-24 hours. After the reaction, the surface is placed in a passivation agent The unreacted NHS group is passivated in the solution of (EG) ₂ NH ₂ , the passivation temperature is room temperature, and the reaction time is 2-12 hours. After this step is completed, the POEGMA-PNHSMA modified surface is taken out from the solution and washed with ethanol or water solvent respectively to obtain a biofunctional surface immobilized with biotin, collagen and heparin bioactive molecules.

The bioactive molecule is biotinyl hydrazine, fibronectin, collagen, heparin, lysine or other biomolecules containing amino groups. The solution of bioactive molecules is ethanol solution, acetic acid solution, phosphate buffer solution or other solutions that can dissolve corresponding bioactive molecules.

The (EG) ₂ NH ₂ solution is an ethanol solution.

After the above steps, a high-density immobilized biofunctional molecular material is finally obtained, and the surface of the material has the function of not adsorbing non-specific proteins.

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

The method provided by the invention is a method for directly surface-initiating polymerization to prepare a bioactive surface, without intermediate group activation process. Compared with the prior art, the present invention has the following prominent features:

1. Conducive to maintaining the activity of immobilized biomolecules: Compared with the traditional method of activating functional groups and then immobilizing biomolecules, this method can immobilize bioactive molecules at a higher density, and the presence of POEGMA as a spacer layer can exclude non-specific protein adsorption , and can provide a hydrophilic environment, which is conducive to maintaining the activity of immobilized biomolecules.

2. Simple and easy to operate: the physical and chemical properties of the material surface can be adjusted conveniently by changing the polymerization conditions of the secondary ATRP.

3. Wide applicability: It can be used to immobilize any biologically active molecules containing amino groups and effectively realize their biological functions. It is suitable for many biomedical fields such as biological detection, tissue engineering, and protein separation and purification. For example, after biotin is immobilized on the active surface obtained by this method, avidin can be selectively bound; there is no cell adhesion phenomenon on the POEGMA modified surface (see Figure 1), and after collagen is immobilized on the surface, it can effectively promote Cell adhesion and spreading (see Figure 2); after immobilizing heparin on the surface, it can specifically bind anticoagulant factor ATIII (see Figure 3), and obtain a surface with anticoagulant function, etc. It can be seen from Figure 3 that the adsorption amount of ATIII on the surface modified by POEGMA is 0.0129ug/cm ² , while the adsorption amount of ATIII on the surface after immobilizing heparin is 0.0942ug/cm ² , that is, the adsorption amount of ATIII on the surface after immobilization increases by 10 times .

Description of drawings

Figure 1 shows the adhesion and growth of POEGMA-modified surface cells.

Figure 2 shows the adhesion and growth of cells on the surface of fixed collagen.

Figure 3 shows the adsorption of anticoagulant factor ATIII on the POEGMA modified surface and the surface after immobilizing heparin.

Detailed ways

The invention provides a method for preparing a biofunctional surface that immobilizes biofunctional molecules with high density and excludes non-specific protein adsorption. After immobilizing the initiator on the surface of the material, POEGMA was grafted on the surface of the material by ATRP technology in the presence of ligand/catalyst and monomer OEGMA, and then PNHSMA was grafted on the end of POEGMA by ATRP secondary initiation. A large number of NHS groups contained in PNHSMA are used to immobilize bioactive molecules containing amino groups (such as proteins, polysaccharides, enzymes, etc.) with high density, and finally realize their physiological functions.

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

Example 1

2.2 mL of methacryloyl chloride in 10 mL of chloroform was added dropwise to a solution of 2.3 g of NHS and 3.3 mL of triethylamine in 20 mL of chloroform at 0°C. After the dropwise addition was completed, the reaction was carried out at room temperature for 4 hours. The reaction solution was washed with saturated ice brine, and the organic phase was dried with anhydrous magnesium sulfate. The precipitate was filtered, the solvent was concentrated, and then recrystallized in a mixed solution of 0.8 mL ethyl acetate and 6 mL n-hexane to obtain NHSMA as a monomer.

Under a nitrogen atmosphere, 7.93g of OEGMA, 312mg of 2,2-bipyridine and 143mg of cuprous bromide were dissolved in a mixed solution of 15mL of methanol and water (4:1). The above solution was ultrasonically transferred to a silicon chip with α-bromobutyryl bromide initiator fixed on the surface for 5 minutes, reacted at room temperature for 2 hours, then stopped the reaction with copper bromide/bipyridyl, and cleaned the surface with a large amount of water and ethanol. Blow dry with nitrogen, and the treated surface has POEGMA immobilized. The above surface was placed in 6mL anisole containing 14mg cuprous bromide, 0.92g NHSMA, 42μL PMDETA, and the reaction was carried out at 90°C for 2h. After the reaction was completed, a large amount of N,N-dimethylformamide (DMF ) to clean the wafer and dry it in a vacuum oven.

The above surface was placed in 1 mg/mL biotin hydrazide in sodium acetate solution (pH 5.5) and reacted overnight at room temperature. After the reaction, the surface was cleaned with sodium acetate solution, water, and ethanol, and the membrane was placed in a 0.1M (EG) ₂ NH ₂ ethanol solution to passivate the remaining active ester groups on the surface. A bioactive surface with high-density biotin immobilization can be obtained.

Example 2

Add 2.2 mL of methacryloyl chloride in 10 mL of chloroform dropwise to a solution of 2.3 g of NHS and 3.3 mL of triethylamine in 20 mL of chloroform at 0°C. After the dropwise addition was completed, the reaction was carried out at room temperature for 4 hours. The reaction solution was washed with saturated ice brine, and the organic phase was dried with anhydrous magnesium sulfate. The precipitate was filtered, the solvent was concentrated, and then recrystallized in a mixed solution of 0.8 mL ethyl acetate and 6 mL n-hexane to obtain NHSMA as a monomer.

Under a nitrogen atmosphere, 7.93g of OEGMA, 312mg of 2,2-bipyridine and 143mg of cuprous bromide were dissolved in a mixed solution of 15mL of methanol and water (1:1). After the above solution was ultrasonicated for 5 minutes, it was transferred to a silicon wafer with α-bromobutyryl bromide initiator fixed on the surface, reacted at room temperature for 2 hours, and the surface was cleaned with a large amount of water and ethanol, and then dried with nitrogen. The above surface was placed in 6mL anisole containing 14mg cuprous bromide, 0.92g NHSMA, 42μL PMDETA, and the reaction was carried out at 70°C for 3h. After the reaction, the silicon wafer was cleaned with a large amount of DMF and placed in a vacuum box. drying.

The acetic acid solution of 4 wt % collagen was adjusted to pH 8.0 with 2.0 M sodium hydroxide solution to obtain a collagen solution. The above surface was placed in collagen solution and reacted overnight at 4°C. After the reaction, the surface was washed with water and ethanol, and the membrane was placed in a 0.1M (EG) ₂ NH ₂ ethanol solution to passivate the remaining active ester groups. A bioactive surface with high-density immobilized collagen can be obtained.

Example 3

Add 2.2 mL of methacryloyl chloride in 10 mL of chloroform dropwise to a solution of 2.3 g of NHS and 3.3 mL of triethylamine in 20 mL of chloroform at 0°C. After the dropwise addition was completed, the reaction was carried out at room temperature for 4 hours. The reaction solution was washed with saturated ice brine, and the organic phase was dried with anhydrous magnesium sulfate. The precipitate was filtered, the solvent was concentrated, and then recrystallized in a mixed solution of 0.8 mL ethyl acetate and 6 mL n-hexane to obtain NHSMA as a monomer.

Under a nitrogen atmosphere, 7.93g of OEGMA, 312mg of 2,2-bipyridine and 143mg of cuprous bromide were dissolved in a mixed solution of 15mL of methanol and water (2:1). The above solution was ultrasonicated for 5 minutes and then transferred to a silicon wafer with α-bromobutyryl bromide initiator fixed on the surface. After reacting for 2 hours at room temperature, the surface was cleaned with a large amount of water and ethanol, and then dried with nitrogen. The above surface was placed in 6 mL anisole containing 14 mg cuprous bromide, 0.92 g NHSMA, 42 μL PMDETA, and the reaction was carried out at 100 °C for 4 h. After the reaction, the silicon wafer was cleaned with a large amount of DMF and dried in a vacuum oven.

The diaphragm was placed in 10 mg/mL heparin-phosphate buffer solution (pH=8.0) and reacted at room temperature for 24 hours. After the reaction, the surface was washed with phosphate buffer solution, water, and ethanol, and the membrane was placed in 0.1M (EG) ₂ NH ₂ ethanol solution to passivate the remaining active ester groups. A bioactive surface with high-density immobilized heparin can be obtained.

Example 4

2.2 mL of methacryloyl chloride in 10 mL of chloroform was added dropwise to a solution of 2.3 g of NHS and 3.3 mL of triethylamine in 20 mL of chloroform at 0°C. After the dropwise addition was completed, the reaction was carried out at room temperature for 4 hours. The reaction solution was washed with saturated ice brine, and the organic phase was dried with anhydrous magnesium sulfate. The precipitate was filtered, the solvent was concentrated, and then recrystallized in a mixed solution of 0.8 mL ethyl acetate and 6 mL n-hexane to obtain NHSMA as a monomer.

Under a nitrogen atmosphere, 7.93g of OEGMA, 312mg of 2,2-bipyridine and 143mg of cuprous bromide were dissolved in a mixed solution of 15mL of methanol and water (2:1). The above solution was ultrasonicated for 5 minutes and then transferred to a silicon wafer with α-bromobutyryl bromide initiator fixed on the surface. After reacting for 4 hours at room temperature, the surface was cleaned with a large amount of water and ethanol, and then dried with nitrogen. The above surface was placed in 6 mL anisole containing 14 mg cuprous bromide, 0.92 g NHSMA, 42 μL PMDETA, and the reaction was carried out at 100 °C for 6 h. After the reaction, the silicon wafer was cleaned with a large amount of DMF and dried in a vacuum oven.

The membrane was placed in a citrate buffer solution (pH=8.0) of 0.1 mg/mL RGD polypeptide and reacted at room temperature for 24 hours. After the reaction, the surface was washed with citrate buffer solution, water, and ethanol, and the membrane was placed in 0.1M (EG) ₂ NH ₂ ethanol solution to passivate the remaining active ester groups. A bioactive surface with high-density immobilization of RGD can be obtained.

Example 5

Add 2.2 mL of methacryloyl chloride in 10 mL of chloroform dropwise to a solution of 2.3 g of NHS and 3.3 mL of triethylamine in 20 mL of chloroform at 0°C. After the dropwise addition was completed, the reaction was carried out at room temperature for 4 hours. The reaction solution was washed with saturated ice brine, and the organic phase was dried with anhydrous magnesium sulfate. The precipitate was filtered, and the solvent was concentrated and then recrystallized in a mixed solution of 0.8 mL ethyl acetate and 6 mL n-hexane to obtain monomer NHSMA.

Under a nitrogen atmosphere, 7.93g of OEGMA, 312mg of 2,2-bipyridine and 143mg of cuprous bromide were dissolved in a mixed solution of 15mL of methanol and water (1:1). The above solution was ultrasonicated for 5 minutes and then transferred to a silicon wafer with α-bromobutyryl bromide initiator fixed on the surface. After reacting for 3 hours at room temperature, the surface was cleaned with a large amount of water and ethanol, and then dried with nitrogen. The above surface was placed in 6 mL anisole containing 14 mg cuprous bromide, 0.92 g NHSMA, 42 μL PMDETA, and the reaction was carried out at 90 °C for 4 h. After the reaction, the silicon wafer was cleaned with a large amount of DMF and dried in a vacuum oven.

The membrane was placed in a citrate buffer solution (pH=8.0) of 0.1 mg/mL RGD polypeptide and reacted at room temperature for 24 hours. After the reaction, the surface was washed with citrate buffer solution, water, and ethanol, and the membrane was placed in 0.1M (EG) ₂ NH ₂ ethanol solution to passivate the remaining active ester groups. A bioactive surface with high-density immobilization of RGD can be obtained.

Example 6

Add 2.2 mL of methacryloyl chloride in 10 mL of chloroform dropwise to a solution of 2.3 g of NHS and 3.3 mL of triethylamine in 20 mL of chloroform at 0°C. After the dropwise addition was completed, the reaction was carried out at room temperature for 4 hours. The reaction solution was washed with saturated ice brine, and the organic phase was dried with anhydrous magnesium sulfate. The precipitate was filtered, the solvent was concentrated, and then recrystallized in a mixed solution of 0.8 mL ethyl acetate and 6 mL n-hexane to obtain NHSMA as a monomer.

Under a nitrogen atmosphere, 7.93g of OEGMA, 312mg of 2,2-bipyridine and 143mg of cuprous bromide were dissolved in a mixed solution of 15mL of methanol and water (2:1). The above solution was ultrasonicated for 5 minutes and then transferred to a silicon wafer with α-bromobutyryl bromide initiator fixed on the surface. After reacting for 4 hours at room temperature, the surface was cleaned with a large amount of water and ethanol, and then dried with nitrogen. The above surface was placed in 6 mL anisole containing 14 mg cuprous bromide, 0.92 g NHSMA, 42 μL PMDETA, and the reaction was carried out at 100 °C for 6 h. After the reaction, the silicon wafer was cleaned with a large amount of DMF and dried in a vacuum oven.

The membrane was placed in 0.1 mg/mL fibronectin in phosphate buffer solution (pH=7.4) and reacted at 0° C. for 24 h. After the reaction, the surface was washed with phosphate buffer solution, water, and ethanol, and the membrane was placed in 0.1M (EG) ₂ NH ₂ ethanol solution to passivate the remaining active ester groups. A bioactive surface with high-density immobilization of fibronectin can be obtained.

Claims (5)

1. A method for preparing a material for high-density immobilization of biofunctional molecules, characterized in that it comprises the following steps: (1) Preparation of POEGMA modified surface: Place the surface of the material immobilized with the initiator in a solution containing the catalyst/ligand system and OEGMA monomer under the protection of nitrogen for ATRP reaction, the reaction temperature is 0-45°C, and the reaction time is 0.5-6 hours to obtain POEGMA Modified surface; the material fixed with the initiator is single crystal silicon or glass; the initiator is α-bromobutyryl bromide, α-bromoisobutyryl bromide or α-bromopropionyl bromide; the described The catalyst/ligand system is cuprous bromide/pentamethyldiethylenetriamine or copper bromide/ascorbic acid; POEGMA is the English abbreviation of poly(polyethylene glycol methacrylate), OEGMA is methacrylic acid oligomer The English abbreviation of ethylene glycol ester, ATRP is the English abbreviation of atom transfer radical polymerization; (2) Preparation of NHSMA: In the reaction device, slowly drop methacryloyl chloride into the NHS solution at a molar ratio of 1:1 to 1.2:1, stir and react in the presence of triethylamine, and the reaction temperature is 0 to 35°C. The reaction time is 1 to 12 hours, and the monomer solution of NHSMA is obtained; then the monomer solution is recrystallized to obtain white crystal NHSMA; the NHSMA is the English abbreviation of methacryloyloxysuccinimide, and the NHS It is the English abbreviation of N-hydroxysuccinimide; (3) Preparation of POEGMA-PNHSMA modified surface: The obtained POEGMA modified surface is placed in the monomer solution containing the catalyst/ligand system and the obtained NHSMA to carry out a second ATRP reaction, the reaction temperature is 70-100 ° C, and the reaction time is 2-6 hours to obtain POEGMA-PNHSMA Modified surface; the catalyst/ligand system is copper bromide/pentamethyldiethylenetriamine, or cuprous bromide/pentamethyldiethylenetriamine; (4) POEGMA-PNHSMA modified surface immobilization bioactive molecules: The obtained POEGMA-PNHSMA modified surface is placed in a solution containing biologically active molecules for reaction, the temperature is controlled at 0-40°C, and the reaction time is 2-24 hours. After the reaction, the surface is placed in a passivation agent (EG) Passivate unreacted NHS groups in a solution of ethanol in ₂ NH ₂ , the passivation temperature is room temperature, and the reaction time is 2 to 12 hours; the biologically active molecules are biotin hydrazide, lysine Acids or other bioactive molecules containing amino groups; After the above steps, a high-density immobilized biofunctional molecular material is finally obtained, and the surface of the material has the function of not adsorbing non-specific proteins. 2. The method according to claim 1, characterized in that: the OEGMA monomer solution is an aqueous solution, a methanol solution, or a mixed solution of water and methanol. 3. the method according to claim 1 is characterized in that: described NHS solution is the chloroform solution of N-hydroxysuccinimide, and by W/V, triethylamine consumption is 10% of described solution. ~25%. 4. The method according to claim 1, characterized in that: the NHSMA monomer solution described in step (3) is an anisole solution or a dimethyl sulfoxide solution. 5. The method according to claim 1, characterized in that: the solution of the biologically active molecules is ethanol solution, acetic acid solution, phosphate buffer solution or other solutions capable of dissolving corresponding biologically active molecules.

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