CN100557099C - A kind of glycosyl-modified acrylonitrile-based nanofiber and its preparation method and application - Google Patents

Abstract

The invention discloses a method for preparing sugar-modified acrylonitrile-based nanofibers through "click chemistry". The method adopts electrospinning technology to prepare acrylonitrile-based copolymer nanofibers with carboxyl functional groups, introduces alkyne groups on its surface through coupling reaction, and then utilizes "click chemistry" to convert sugar groups containing azide groups into nanofibers. fixed to the surface of the nanofibers. The invention combines the characteristics of high specific surface area of nanofibers and high specificity of "click chemistry" reaction, effectively improves the immobilization rate of sugar groups on the surface of nanofibers, and is beneficial to exert the clustering effect of sugar groups, thereby significantly improving the protein separation efficiency. The glycosyl-modified acrylonitrile-based nanofiber has the advantages of simple preparation method, repeated use, low cost and the like.

Description

A kind of glycosyl-modified acrylonitrile-based nanofiber and its preparation method and application

technical field

The invention relates to a preparation method and application of nanofibers, in particular to a preparation method and application of sugar-modified acrylonitrile-based copolymer nanofibers.

Background technique

Sugar, like protein, lipid and nucleic acid, is an important component of living organisms. Sugar plays an important role in the construction of cells, the biosynthesis of cells and the regulation of cell life activities. Glycoconjugates widely exist, including glycoproteins, proteoglycans, and glycolipids, are involved in important physiological processes such as protein targeting, cell recognition, and antibody-antigen interactions. Another important subject after DNA and protein has attracted people's attention. In 1988, a new discipline of glycobiology emerged to study the structure, biosynthesis and biological functions of glycoconjugate sugar chains.

Glycoconjugates exist on the outer surface of the cell membrane and play a role in protecting the cell membrane and communicating with the outside world. In recent years, people have attempted to immobilize sugar groups on the surface of different carriers, such as silica, gold nanoparticles, and membrane separation materials, to simulate various biological functions of sugars, so as to deeply study the affinity interactions between sugars and proteins, and realize detection and separation of proteins. The patent document with the publication number CN1200739 discloses a method for fixing α-allyl glucose to a hydrophobic intraocular lens by using a plasma method. The introduction of sugar groups improves the biocompatibility of the intraocular lens. U.S. Patent US 20010017270 discloses a technology for immobilizing sugar groups on gold surfaces, which can be used for detection of proteins, viruses or cells. The patent documents with publication numbers CN 1935342 and CN 101070401 respectively disclose methods for immobilizing sugar groups on the surface of polypropylene membranes and polypropylene microspheres, which are beneficial to the separation, concentration or targeted removal of proteins. Recently, the specific recognition function of surface sugar probes has also been used to expand the sugar-modified materials to the application fields of glycan microarrays and biochips.

Electrospinning, a method to obtain nanometer to micrometer-sized fiber materials, has attracted attention in recent years. Currently, electrospinning can be used to prepare more than 100 polymer nanofibers. Common polymers such as polyvinyl alcohol, polyethylene, polypropylene, polystyrene, polysulfone, polycarbonate, polyurethane, polymethacrylate, polyvinyl chloride, polyamide in US 20030215624 and US 20040013873 , polyacrylate, polyvinylpyrrolidone, etc. are prepared into nanofibers by electrospinning. The patent document with the publication number CN 1843592 discloses that glycosylated acrylonitrile copolymers are used as raw materials to prepare sugar-containing nanofiber membranes by electrospinning. The preparation method is simple and has a certain specific recognition effect on proteins. Due to the advantages of high specific surface area and high porosity, nanofibrous membranes with excellent mechanical properties are often also a good carrier material. Publication Nos. CN 1837266 and CN 1948474 respectively disclose the use of electrospinning technology to prepare phospholipid-modified acrylic nanofibers and chitosan nanofibers for enzyme immobilization. quantity and catalytic efficiency. Hai-Quan Mao's group prepared galactose-modified nanofiber membranes as scaffold materials for hepatocyte culture (Chua KN, Lim WS, Zhang PC, Lu HF, Wen J, Ramakrishna S, Leong KW, Mao HQ "Stableimmobilization of rat Hepatocyte spheroids on galactosylated nanofiberscaffold" "Biomaterials" 2005, 26: 2537-2547), experiments found that it is beneficial to promote the aggregation of liver cells, and has potential application prospects in liver tissue engineering.

In order to improve the immobilization rate of sugar groups, give full play to the clustering effect of sugar groups (meaning that a large number of sugar groups gather together in space in a certain way, and their specific interaction with proteins is much higher than that of an equal number of sugar groups. Addition of individual effects), the introduction of "click chemistry" (Click chemistry) method to achieve glycosyl modification is a more feasible method. "Click chemistry" was first proposed by Sharpless (Kolb HC, Finn MG, Sharpless KB Click chemistry: Diversechemical function from a few good reactions. Angew. Chem. Int. Ed. 2001, 40: 2004-2021), a commonly used reaction The type is a 1,3-dipolar cycloaddition reaction, that is, the reaction of an alkynyl group and an azido group to form a 1,2,3-triazole. Due to the advantages of rapidity, efficiency, high selectivity, and high yield, it has been used more and more in the field of polymer synthesis chemistry. Patent application WO 2005087818 discloses that a new functional group is introduced into a polymer with a specific functional group through a "click chemistry" method to obtain a new functional polymer. The publication number is that in the patent literature disclosed by CN 101022895, a polymer coating for bonding is formed on the metal surface by a "click chemistry" method. Patent application WO2007011967 discloses the preparation of functionalized hyperbranched macromolecules by dipolar cycloaddition reaction, the reaction does not require protective groups, the yield is high, and the purification steps are few. Patent application WO 2007003054 discloses the immobilization of biomacromolecules to synthetic polymer nano/micro ionic surfaces by "click chemistry", which can be used for drug release, biosensors, medical implant materials, etc. In recent years, "click chemistry" has also made some progress in the research of glycochemistry, especially recently, Haddleton group (Chen GJ, Tao L, Mantovani G, Geng J, Nystrom D, Haddleton DM "Amodular click approach to glycosylated polymeric beads : Design, synthesis and preliminary lectin, recognition studies" "Macromolecules" 2007, 40: 7513-7520) reported the use of "click chemistry" method to immobilize mannose groups on the surface of polymer microspheres, and the results showed that 75% moles of microspheres surface The hydroxyl groups in the content can be converted into sugar groups.

Contents of the invention

The invention provides a method for obtaining glycosyl-modified acrylonitrile-based copolymer nanofibers and applying the method to the adsorption and separation of proteins.

A method for preparing sugar-modified acrylonitrile-based nanofibers, the specific steps comprising:

(1) A solution A is obtained by dissolving an acrylonitrile-based copolymer in a solvent.

The mass percent content of the acrylonitrile-based copolymer in the solution A is 2-8%.

The acrylonitrile-based copolymer is acrylonitrile/acrylic acid copolymer, acrylonitrile/methacrylic acid copolymer or acrylonitrile/maleic acid copolymer, with a viscosity-average molecular weight of 80,000 to 300,000 and a carboxyl molar content of 5 to 300,000. 25%.

The solvent is one of dimethylsulfoxide, dimethylformamide, dimethylacetamide or a mixed solvent mixed in any proportion.

(2) Using the solution A as the spinning liquid, the acrylonitrile-based copolymer nanofibers were prepared by electrospinning.

The spinning voltage of the electrospinning is 6-25 kV, the flow rate of the spinneret solution is 0.5-2.0 ml/hour, the receiving distance is 8-20 cm, and the obtained fiber diameter is 60-1000 nanometers.

(3) Submerge the acrylonitrile-based copolymer nanofibers in disodium hydrogen phosphate/potassium dihydrogen phosphate buffer solution (pH 5.5) containing composite activators and alkyne compounds, react for 5 to 20 hours, take it out and use it Ion water and ethanol are washed and dried repeatedly in sequence to obtain alkyne-modified acrylonitrile-based copolymer nanofibers, and the conversion rate of carboxyl groups on the fiber surface is 10-85%.

The alkynyl compounds are propargylamine, propynyl alcohol, and pentynyl alcohol, and the addition amount (molar amount) is 1 to 20 times the amount of carboxyl substances on the fiber surface.

Described composite activator is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxyl succinimide (NHS) with a molar ratio of 1:1 ), the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the buffer solution is 5-20 mg/ml.

(4) Submerge the above alkyne-modified acrylonitrile-based copolymer nanofibers in an aqueous solution containing a composite catalyst and azide sugar, and react under nitrogen protection and oscillation for 10 to 20 hours at 25°C, and then use deionized water and ethanol sequentially Washing and drying are repeated to obtain sugar-modified acrylonitrile-based nanofibers, and the conversion rate of alkyne groups on the fiber surface is 70-90%.

The composite catalyst is copper sulfate pentahydrate and sodium ascorbate, wherein the addition amount (molar amount) of copper sulfate pentahydrate is 10% to 20% of the amount of alkyne groups on the fiber surface, and the molar ratio of copper sulfate pentahydrate to sodium ascorbate is It is 1:5.

The azido sugar is 2-azidoethyl-α-glucose, 2-azidoethyl-β-glucose, 2-azidoethyl-α-mannose, 2-azidoethyl-β- Galactose, 2-azidoethyllactose, 1-azido-α-glucose, 1-azido-β-glucose, 1-azido-α-mannose, 1-azido-β-galactose or 1 - Azidolactose, the added amount (molar amount) of azidosugar is 2 times of the amount of alkyne group substances on the fiber surface.

Application of glycosyl-modified acrylonitrile-based nanofibers in protein separation and purification.

Immerse the sugar-modified acrylonitrile-based nanofibers in the mixed protein solution, shake and adsorb at 25° C. for 2 hours, then filter out and wash. Using the specific recognition of different sugar groups and specific proteins, the nanofibers will selectively adsorb proteins, and the proteins can be eluted from the fibers with a high-concentration sugar solution, thereby realizing the separation of proteins in the mixture.

The invention mainly utilizes the advantages of high specific surface area and excellent mechanical properties of the acrylonitrile-based copolymer nanofiber, and uses it as a carrier material for immobilizing sugar groups. Aiming at the relatively low immobilization rate of glycosyl groups in heterogeneous reactions, the "click chemistry" method with the characteristics of good specific selectivity, high conversion rate and mild operating conditions was introduced, which greatly improved the immobilization of glycosyl groups on the fiber surface. The density helps to exert the clustering effect of sugar groups, thereby increasing the binding capacity of sugar groups to specific proteins and effectively improving the separation efficiency.

The advantages of the present invention are:

(1) The preparation of acrylonitrile-based copolymer nanofibers is simple, and the diameter range can be adjusted within 60-1000 nanometers, which is a kind of carrier material with high specific surface;

(2) The "click chemistry" method is simple to operate and has strong specificity, which can greatly improve the immobilization rate of sugar groups on the fiber surface, and is economical and practical;

(3) The immobilization method is applicable to any azide-containing monosaccharide and polysaccharide, and there is no need to protect the hydroxyl group on the sugar;

(4) The sugar group is fixed to the surface of the nanofiber by chemical bonding, and has good stability and durability;

(5) The glycosyl-modified acrylonitrile-based nanofibers are used for the separation and purification of proteins, and can be reused many times with low cost.

Description of drawings

FIG. 1 is a schematic diagram of the structure of the glycosyl-modified nanofiber obtained in Example 6.

FIG. 2 is a schematic diagram of the structure of the glycosyl-modified nanofiber obtained in Example 9. FIG.

Detailed ways

The viscosity-average molecular weight of the acrylonitrile-based copolymer in the present invention is measured by the one-point method in the viscosity method; the carboxyl content in the copolymer is measured by ¹ H nuclear magnetic resonance; the diameter of the nanofiber is measured by a field emission scanning electron microscope; The content of alkyne groups on the surface of the nanofiber membrane is calculated by the carboxyl staining method through the variation of the carboxyl content; the sugar group content on the surface of the glycosyl-modified nanofiber membrane is determined by elemental analysis; the separation of protein by the glycosyl-modified nanofiber membrane is Measured with a UV-Vis spectrophotometer.

Preparation of sugar-modified acrylonitrile-based copolymer nanofibers

Example 1

The acrylonitrile/acrylic acid copolymer (viscosity average molecular weight is 80,000, carboxyl molar content is 25.0%) that mass fraction is 6% is dissolved in the dimethylformamide solvent, is 15 kilovolts at spinning voltage, spinneret Electrospinning was carried out under the conditions of a solution flow rate of 0.5 ml/hour and a receiving distance of 15 cm to prepare acrylonitrile/acrylic acid copolymer nanofibers with a diameter of 78±23 nm.

10 mg of acrylonitrile/acrylic acid copolymer nanofibers were immersed in 10 ml of disodium hydrogen phosphate/potassium dihydrogen phosphate buffer solution (pH 5.5) with an EDC concentration of 20 mg/ml (EDC/NHS molar ratio 1:1), The amount of propargylamine added is 20 times of the amount of carboxyl substances on the surface of the fiber, reacted for 20 hours, after taking out, it is washed and dried repeatedly with deionized water and ethanol successively, and the conversion rate of carboxyl groups on the surface of the fiber is 75%.

Immerse the acrylonitrile/acrylic acid copolymer nanofibers modified by the above-mentioned alkyne group in 10 milliliters of 2-azidoethyl-α-glucose aqueous solution, then add copper sulfate pentahydrate and sodium ascorbate composite catalyst (alkyne group on the fiber surface, azido sugar , copper sulfate pentahydrate, and sodium ascorbate in a molar ratio of 1:2:0.15:0.75), under nitrogen protection and shaking reaction for 40 hours at 25°C, after taking it out, wash it repeatedly with deionized water and ethanol in sequence, and dry it to obtain glycosyl-modified Nanofibers, the alkyne group conversion rate on the fiber surface is 88%, and the immobilization rate is 835 mg/g fiber.

Example 2

The acrylonitrile/acrylic acid copolymer (viscosity average molecular weight is 270,000, carboxyl molar content is 5.0%) that mass fraction is 8% is dissolved in dimethylformamide solvent, is 20 kilovolts at spinning voltage, spinneret Electrospinning was carried out under the conditions of a solution flow rate of 0.5 ml/hour and a receiving distance of 15 cm to prepare acrylonitrile/acrylic acid copolymer nanofibers with a diameter of 897±36 nm.

10 mg of acrylonitrile/acrylic acid copolymer nanofibers were immersed in 10 ml of disodium hydrogen phosphate/potassium dihydrogen phosphate buffer solution (pH 5.5) with an EDC concentration of 5 mg/ml (EDC/NHS molar ratio 1:1), The amount of propargylamine added and the amount of carboxyl groups on the surface of the fiber were reacted for 10 hours. After taking it out, it was washed and dried repeatedly with deionized water and ethanol successively. The conversion rate of carboxyl groups on the fiber surface was 10%.

Immerse the acrylonitrile/acrylic acid copolymer nanofibers modified by the above-mentioned alkyne group in 10 milliliters of 2-azidoethyl-α-glucose aqueous solution, then add copper sulfate pentahydrate and sodium ascorbate composite catalyst (alkyne group on the fiber surface, azido sugar , copper sulfate pentahydrate, and sodium ascorbate in a molar ratio of 1:2:0.15:0.75), under nitrogen protection and shaking reaction for 40 hours at 25°C, after taking it out, wash it repeatedly with deionized water and ethanol in sequence, and dry it to obtain glycosyl-modified Nanofibers, the alkyne group conversion rate on the fiber surface is 90%, and the immobilization rate is 21 mg/g fiber.

Example 3

The acrylonitrile/acrylic acid copolymer (viscosity average molecular weight is 80,000, carboxyl molar content is 25.0%) that mass fraction is 6% is dissolved in the dimethylformamide solvent, is 15 kilovolts at spinning voltage, spinneret Electrospinning was carried out under the conditions of a solution flow rate of 0.5 ml/hour and a receiving distance of 15 cm to prepare acrylonitrile/acrylic acid copolymer nanofibers with a diameter of 78±23 nm.

10 mg of acrylonitrile/acrylic acid copolymer nanofibers were immersed in 10 ml of disodium hydrogen phosphate/potassium dihydrogen phosphate buffer solution (pH 5.5) with an EDC concentration of 20 mg/ml (EDC/NHS molar ratio 1:1), The amount of propynyl alcohol added is 20 times of the amount of carboxyl substances on the fiber surface, reacted for 20 hours, after taking out, repeated cleaning and drying with deionized water and ethanol successively, the conversion rate of carboxyl groups on the fiber surface is 84%.

Immerse the acrylonitrile/acrylic acid copolymer nanofibers modified by the above-mentioned alkyne group in 10 milliliters of 2-azidoethyl-α-glucose aqueous solution, then add copper sulfate pentahydrate and sodium ascorbate composite catalyst (alkyne group on the fiber surface, azido sugar , copper sulfate pentahydrate, and sodium ascorbate in a molar ratio of 1:2:0.1:0.5), under nitrogen protection and shaking reaction for 10 hours at 25°C, after taking it out, wash it repeatedly with deionized water and ethanol in sequence, and dry it to obtain glycosyl-modified The conversion rate of alkyne group on the fiber surface is 72%, and the immobilization rate is 651 mg/g fiber.

Example 4

The acrylonitrile/acrylic acid copolymer (viscosity average molecular weight is 80,000, carboxyl molar content is 25.0%) that mass fraction is 6% is dissolved in the dimethylformamide solvent, is 15 kilovolts at spinning voltage, spinneret Electrospinning was carried out under the conditions of a solution flow rate of 0.5 ml/hour and a receiving distance of 15 cm to prepare acrylonitrile/acrylic acid copolymer nanofibers with a diameter of 78±23 nm.

10 mg of acrylonitrile/acrylic acid copolymer nanofibers were immersed in 10 ml of disodium hydrogen phosphate/potassium dihydrogen phosphate buffer solution (pH 5.5) with an EDC concentration of 20 mg/ml (EDC/NHS molar ratio 1:1), The amount of propynyl alcohol added is 20 times of the amount of carboxyl substances on the fiber surface, reacted for 20 hours, after taking out, repeated cleaning and drying with deionized water and ethanol successively, the conversion rate of carboxyl groups on the fiber surface is 84%.

Immerse the acrylonitrile/acrylic acid copolymer nanofibers modified by the above-mentioned alkyne group in 10 milliliters of 2-azidoethyl-β-glucose aqueous solution, then add copper sulfate pentahydrate and sodium ascorbate composite catalyst (alkyne group on the surface of the fiber, azido sugar , copper sulfate pentahydrate, and sodium ascorbate in a molar ratio of 1:2:0.15:0.75), under nitrogen protection and shaking reaction for 40 hours at 25°C, after taking it out, wash it repeatedly with deionized water and ethanol in sequence, and dry it to obtain glycosyl-modified The conversion rate of alkyne groups on the fiber surface is 89%, and the immobilization rate is 806 mg/g fiber.

Example 5

The mass fraction is 4% acrylonitrile/methacrylic acid copolymer (viscosity-average molecular weight is 180,000, carboxyl molar content is 11.2%) is dissolved in the dimethylacetamide solvent, under the spinning voltage of 16 kV, spray Electrospinning was carried out under the condition that the flow rate of the filament solution was 0.5 ml/hour and the receiving distance was 15 cm, and acrylonitrile/methacrylic acid copolymer nanofibers with a diameter of 218±20 nm were prepared.

Immerse 10 mg of acrylonitrile/methacrylic acid copolymer nanofibers into 10 mL of disodium hydrogen phosphate/potassium dihydrogen phosphate buffer solution (pH 5.5) with an EDC concentration of 5 mg/mL (EDC/NHS molar ratio 1:1) In the process, the amount of propargylamine added and the amount of carboxyl groups on the surface of the fiber were reacted for 20 hours. After taking it out, it was washed and dried repeatedly with deionized water and ethanol successively. The conversion rate of carboxyl groups on the fiber surface was 15%.

The acrylonitrile/methacrylic acid copolymer nanofibers modified by the above-mentioned alkyne group are immersed in 10 milliliters of 2-azidoethyl-α-mannose aqueous solution, then add copper sulfate pentahydrate and sodium ascorbate composite catalyst (fiber surface alkynyl group, The molar ratio of sugar azide, copper sulfate pentahydrate and sodium ascorbate is 1:2:0.1:0.5), under nitrogen protection and shaking reaction for 20 hours at 25°C, after taking it out, wash it repeatedly with deionized water and ethanol successively, and dry it to obtain The glycosyl-modified nanofiber has an alkyne conversion rate of 83% on the surface of the fiber and an immobilization rate of 205 mg/g fiber.

Example 6

The mass fraction is 4% acrylonitrile/methacrylic acid copolymer (viscosity-average molecular weight is 180,000, carboxyl molar content is 11.2%) is dissolved in the dimethylacetamide solvent, under the spinning voltage of 16 kV, spray Electrospinning was carried out under the condition that the flow rate of the filament solution was 0.5 ml/hour and the receiving distance was 15 cm, and acrylonitrile/methacrylic acid copolymer nanofibers with a diameter of 218±20 nm were prepared.

Immerse 10 mg of acrylonitrile/methacrylic acid copolymer nanofibers into 10 mL of disodium hydrogen phosphate/potassium dihydrogen phosphate buffer solution (pH 5.5) with an EDC concentration of 10 mg/mL (EDC/NHS molar ratio 1:1) In the method, the amount of propargylamine added is 10 times the amount of carboxyl substances on the surface of the fiber, reacted for 20 hours, and after taking it out, it was washed and dried repeatedly with deionized water and ethanol in sequence, and the conversion rate of carboxyl groups on the fiber surface was 49%.

The acrylonitrile/methacrylic acid copolymer nanofibers modified by the above-mentioned alkyne group are immersed in 10 milliliters of 2-azidoethyl-beta-galactose aqueous solution, then add copper sulfate pentahydrate and sodium ascorbate composite catalyst (fiber surface alkynyl group, The molar ratio of sugar azide, copper sulfate pentahydrate and sodium ascorbate is 1:2:0.1:0.5), under nitrogen protection and shaking reaction for 20 hours at 25°C, after taking it out, wash it repeatedly with deionized water and ethanol successively, and dry it to obtain The glycosyl-modified nanofiber has an alkyne conversion rate of 79% on the surface of the fiber and an immobilization rate of 61 mg/g fiber.

Example 7

The acrylonitrile/maleic acid copolymer (viscosity-average molecular weight is 240,000, carboxyl molar content is 7.8%) that mass fraction is 3% is dissolved in the dimethylacetamide solvent, and spinning voltage is 20 kilovolts, spray Electrospinning was carried out under the condition that the flow rate of the filament solution was 0.5 ml/hour and the receiving distance was 15 cm, and acrylonitrile/maleic acid copolymer nanofibers with a diameter of 243±26 nm were prepared.

Immerse 10 mg of acrylonitrile/maleic acid copolymer nanofibers into 10 ml of disodium hydrogen phosphate/potassium dihydrogen phosphate buffer solution (pH 5.5) with an EDC concentration of 10 mg/ml (EDC/NHS molar ratio 1:1) In the method, the amount of propargylamine added is 10 times the amount of carboxyl substances on the surface of the fiber, reacted for 20 hours, and after taking it out, it was washed and dried repeatedly with deionized water and ethanol in sequence, and the conversion rate of carboxyl groups on the fiber surface was 49%.

The acrylonitrile/maleic acid copolymer nanofibers modified by the above-mentioned alkyne group are immersed in 10 milliliters of 2-azidoethyl lactose aqueous solution, then add copper sulfate pentahydrate and sodium ascorbate composite catalyst (alkyne group on fiber surface, azido sugar, The molar ratio of copper sulfate pentahydrate and sodium ascorbate is 1:2:0.1:0.5), and the reaction was carried out under nitrogen protection and shaking for 20 hours at 25°C. After taking it out, it was repeatedly washed with deionized water and ethanol, and dried to obtain sugar-modified nano Fiber, the alkyne group conversion rate on the fiber surface is 79%, and the immobilization rate is 132 mg/g fiber.

Example 8

The acrylonitrile/acrylic acid copolymer (viscosity-average molecular weight is 80,000, carboxyl molar content is 25.0%) that mass fraction is 6% is dissolved in the dimethyl sulfoxide solvent, and spinning voltage is 15 kilovolts, spinneret Electrospinning was carried out under the conditions of a solution flow rate of 0.5 ml/hour and a receiving distance of 15 cm to prepare acrylonitrile/acrylic acid copolymer nanofibers with a diameter of 78±23 nm.

10 mg of acrylonitrile/acrylic acid copolymer nanofibers were immersed in 10 ml of disodium hydrogen phosphate/potassium dihydrogen phosphate buffer solution (pH 5.5) with an EDC concentration of 20 mg/ml (EDC/NHS molar ratio 1:1), The amount of propargylamine added is 20 times the amount of carboxyl substances on the fiber surface, reacted for 20 hours, and after taking out, it is repeatedly washed and dried with deionized water and ethanol successively, and the conversion rate of carboxyl groups on the fiber surface is 80%.

Immerse the acrylonitrile/acrylic acid copolymer nanofibers modified by the above-mentioned alkyne group in 10 milliliters of 1-azide-α-glucose aqueous solution, then add copper sulfate pentahydrate and sodium ascorbate composite catalyst (alkyne group on the fiber surface, sugar azido, penta The molar ratio of copper sulfate in water and sodium ascorbate is 1:2:0.15:0.75), under nitrogen protection and shaking reaction for 40 hours at 25°C, after taking it out, wash it repeatedly with deionized water and ethanol, and dry it to obtain sugar-modified nanofibers , the conversion rate of alkyne groups on the fiber surface was 86%, and the immobilization rate was 532 mg/g fiber.

Example 9

The acrylonitrile/methacrylic acid copolymer (viscosity average molecular weight is 180,000, carboxyl molar content is 11.2%) that mass fraction is 4% is dissolved in the dimethyl sulfoxide solvent, is 16 kilovolts of spinning voltage, spray Electrospinning was carried out under the condition that the flow rate of the filament solution was 0.5 ml/hour and the receiving distance was 15 cm, and acrylonitrile/methacrylic acid copolymer nanofibers with a diameter of 218±20 nm were prepared.

Immerse 10 mg of acrylonitrile/methacrylic acid copolymer nanofibers into 10 mL of disodium hydrogen phosphate/potassium dihydrogen phosphate buffer solution (pH 5.5) with an EDC concentration of 20 mg/mL (EDC/NHS molar ratio 1:1) In the process, the amount of propargylamine added is 20 times the amount of carboxyl substances on the surface of the fiber, reacted for 20 hours, after taking it out, it is washed and dried repeatedly with deionized water and ethanol in sequence, and the conversion rate of carboxyl groups on the fiber surface is 80%.

Immerse the acrylonitrile/methacrylic acid copolymer nanofibers modified by the above-mentioned alkyne group in 10 milliliters of 1-azido-beta-galactose aqueous solution, then add copper sulfate pentahydrate and sodium ascorbate composite catalyst (alkyne group on the fiber surface, azide The molar ratio of sugar, copper sulfate pentahydrate and sodium ascorbate is 1:2:0.15:0.75), under nitrogen protection and shaking reaction for 40 hours at 25°C, after taking it out, wash it repeatedly with deionized water and ethanol in sequence, and dry it to obtain the sugar base After modifying the nanofibers, the alkyne conversion rate on the fiber surface is 84%, and the immobilization rate is 272 mg/g fiber.

Example 10

The acrylonitrile/maleic acid copolymer (viscosity-average molecular weight is 240,000, carboxyl molar content is 7.8%) that mass fraction is 3% is dissolved in the dimethyl sulfoxide solvent, and spinning voltage is 20 kilovolts, spray Electrospinning was carried out under the condition that the flow rate of the filament solution was 0.5 ml/hour and the receiving distance was 15 cm, and acrylonitrile/maleic acid copolymer nanofibers with a diameter of 243±26 nm were prepared.

Immerse 10 mg of acrylonitrile/maleic acid copolymer nanofibers into 10 ml of disodium hydrogen phosphate/potassium dihydrogen phosphate buffer solution (pH 5.5) with an EDC concentration of 10 mg/ml (EDC/NHS molar ratio 1:1) In the method, the amount of propargylamine added is 5 times of the amount of carboxyl substances on the surface of the fiber, reacted for 20 hours, and after taking it out, it was washed and dried repeatedly with deionized water and ethanol in sequence, and the conversion rate of carboxyl groups on the fiber surface was 39%.

Immerse the acrylonitrile/maleic acid copolymer nanofibers modified by the above-mentioned alkyne group in 10 mg of 1-azide lactose aqueous solution, then add copper sulfate pentahydrate and sodium ascorbate composite catalyst (alkyne group on the fiber surface, sugar azide, pentahydrate The molar ratio of copper sulfate and sodium ascorbate is 1:2:0.1:0.5), and the reaction was carried out under nitrogen protection and oscillation for 10 hours at 25°C, after taking it out, it was repeatedly washed with deionized water and ethanol, and dried to obtain sugar-modified nanofibers. The alkyne group conversion rate on the fiber surface was 73%, and the immobilization rate was 87 mg/g fiber.

2. Application examples of glycosyl-modified acrylonitrile-based copolymer nanofibers

1. Adsorption and separation of concanavalin by sugar-modified acrylonitrile-based copolymer nanofibers

50 mg of glycosyl-modified nanofibers (see Table 1 for immobilization rate) prepared in Examples 1, 4, and 8 were immersed in 10 ml of protein phosphate buffered saline solution (pH 7.4, 0.1 mole/liter), wherein the protein solution was accompanied by The mixed solution of concanavalin/peanut lectin has a concentration of 2 mg/ml. After shaking and adsorption at 25°C for 2 hours, the glycosyl-modified nanofibers were filtered off. The adsorbed concanavalin was eluted with 1 mol/L glucose solution, so as to realize the separation of concanavalin and peanut lectin. The eluted glycosyl-modified nanofibers can be reused.

2. Adsorption and separation of peanut lectin by sugar-modified acrylonitrile-based copolymer nanofibers

50 mg of glycosyl-modified nanofibers prepared in Examples 6 and 7 (see Table 1 for the immobilization rate) were immersed in 10 ml of protein phosphate buffer solution (pH 7.4, 0.1 mol/liter), wherein the protein solution was The mixed solution of legumin/peanut lectin has a concentration of 2 mg/ml. After shaking and adsorption at 25°C for 2 hours, the glycosyl-modified nanofibers were filtered off. The adsorbed peanut agglutinin was eluted with 1 mol/L galactose solution, so as to realize the separation of concanavalin and peanut agglutinin. The eluted glycosyl-modified nanofibers can be reused.

The performance parameters of the glycosyl-modified acrylonitrile-based copolymer nanofibers prepared in Examples 1 to 10 of the present invention are shown in Table 1; The adsorption separation effect is shown in Table 2; the comparison table of different sugar groups and the proteins they recognize is shown in Table 3.

Table 2

table 3

Claims (6)

1. A preparation method for glycosyl-modified acrylonitrile-based nanofibers, the steps are as follows: (1) acrylonitrile-based copolymer is dissolved in a solvent to obtain solution A; (2) using solution A as the spinning solution, adopting an electrospinning method to prepare acrylonitrile-based copolymer nanofibers; (3) Immerse acrylonitrile-based copolymer nanofibers in disodium hydrogen phosphate/potassium dihydrogen phosphate buffer solution containing composite activators and alkyne compounds, and react for 5 to 20 hours to obtain alkyne-modified acrylonitrile-based copolymers nanofibers; The alkynyl compound is propargyl amine, propynyl alcohol or pentynyl alcohol; the amount of the alkynyl compound added is 1 to 20 times the amount of carboxyl substances on the surface of the acrylonitrile-based copolymer nanofiber; Described composite activator is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, wherein, 1-(3-dimethylaminopropyl The molar ratio of -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:1; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide The concentration of hydrochloride in the buffer solution is 5-20 mg/ml; (4) immerse the alkyne-modified acrylonitrile-based copolymer nanofibers in water containing a composite catalyst and azide sugar, and react under nitrogen protection for 10 to 20 hours to obtain sugar-modified acrylonitrile-based nanofibers; Described composite catalyst is copper sulfate pentahydrate and sodium ascorbate, wherein, the molar ratio of copper sulfate pentahydrate and sodium ascorbate is 1:5; 10-20% of the amount of alkyne-based substances; The azido sugar is 2-azidoethyl-α-glucose, 2-azidoethyl-β-glucose, 2-azidoethyl-α-mannose, 2-azidoethyl-β- Galactose, 2-azidoethyllactose, 1-azido-α-glucose, 1-azido-β-glucose, 1-azido-α-mannose, 1-azido-β-galactose or 1 - azidolactose; the added amount of azidosugar is 2 times of the amount of alkyne group substance on the surface of the acrylonitrile-based copolymer nanofiber modified by the alkyne group. 2. The preparation method according to claim 1, characterized in that: the acrylonitrile-based copolymer described in step (1) is acrylonitrile/acrylic acid copolymer, acrylonitrile/methacrylic acid copolymer or acrylonitrile/malay Acid copolymer, the viscosity-average molecular weight of the acrylonitrile-based copolymer is 80,000 to 300,000, and the molar content of the functional monomer in the acrylonitrile-based copolymer, that is, the carboxyl group, is 5-25%; The mass percent content of the acrylonitrile-based copolymer in the solution A is 2-8%. 3. The preparation method according to claim 1, characterized in that: the solvent described in step (1) is one of dimethylsulfoxide, dimethylformamide, dimethylacetamide or any Mixed in various proportions. 4. The preparation method according to claim 1, characterized in that: the spinning voltage of the electrospinning described in step (2) is 6-25 kV, the flow rate of the spinneret solution is 0.5-2.0 ml/hour, The receiving distance is 8-20 centimeters, and the fiber diameter is 60-1000 nanometers. 5. The sugar-modified acrylonitrile-based nanofiber prepared by the preparation method as claimed in claim 1. 6. The application of the sugar-modified acrylonitrile-based nanofibers according to claim 5 in the separation and purification of proteins.

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