CN105734827A - Preparation method of sugary temperature-sensitive copolymer nanofiber membrane - Google Patents
Preparation method of sugary temperature-sensitive copolymer nanofiber membrane Download PDFInfo
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- D—TEXTILES; PAPER
- D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
- D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
- D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
- D04H1/40—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres from fleeces or layers composed of fibres without existing or potential cohesive properties
- D04H1/42—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres from fleeces or layers composed of fibres without existing or potential cohesive properties characterised by the use of certain kinds of fibres insofar as this use has no preponderant influence on the consolidation of the fleece
- D04H1/4326—Condensation or reaction polymers
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
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- D—TEXTILES; PAPER
- D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
- D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
- D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
- D04H1/70—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres
- D04H1/72—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged
- D04H1/728—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged by electro-spinning
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/10—Esters
- C08F220/26—Esters containing oxygen in addition to the carboxy oxygen
- C08F220/28—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety
- C08F220/282—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety and containing two or more oxygen atoms
Abstract
The invention discloses a preparation method of a sugary temperature-sensitive copolymer nanofiber membrane. The preparation method comprises the following steps: mixing diacid, vinyl acetate, mercuric acetate and copper acetate with different chain lengths into a flask, stirring for 1-15 minutes at a room temperature, adding concentrated sulfuric acid, stirring for reaction for 8 hours at a constant temperature of 50-70 DEG C, adding anhydrous sodium acetate and concentrated sulfuric acid and performing separation and purification to obtain diacid divinyl ester with different chain lengths. A glucose vinyl ester homopolymer is prepared by adopting an aqueous-phase precipitation polymerization method; the preparation method is simple to operate, and a product is easy to dispose, economic and environment-friendly; glycosylated nanofibers are used for protein specificity recognition, high efficiency and quickness are realized, the activities of biological macromolecules are favorably maintained, a specificity recognition mechanism between sugar and protein is researched, and repeated use for multiple times can be realized; the industrial production is easy.
Description
Technical field
The invention belongs to the preparation of nano fibrous membrane and application, particularly to the preparation method of a kind of sugary temperature sensitive copolymer nano fibrous membrane.
Background technology
Sugar and protein, lipid is the same with nucleic acid, is the important component constituting organism.The glycoconjugate being widely present, including glycoprotein, Dan Baiduotang proteoglycan PG and glycolipid, take part in the important physiological process such as the targeting of albumen, cell recognition, antibody-antigene interaction.Glycosylation film has high hydrophilic and the characteristic such as anti-non-specific protein absorption and high biocompatibility, therefore protein separation it is widely used in very much, the aspects such as the cultivation of the specific capture of antibacterial, bio-medical material and cell, it is also possible to prepare surface-functionalized separation film.People attempt that glycosyl is fixed to different carriers surface and simulate the various biological functions of sugar, interact thus furtheing investigate the sugared affinity with protein, it is achieved that detection and the separation to protein, the molecule mechanism of research saccharide and protein specific identification.At present, the preparation of glycosylation film mainly has three kinds of approach, a kind of immobilization being to carry out on film saccharide macromole；Two is cause the glycerol polymerization containing sugar monomer on film surface；Three is the reaction of sugary little molecule and macromolecule side base.Saccharide can be made directly specific identification with protein, completes the transmission of bio information.Therefore, preparation has the glycosylation material of protein specific identification, has the prospect that is widely applied very much.
NIPA is owing to having hydrophilic amide groups and hydrophobic isopropyl in molecule, its homogeneous polymer has the good characteristics such as relatively low critical solution temperature (LCST).Owing to its polymer has more than 32 DEG C in this special dissolution characteristics of water-insoluble, developing the raw material for manufacturing some response to temperature polymer gel.NIPA is easily individually polymerized with common radical polymerization initiator, also easily copolymerization.Therefore, it becomes the focus of current thermoresponsive investigation of materials.
Sugary nano fibrous membrane prepared by electrostatic spinning technique is utilized to have the potentiality of specific adsorption mechanism between follow-up study protein.
Summary of the invention
The preparation method that the technical problem to be solved is to provide a kind of sugary temperature sensitive copolymer nano fibrous membrane, the method is simple to operate, product is disposable and economic and environment-friendly, this nano fibrous membrane contain abundant can reactive hydrophilic activated functional groups and there is the phase transition temperature close to body temperature, while the specific adsorption that improve material surface and the biocompatibility improving material surface, the basis of phase-change characteristic can have much potential application, realize the temperature sensitive of tumor tissues is controlled release as hot-spot processing method can be passed through, the tissue engineering bracket with phase transformation can also be prepared.Simultaneously this sugary temperature sensitive copolymer nano fibrous membrane can also specific recognition Con A Concanavalin (ConA), thus completing separation and the identification of protein.
The present invention is achieved through the following technical solutions:
The preparation method that the invention discloses a kind of sugary temperature sensitive copolymer nano fibrous membrane, specifically comprises the following steps that
(1) by the diacid of different chain length, vinylacetate, mercuric acetate, in Schweinfurt green mixing flask, after 1～15min is stirred at room temperature, add concentrated sulphuric acid, constant temperature stirring reaction 8h at 50 DEG C～70 DEG C, add in anhydrous sodium acetate and concentrated sulphuric acid, separating-purifying, obtain the diacid divinyl fat of different chain length；
(2) by the diacid divinyl ester of different chain length and glucose (1:1～4:1 according to a certain percentage, mol ratio), it is dissolved in 50～100mL anhydrous pyridine, add 0.5～1.5g alkaline protease, putting in 40～60 DEG C of constant-temperature shaking incubators and react 3～5 days, rotating speed is 210rpm synthesis of glucose ethylene fat；
(3) in above-mentioned glucose ethylene fat, add the NIPA that ratio is 1:1～50:1 with glucose ethylene fat; account for glucose ethylene fat and NIPA mass fraction 0.2～2% azodiisobutyronitrile (AIBN) as initiator; adding monomer concentration is 2-3mol/L ethanol as solvent; then reacting 3-8h in 55～60 DEG C of stirred under nitrogen atmosphere, polyreaction obtains glucose ethylene fat/N-isopropylacrylamide copolymer after terminating；
(4) in dehydrated alcohol, add above-mentioned glucose ethylene fat/N-isopropylacrylamide copolymer and account for the PLCL that mass volume ratio is 3wt%～30wt% of spinning solution, stirring, to being completely dissolved, obtains glucose ethylene fat/N-isopropylacrylamide copolymer spinning fluid；
(5) above-mentioned glucose ethylene fat/N-isopropylacrylamide copolymer and PLCL blend spinning liquid are carried out electrostatic spinning and obtain glucose ethylene fat/N-isopropylacrylamide copolymer superfine nano fibrous membrane, final drying, obtains the sugary copolymer nano fibrous membrane of Thermo-sensitive.
As improving further, step 1 of the present invention) in the diacid of different chain length can be adipic acid, Azelaic Acid and decanedioic acid, the mol ratio of diacid and vinylacetate is 1:1～1:5.
As improving further, step 1 of the present invention) in mercuric acetate quality 1g～3g, concentrated sulphuric acid volume is 0.1-0.25mL.
As improving further, step 1 of the present invention) in the quality of anhydrous sodium acetate be 0.1～1.0g, the remove impurity mode of excessive vinylacetate is rotary evaporation, and purification mode is silica gel column chromatography (300～400 order).
As improving further, step 2 of the present invention) described in glucose ethylene fat, its crude product purified by silica gel chromatographic column separating-purifying, eluant is ethyl acetate, and developing solvent is volume ratio is the ethyl acetate/methanol/water of 17:3:1, uses I2Colour developing.
As improving further, step 3 of the present invention) described in polyreaction terminate after, product is removed through 3500D dialyzer the glucose ethylene fat of no reaction.
As improving further, step 4 of the present invention) described in glucose ethylene fat/N-isopropylacrylamide copolymer spinning fluid in the mass fraction of glucose ethylene resin copolymer be 1%～90%, in described glucose ethylene fat/N-isopropylacrylamide copolymer, glucose ethylene fat is 5:1 with the mol ratio of N-isopropylacrylamide；10:1；15:1.
As improving further, the technological parameter of the electrostatic spinning described in step of the present invention (5) is: specification of syringe is 5ml, syringe needle internal diameter is 0.4～0.7mm, ejection flow velocity 0.8～2ml/h, electrostatic pressure 10～18kV, receiving screen adopts aluminium foil ground connection to receive, and accepting distance is 10～20cm, adopting orthogonal method to regulate spinning parameter and carry out electrospinning, described dries as putting into 30～60 DEG C of freeze-day with constant temperature 24～48h in vacuum drying oven.
As improving further, the present invention utilizes the glucosyl group specific adsorption to Con A Concanavalin (ConA), the described sugary copolymer nano fibrous membrane of Thermo-sensitive is mixed with the Con A Concanavalin (FL-ConA) of fluorescent decoration, observes its specific adsorption to ConA by laser tube focusing microscope (CLSM)；Utilize high concentration sugar solution can release the glucosyl group specific adsorption to Con A Concanavalin ConA, utilize the sugary copolymer nano fibrous membrane of wash buffer Thermo-sensitive, observe its De contamination ability.
As improving further, the protein adsorption time of the present invention is 2h, and sugar juice is glucose, and elution buffer is HEPES buffer.
Sugar is the one in biomolecule, is widely present in organism, is polyhydroxyl compound, has extremely strong hydrophilic, therefore, sugar is introduced the surface of material, will be effectively improved the biocompatibility of material surface.Therefore, sugar-containing polymer is usually utilized to the absorption that improves the materials such as the hydrophilic of material and specific protein at material surface.
Thermo-sensitive is containing sugar copolymer application in oncotherapy.
Thermo-sensitive material is introduced containing in sugar substance, the Thermo-sensitive to material can be realized control, its block copolymer with hydrophobic polymeric is often used to prepare Micellar drug carrier, the LCST that can make copolymer reaches 37 DEG C even higher, by its with after aliphatic polyester block copolymerization and prepare into carrier micelle, can realize the temperature sensitive of tumor tissues is controlled release in conjunction with hot-spot processing method.And proving on evidence, ConA has the functions such as the adhesion regulating tumor cell and apoptosis, therefore, it can the specific adsorption character utilizing material to ConA, for the apoptosis-promoting effect to tumor cell.
NIPA and glucose ethylene fat copolymerization are carried out electrospinning by the present invention, it is thus achieved that diameter and aperture are at nanoscale glucose ethylene fat/acrylonitrile copolymer nano fibrous membrane.This membrane material contain abundant can reactive hydrophilic activated functional groups, improve the non-specific adsorption of material surface and improve the biocompatibility of material surface, it does the potentiality that follow-up related experiment is analyzed to have application, and there is controlled LCST value, there is the application potentials such as the Targeting delivery that realizes medicine.
Beneficial effects of the present invention is as follows:
1), the present invention adopt aqueous deposited polymerization method to be prepared for glucose ethylene fat homopolymer, preparation method is simple to operate, product is disposable and economic and environment-friendly.
2), NIPA copolymer nano fiber is prepared simple, is a class specific surface, the carrier material of high porosity.
3), the membrane material of the present invention contain abundant can reactive hydrophilic activated functional groups, improve the non-specific adsorption of material surface and improve the biocompatibility of material surface, and due to containing substantial amounts of glucose group, therefore can carrying out specific recognition with specific agglutinin, it does the potentiality that follow-up related experiment is analyzed to have application.
4), glycosylation nanofiber for the specific recognition of protein, efficiently, quickly, be conducive to keeping the activity of biomacromolecule, the mechanism of specific recognition between research saccharide and protein, and can repeatedly using, it is easy to industrialized production.
After the fixing glucose of surface, it is possible to for separating and protein purification, the recognition mechanism between research saccharide and protein, and there is temperature-sensitive, there is more potential application.
Accompanying drawing explanation
Fig. 1 is the stereoscan photograph (spinning voltage is 10kV, and receiving range is 15cm, and injection flow velocity is 1.0ml/h) of the sugary copolymer nano fibrous membrane of Thermo-sensitive of embodiment 1 gained；
Fig. 2 is the infared spectrum of the sugary copolymer nano fibrous membrane of Thermo-sensitive of embodiment 2 gained；
Fig. 3 is the Thermo-sensitive of the embodiment 3 gained nuclear magnetic spectrum containing sugar copolymer, and wherein, A is vinylacetate nuclear magnetic spectrum, and B is glucose vinyl acetate nuclear magnetic spectrum, and C is the sugary copolymer nano fibrous membrane nuclear magnetic spectrum of Thermo-sensitive；
Fig. 4 is laser tube focusing microscope (CLSM) picture after the ConA absorption under 37 DEG C of conditions of the sugary copolymer nano fibrous membrane of Thermo-sensitive of embodiment 4 gained；
Fig. 5 is laser tube focusing microscope (CLSM) picture after the sugary copolymer nano fibrous membrane of Thermo-sensitive of embodiment 4 gained ConA De contamination under 37 DEG C of conditions.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention rather than restriction the scope of the present invention.In addition, it is to be understood that after having read the content that the present invention lectures, the present invention can be made various changes or modifications by those skilled in the art, and these equivalent form of values fall within the application appended claims limited range equally.Embodiment 1
(1) by the decanedioic acid of 1 mole, vinylacetate, 1g mercuric acetate, acetate in minute copper is mixed in 250mL flask, adds 0.2mL concentrated sulphuric acid, constant temperature stirring reaction 8h at 50 DEG C～70 DEG C after 15min is stirred at room temperature.React complete and obtain blue settled solution.Adding 0.5g anhydrous sodium acetate after reactant liquor cooling, fully concussion is to remove sulphuric acid therein, stands rotary evaporation after filtering and removes excessive vinylacetate.Surplus solution, through silica gel column chromatography (300～400 order) separating-purifying, obtains decanedioic acid divinyl fat.
(2) decanedioic acid divinyl fat and glucose (4:1, mol ratio) according to a certain percentage are dissolved in anhydrous pyridine, utilize enzyme' s catalysis technology synthesis of glucose ethylene fat.After reaction terminates, crude product purified by silica gel chromatographic column separating-purifying, eluant is ethyl acetate, and developing solvent is ethyl acetate, first alcohol and water (17:3:1, v/v/v), uses I2Colour developing.Above-mentioned glycolipid is put in trace polymerization pipe, add a certain amount of NIPA (being 7:1 with the mol ratio of glucose ethylene fat), with azodiisobutyronitrile (AIBN) (mass fraction accounting for glucose ethylene fat and NIPA gross mass is 2.0) as initiator, add dehydrated alcohol and make solvent (monomer concentration is 2.5mol/L dehydrated alcohol), seal, with nitrogen logical after oil pump evacuation, repeated multiple times.System is placed in 50-60 DEG C of stirred under nitrogen atmosphere reaction 6h.
(3) it is that 1:1 adds in dehydrated alcohol by the Thermo-sensitive glucose ethylene resin copolymer of certain mass and PLCL according to mol ratio, in copolymer, glucose lipid is 1:10 with the mol ratio of NIPAM, stirred for several hour is to being completely dissolved, standing a few hours make bubble in spinning liquid be wholly absent, and stirring forms homogeneous spinning liquid.
(4) Thermo-sensitive is extracted containing sugar copolymer with syringe, it is fixed on electrostatic spinning apparatus, control ejection flow velocity 1.0ml/h, electrostatic pressure 10kV, receiving screen adopts aluminium foil ground connection to receive, and the distance of syringe needle and receiving screen is 10-20cm, adopts orthogonal method (to change a certain parameter, fix other parameter) regulate different spinning parameter and carry out electrospinning, obtain Thermo-sensitive containing sugar copolymer superfine nano fibrous membrane；Putting into 30-60 DEG C of freeze-day with constant temperature 36h in vacuum drying oven, make sugar-containing polymer nano fibrous membrane, its electromicroscopic photograph is as it is shown in figure 1, it appeared that fiber is evenly distributed, without beading, it was demonstrated that its pattern is good.
Embodiment 2
Azelaic Acid divinyl fat and glucose (4:1, mol ratio) according to a certain percentage are dissolved in anhydrous pyridine, utilize enzyme' s catalysis technology synthesis of glucose ethylene fat.After reaction terminates, crude product purified by silica gel chromatographic column separating-purifying, eluant is ethyl acetate, and developing solvent is ethyl acetate, first alcohol and water (17:3:1, v/v/v), uses I2Colour developing.Add a certain amount of NIPA (being 7:1 with the mol ratio of glucose ethylene fat), with azodiisobutyronitrile (AIBN) (mass fraction accounting for glucose ethylene fat and NIPA gross mass is 2.0) as initiator, add dehydrated alcohol and make solvent (monomer concentration is 2.5mol/L dehydrated alcohol), seal, with nitrogen logical after oil pump evacuation, repeated multiple times.System is placed in 55-60 DEG C of stirred under nitrogen atmosphere reaction 6h.
(2) it is that 1:1 adds in dehydrated alcohol by the Thermo-sensitive glucose ethylene resin copolymer of certain mass and PLCL according to mol ratio, in copolymer, glucose lipid is 1:15 with the mol ratio of NIPAM, stirred for several hour is to being completely dissolved, standing a few hours make bubble in spinning liquid be wholly absent, and stirring forms homogeneous spinning liquid.
(3) Thermo-sensitive is extracted containing sugar copolymer with syringe, it is fixed on electrostatic spinning apparatus, control ejection flow velocity 1.0ml/h, electrostatic pressure 10kV, receiving screen adopts aluminium foil ground connection to receive, and the distance of syringe needle and receiving screen is 10-20cm, adopts orthogonal method (to change a certain parameter, fix other parameter) regulate different spinning parameter and carry out electrospinning, obtain Thermo-sensitive containing sugar copolymer superfine nano fibrous membrane；Putting into 60-80 DEG C of freeze-day with constant temperature 24h in vacuum drying oven, make sugar-containing polymer nano fibrous membrane, its infared spectrum is as shown in Figure 2.
Embodiment 3
(1) vinyl hexanediacetate and glucose (4:1, mol ratio) according to a certain percentage are dissolved in anhydrous pyridine, utilize enzyme' s catalysis technology synthesis of glucose ethylene fat.After reaction terminates, crude product purified by silica gel chromatographic column separating-purifying, eluant is ethyl acetate, and developing solvent is ethyl acetate, first alcohol and water (17:3:1, v/v/v), uses I2Colour developing.Add a certain amount of NIPA (being 7:1 with the mol ratio of glucose ethylene fat), with azodiisobutyronitrile (AIBN) (mass fraction accounting for glucose ethylene fat and NIPA gross mass is 2.0) as initiator, add dehydrated alcohol and make solvent (monomer concentration is 2.5mol/L dehydrated alcohol), seal, with nitrogen logical after oil pump evacuation, repeated multiple times.System is placed in 55～60 DEG C of stirred under nitrogen atmosphere reaction 6h.Obtaining sugar-containing polymer, the nuclear magnetic spectrum of vinyl hexanediacetate, glucose ethylene fat and sugar-containing polymer is as shown in Figure 3.
(2) it is that 1:1 adds in dehydrated alcohol by the Thermo-sensitive glucose ethylene resin copolymer of certain mass and PLCL according to mol ratio, in copolymer, glucose lipid is 10:1 with the mol ratio of NIPAM, stirred for several hour is to being completely dissolved, standing a few hours make bubble in spinning liquid be wholly absent, and stirring forms homogeneous spinning liquid.
(3) Thermo-sensitive is extracted containing sugar copolymer with syringe, it is fixed on electrostatic spinning apparatus, control ejection flow velocity 1.0ml/h, electrostatic pressure 10kV, receiving screen adopts aluminium foil ground connection to receive, and the distance of syringe needle and receiving screen is 10～20cm, adopts orthogonal method (to change a certain parameter, fix other parameter) regulate different spinning parameter and carry out electrospinning, obtain Thermo-sensitive that diameter is 80～250nm containing sugar copolymer superfine nano fibrous membrane；Put into 60～80 DEG C of freeze-day with constant temperature 24h in vacuum drying oven, make regeneration nano composite membrane.
Embodiment 4
(1) absorption of ConA need to be configured HEPES buffer solution by glucose ethylene fat/N-isopropylacrylamide copolymer and PLCL blending nano fibrous membrane, and pH is 7.5, wherein 10mMHEPES, 0.15MNaCl, 0.1mMCa2+, 0.01mMMn2+.
(2) the glucose ethylene fat/N-isopropylacrylamide copolymer prepared by embodiment 3 and PLCL blending nano fibrous membrane priority PBS buffer solution pretreatment (20～30min), to remove the pollutant on film surface, take 0.5mg film to be measured, put into after shredding in test tube, it is separately added into ConA (FL-ConA) solution modified containing Fluorescein isothiocyanate of 1～10mL same concentrations, at 25 DEG C and 37 DEG C round waters constant temperature oscillator lucifuge constant temperature 2h.After balance upon adsorption, take out film, clean 4-8 time with HEPES buffer solution, after at room temperature vacuum drying.The CLSM fluorescence intensity measuring material surface is utilized after dry end.Such as Fig. 4, it is possible to finding that the surface of fibrous membrane also has a large amount of green fluorescence, surface C onA is adsorbed on the surface of fibrous membrane in a large number, it was demonstrated that sugar-containing polymer fibrous membrane can specific recognition ConA.
(5) membrane material of above-mentioned absorption FL-ConA need to be immersed in the glucose solution that concentration is 1M of 200 μ L by glucose ethylene fat/N-isopropylacrylamide copolymer and PLCL blending nano fibrous membrane by the De contamination of ConA, De contamination 24h under 37 DEG C of conditions.Then, membrane material HEPES is cleaned 4-8 time, after at room temperature vacuum drying.The CLSM fluorescence intensity measuring material surface is utilized after dry end.Such as Fig. 5, it is possible to find that green fluorescence originally disappears, it was demonstrated that ConA is eluted, it was demonstrated that sugar-containing polymer fibrous membrane has reproducibility, it is possible to for separation and the collection of albumen.
Embodiment 1～4 of the present invention is only the description that the preferred embodiment of the present invention is carried out; not present inventive concept and scope are defined; under the premise without departing from design philosophy of the present invention; various modification that in this area, technical scheme is made by engineering and improvement, all should fall into protection scope of the present invention.
Claims (10)
1. the preparation method of a sugary temperature sensitive copolymer nano fibrous membrane, it is characterised in that specifically comprise the following steps that
(1) by the diacid of different chain length, vinylacetate, mercuric acetate, in Schweinfurt green mixing flask, after 1～15min is stirred at room temperature, add concentrated sulphuric acid, constant temperature stirring reaction 8h at 50 DEG C～70 DEG C, add in anhydrous sodium acetate and concentrated sulphuric acid, separating-purifying, obtain the diacid divinyl fat of different chain length；
(2) by the diacid divinyl ester of different chain length and glucose (1:1～4:1 according to a certain percentage, mol ratio), it is dissolved in 50～100mL anhydrous pyridine, add 0.5～1.5g alkaline protease, putting in 40～60 DEG C of constant-temperature shaking incubators and react 3～5 days, rotating speed is 210rpm synthesis of glucose ethylene fat；
(3) in above-mentioned glucose ethylene fat, add the NIPA that ratio is 1:1～50:1 with glucose ethylene fat; account for glucose ethylene fat and NIPA mass fraction 0.2～2% azodiisobutyronitrile (AIBN) as initiator; adding monomer concentration is 2-3mol/L ethanol as solvent; then reacting 3-8h in 55～60 DEG C of stirred under nitrogen atmosphere, polyreaction obtains glucose ethylene fat/N-isopropylacrylamide copolymer after terminating；
(4) in dehydrated alcohol, add above-mentioned glucose ethylene fat/N-isopropylacrylamide copolymer and account for the PLCL that mass volume ratio is 3wt%～30wt% of spinning solution, stirring, to being completely dissolved, obtains glucose ethylene fat/N-isopropylacrylamide copolymer spinning fluid；
(5) above-mentioned glucose ethylene fat/N-isopropylacrylamide copolymer and PLCL blend spinning liquid are carried out electrostatic spinning and obtain glucose ethylene fat/N-isopropylacrylamide copolymer superfine nano fibrous membrane, final drying, obtains the sugary copolymer nano fibrous membrane of Thermo-sensitive.
2. the preparation method of sugary temperature sensitive copolymer nano fibrous membrane according to claim 1, it is characterized in that, described step 1) in the diacid of different chain length can be adipic acid, Azelaic Acid and decanedioic acid, the mol ratio of diacid and vinylacetate is 1:1～1:5.
3. the preparation method of sugary temperature sensitive copolymer nano fibrous membrane according to claim 1, it is characterised in that step 1) in mercuric acetate quality 1g～3g, concentrated sulphuric acid volume is 0.1-0.25mL.
4. the preparation method of sugary temperature sensitive copolymer nano fibrous membrane according to claim 1, it is characterized in that: step 1) in the quality of anhydrous sodium acetate be 0.1～1.0g, the remove impurity mode of excessive vinylacetate is rotary evaporation, and purification mode is silica gel column chromatography (300～400 order).
5. the preparation method of the sugary temperature sensitive copolymer nano fibrous membrane according to claim 1 or 2 or 3 or 4, it is characterized in that: step 2) described in glucose ethylene fat, its crude product purified by silica gel chromatographic column separating-purifying, eluant is ethyl acetate, developing solvent is volume ratio is the ethyl acetate/methanol/water of 17:3:1, uses I2Colour developing.
6. the preparation method of sugary temperature sensitive copolymer nano fibrous membrane according to claim 5, it is characterised in that step 3) described in polyreaction terminate after, product is removed through 3500D dialyzer the glucose ethylene fat of no reaction.
7. the preparation method of the sugary temperature sensitive copolymer nano fibrous membrane according to claim 1 or 2 or 3 or 4 or 6, it is characterized in that, step 4) described in glucose ethylene fat/N-isopropylacrylamide copolymer spinning fluid in the mass fraction of glucose ethylene resin copolymer be 1%～90%, in described glucose ethylene fat/N-isopropylacrylamide copolymer, glucose ethylene fat is 5:1 with the mol ratio of N-isopropylacrylamide；10:1；15:1.
8. the preparation method of sugary temperature sensitive copolymer nano fibrous membrane according to claim 7, it is characterized in that: the technological parameter of the electrostatic spinning described in step (5) is: specification of syringe is 5ml, syringe needle internal diameter is 0.4～0.7mm, ejection flow velocity 0.8～2ml/h, electrostatic pressure 10～18kV, receiving screen adopts aluminium foil ground connection to receive, accepting distance is 10～20cm, adopting orthogonal method to regulate spinning parameter and carry out electrospinning, described dries as putting into 30～60 DEG C of freeze-day with constant temperature 24～48h in vacuum drying oven.
9. the preparation method of sugary temperature sensitive copolymer nano fibrous membrane according to claim 1, it is characterized in that, utilize the glucosyl group specific adsorption to Con A Concanavalin (ConA), the described sugary copolymer nano fibrous membrane of Thermo-sensitive is mixed with the Con A Concanavalin (FL-ConA) of fluorescent decoration, observes its specific adsorption to ConA by laser tube focusing microscope (CLSM)；Utilize high concentration sugar solution can release the glucosyl group specific adsorption to Con A Concanavalin ConA, utilize the sugary copolymer nano fibrous membrane of wash buffer Thermo-sensitive, observe its De contamination ability.
10. the preparation method of sugary temperature sensitive copolymer nano fibrous membrane according to claim 9, it is characterised in that the described protein adsorption time is 2h, and sugar juice is glucose, and elution buffer is HEPES buffer.
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