CN104945579A - Carbon nano tube surface lysozyme molecularly-imprinted polymer and preparation method thereof - Google Patents
Carbon nano tube surface lysozyme molecularly-imprinted polymer and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a carbon nano tube surface lysozyme molecularly-imprinted polymer. The carbon nano tube surface lysozyme molecularly-imprinted polymer adopts lysozyme as a template molecule, adopts 1-allyl-3-methylimidazolium hexafluorophate ionic liquid and N-tert-butyl acrylamide as a mixing functional monomer, adopts an acrylamide modified carbon nanotube as a matrix, adopts a Tris-HCl buffer solution with the pH being 9 as a reaction system, is obtained under the effect of a cross-linking agent, an initiating agent and a catalyst and has the selective recognition capacity for lysozyme. The number of acting sites is multiple when the template molecule and the functional monomer are combined, the acting force is higher, more effective recognition sites can be formed in the preparation process of the molecularly-imprinted polymer, and therefore the printing effect and the preferential adsorption capacity for lysozyme are better.
Description
Technical field
The invention belongs to chemistry and Material Field, relate to a kind of carbon nano tube surface lysozyme molecular imprinting polymkeric substance and preparation method thereof.
Background technology
Molecular imprinting is that a kind of analogue antigen-antibody interacts, and adopts artificial synthesis to obtain the technology of the polymkeric substance that space structure and binding site and template molecule match.First, produce complementary interaction between template molecule and function monomer and form mixture, then under linking agent effect, around template-monomer mixture, there is bulk polymerization form polymkeric substance, the last template molecule removed under certain condition in polymkeric substance, the selectivity molecular recognition material with particular space structure hole and the recognition site mated with template molecule can be obtained, namely usually said molecularly imprinted polymer.Compared with natural biomacromolecule recognition system, the molecularly imprinted polymer of synthesis has the advantages such as conformation precordainment, specific recognition, permanent stability and being easy to implement property, has been widely used in chromatographic separation, Solid-Phase Extraction, mimetic enzyme catalysis and sensor numerous areas.But, due to exist as the protein of imprinted templates itself bulky, complex structure, can binding site mainly with and the problem such as conformation is unstable, up to now, for the trace still faces enormous challenge of protein and other.
Surface molecule print technology is a kind of molecular imprinting method built on the substrate surface that latest developments are got up.The method in polymeric solution, introduces matrix or is coated in matrix by polymeric solution, makes the polyreaction of template molecule and function monomer occur in stromal surface to form molecular imprinting polymer membrane, thus make molecular recognition site be exposed to stromal surface.Surface molecule print technology can overcome the sterically hindered large problem that traditional entrapping method exists, and enables template molecule freely pass in and out specific recognition site in stromal surface polymkeric substance.Meanwhile, because matrix has stronger mechanical stability, therefore can the intensity of effective telomerized polymer to adapt to the needs of practical application.Take carbon nanotube as matrix, method that is pre-assembled or self-assembly is adopted to modify its surface, then at its surface grafting one deck molecular imprinting polymer membrane, a series of carbon nano tube surface molecularly imprinted polymer with different recognition site and cavity structure can be prepared, thus the technical problem solving protein molecule engram Macroscopic single crystal and exist in identifying.
Ionic liquid by organic cation and inorganic anion form at room temperature or the organic salt that is in a liquid state under being bordering on room temperature, have nontoxic, low temperature is non-volatile, stablize water and air, solvability is good, stability is high, polarity is strong, film forming properties is good and be easy to the advantage of modification.Ionic liquid is due to the uniqueness of molecular structure and chemical property, application in separation science field is very wide, as being used as the solvent of liquid-liquid extraction, the pore-creating agent of the sorbent material of Solid-Phase Extraction, the coated material of chromatographic column and molecularly imprinted polymer, solubility promoter and function monomer.In numerous ionic liquid, imidazole type ion liquid is most study is also most widely used ionic liquid.Because imidazole ion liquid has special glyoxaline cation and inorganic anion structure, stronger multiplephase mutual effect complementary with it can be provided with protein and other compound tense, comprise electrostatic, hydrogen bond and pi-pi accumulation effect.N tert butyl acrylamide can provide stronger hydrophobic interaction in the preparation and recognition process of molecularly imprinted polymer.Therefore, with hydrophilic ionic liquid and hydrophobic N tert butyl acrylamide for mixing functions monomer, by regulating the proportioning of two kinds of function monomers to obtain the molecularly imprinted polymer with template protein matched on space structure and reactive force, application prospect is good.
Summary of the invention
The object of the present invention is to provide a kind of carbon nano tube surface lysozyme molecular imprinting polymkeric substance and preparation method thereof.
For achieving the above object, the technical solution adopted in the present invention is as follows:
Carbon nano tube surface lysozyme molecular imprinting polymkeric substance provided by the present invention, it take N,O-Diacetylmuramidase as template molecule, 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid and N tert butyl acrylamide are mixing functions monomer, acrylamide is carbon nano-tube modified is matrix, the Tris-HCl buffered soln of pH9 is reaction system, molecular imprinted polymer on surface N,O-Diacetylmuramidase to Selective recognition ability obtained under linking agent, initiator and catalyst action.
Preferably, carbon nano-tube modified, N,O-Diacetylmuramidase, N tert butyl acrylamide, 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid the weight ratio of described acrylamide is followed successively by (20 ~ 40): (15 ~ 25): (30 ~ 120): (160 ~ 400).
Further preferably, carbon nano-tube modified, N,O-Diacetylmuramidase, N tert butyl acrylamide, 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid the weight ratio of described acrylamide is followed successively by 30: 18: (80 ~ 100): (180 ~ 220).
Preferably, described linking agent is N, N '-methylene-bisacrylamide.
Further preferably, described linking agent and the carbon nano-tube modified weight ratio of acrylamide are 3.3 ~ 5: 1.
Preferably, described initiator is ammonium persulphate.
Preferably, described catalyzer is N, N, N', N'-Tetramethyl Ethylene Diamine.
The present invention further provides a kind of method preparing carbon nano tube surface lysozyme molecular imprinting polymkeric substance, it comprises the following steps:
1) be dissolved in the Tris-HCl buffered soln of 30mL pH9 by 15 ~ 25mg N,O-Diacetylmuramidase, add 30 ~ 120mg N tert butyl acrylamide and 160 ~ 400mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h;
2) add 20 ~ 40mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 60 ~ 200mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15-20 minute;
3) add 20-40mg ammonium persulphate, 50-100 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15-20 minute, then normal temperature magnetic agitation 12-48h under anaerobic;
4) by solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH9 to wash 3-5 time to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, to obtain final product.
Preferably, said method comprising the steps of:
1) be dissolved in the Tris-HCl buffered soln of 30mL pH9 by 18mg N,O-Diacetylmuramidase, add 80 ~ 100mg N tert butyl acrylamide and 180 ~ 220mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h;
2) add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 100 ~ 150mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15-20 minute;
3) add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15-20 minute, then normal temperature magnetic agitation 12-48h under anaerobic;
4) by solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH9 to wash 3-5 time to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, to obtain final product.
Preferably, described acrylamide is carbon nano-tube modified to carry out surface chemical modification to carboxylic carbon nano-tube and obtains, caliber≤the 8nm of described carboxylic carbon nano-tube, length is 20 ~ 30 μm, carboxyl-content is 3.86wt%, purity >95%, the method of surface chemical modification is: take 0.6g carboxylic carbon nano-tube, add 60mL thionyl chloride and 10 dimethyl formamides, 70 DEG C of backflow 24h, after solvent evaporate to dryness, continue to add 3g acrylamide and 50mL dimethyl formamide, ultrasonic dissolution 10min, in 45 DEG C of backflow 24h, product ultrapure water is washed till neutrality, 60 DEG C of oven dry.
The invention has the beneficial effects as follows:
1) owing to introducing 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid and hydrophobic N tert butyl acrylamide two kinds of function monomers of polarity, make template molecule and function monomer compound tense action site more, reactive force is stronger, can form more effective recognition site in the preparation process of molecularly imprinted polymer.Therefore, to the imprinting effect of N,O-Diacetylmuramidase and selective adsorption capacity than being only that molecularly imprinted polymer prepared by function monomer is better with ionic liquid.
2) specific surface area due to carbon nanotube is large, physical strength is high, the trace film spreading formed is in the surface of carbon nanotube, present the reticulated structure of porous, make the imprinted sites being exposed to surface more, sterically hindered less, template molecule elution is more complete, absorption is rapider, and imprinting effect is better, and selective adsorption capacity is stronger.
3) because polyreaction is carried out in the Tris-HCl buffered soln of pH 9, the electronegative amino acid that N,O-Diacetylmuramidase surface exposes can form the higher mixture of stability with the glyoxaline cation of ionic liquid by electrostatic, hydrogen bond and pi-pi accumulation effect, thus forms in carbon nano tube surface effective imprinted sites that more multipair N,O-Diacetylmuramidase has specific recognition capability.
These excellent performances make it have good application prospect in N,O-Diacetylmuramidase identification, abstraction and purification field above, also lay a good foundation for solving Problems existing in the synthesis of other oroteins molecularly imprinted polymer and identification simultaneously.
Accompanying drawing explanation
Fig. 1: embodiment 1-11 carbon nano tube surface lysozyme molecular imprinting Macroscopic single crystal route schematic diagram.
Fig. 2: embodiment 12-19 carbon nano tube surface lysozyme molecular imprinting Macroscopic single crystal route schematic diagram.
Fig. 3: 1-allyl group-3-Methylimidazole phosphofluoric acid function monomer content is to the influence curve figure of carbon nano tube surface lysozyme molecular imprinting polymkeric substance and blank molecularly imprinted polymer adsorption effect.
The scanning electron microscope (SEM) photograph of the molecularly imprinted polymer that Fig. 4: 1-allyl group-3-Methylimidazole phosphofluoric acid function monomer content is different and blank molecularly imprinted polymer.(a) function monomer 200mg molecularly imprinted polymer; The blank molecularly imprinted polymer of (b) function monomer 200mg; (c) function monomer 300mg molecularly imprinted polymer; The blank molecularly imprinted polymer of (d) function monomer 300mg.
The molecularly imprinted polymer that Fig. 5: N, N '-methylene-bisacrylamide content of crosslinking agent is different and blank molecularly imprinted polymer are to the adsorptive capacity (polymerization system being function monomer with 1-allyl group-3-Methylimidazole phosphofluoric acid) of N,O-Diacetylmuramidase.
The scanning electron microscope (SEM) photograph of the molecularly imprinted polymer that Fig. 6: N, N '-methylene-bisacrylamide content of crosslinking agent is different and blank molecularly imprinted polymer.(a) linking agent 80mg molecularly imprinted polymer; The blank molecularly imprinted polymer of (b) linking agent 80mg; (c) linking agent 100mg molecularly imprinted polymer; The blank molecularly imprinted polymer of (d) linking agent 100mg; (e) linking agent 150mg molecularly imprinted polymer; The blank molecularly imprinted polymer of (f) linking agent 150mg.
Fig. 7: the molecularly imprinted polymer that N tert butyl acrylamide is different with 1-allyl group-3-Methylimidazole phosphofluoric acid function monomer mol ratio and blank molecularly imprinted polymer are to the adsorptive capacity of N,O-Diacetylmuramidase.
Fig. 8: the scanning electron microscope (SEM) photograph of the molecularly imprinted polymer that N tert butyl acrylamide is different with 1-allyl group-3-Methylimidazole phosphofluoric acid mol ratio and blank molecularly imprinted polymer.(a) function monomer mol ratio 20%:80% molecularly imprinted polymer; The blank molecularly imprinted polymer of (b) function monomer mol ratio 20%:80%; (c) function monomer mol ratio 40%:60% molecularly imprinted polymer; The blank molecularly imprinted polymer of (d) function monomer mol ratio 40%:60%; (e) function monomer mol ratio 50%:50% molecularly imprinted polymer; The blank molecularly imprinted polymer of (f) function monomer mol ratio 50%:50%.
The molecularly imprinted polymer that Fig. 9: N, N '-methylene-bisacrylamide content of crosslinking agent is different and blank molecularly imprinted polymer are to the adsorptive capacity (polymerization system being function monomer with N tert butyl acrylamide and 1-allyl group-3-Methylimidazole phosphofluoric acid) of N,O-Diacetylmuramidase.
The scanning electron microscope (SEM) photograph (polymerization system being function monomer with N tert butyl acrylamide and 1-allyl group-3-Methylimidazole phosphofluoric acid) of the molecularly imprinted polymer that Figure 10: N, N '-methylene-bisacrylamide content of crosslinking agent is different and blank molecularly imprinted polymer.(a) linking agent 100mg molecularly imprinted polymer; The blank molecularly imprinted polymer of (b) linking agent 100mg; (c) linking agent 150mg molecularly imprinted polymer; The blank molecularly imprinted polymer of (d) linking agent 150mg; (e) linking agent 200mg molecularly imprinted polymer; The blank molecularly imprinted polymer of (f) linking agent 200mg.
Figure 11: the carbon nano tube surface lysozyme molecular imprinting polymkeric substance of synthesis and blank molecularly imprinted polymer are to the adsorption isothermal curve of N,O-Diacetylmuramidase.
Figure 12: the molecularly imprinted polymer being mixing functions monomer synthesize with 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid and N tert butyl acrylamide is to the selective adsorption capacity of N,O-Diacetylmuramidase and cytochrome C.
Figure 13: function monomer, linking agent and the carbon nano tube surface lysozyme molecular imprinting of synthesis and the infrared spectrogram of blank imprinted polymer.(a) N tert butyl acrylamide; (b) N, N '-methylene-bisacrylamide; (c) 1-allyl group-3-Methylimidazole phosphofluoric acid; (d) molecularly imprinted polymer (ionic liquid function monomer); (e) blank molecularly imprinted polymer (ionic liquid function monomer); (f) molecularly imprinted polymer (mixing functions monomer); (g) blank molecularly imprinted polymer (mixing functions monomer).
Figure 14: the carbon nano tube surface lysozyme molecular imprinting of function monomer and synthesis and the thermogravimetric analysis figure of blank imprinted polymer.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is further described.Table 1 uses scale for each reactant in embodiment 1 ~ 19 molecularly imprinted polymer building-up process.
1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, purchased from the prompt Chemical Co., Ltd. of upper marine origin; The carbon nanotube that acrylamide is modified, carry out surface chemical modification to carboxylic carbon nano-tube and obtain, carboxylic carbon nano-tube is purchased from Chengdu Organical Chemical Co., Ltd., Chinese Academy of Sciences, caliber≤8nm, length 20 ~ 30 μm, carboxyl-content 3.86wt%, purity >95%.The method of surface chemical modification is: take 0.6g carboxylic carbon nano-tube, add 60mL thionyl chloride and 10 dimethyl formamides, 70 DEG C of backflow 24h, after solvent evaporate to dryness, continue to add 3g acrylamide and 50mL dimethyl formamide, ultrasonic dissolution 10min, in 45 DEG C of backflow 24h, product ultrapure water is washed till neutrality, 60 DEG C of oven dry.
The material usage sheet of table 1 embodiment 1 ~ 19
Embodiment 1:
Be dissolved in the Tris-HCl buffered soln of 30mL pH 9 by 18mg N,O-Diacetylmuramidase, add 200mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h; Add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 100mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15 minutes; Add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15 minutes, then normal temperature magnetic agitation 48h under anaerobic.By solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH 9 to wash 5 times to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, obtains carbon nano tube surface lysozyme molecular imprinting polymkeric substance.The synthesis of blank molecularly imprinted polymer is identical therewith with elution process, just in building-up process, does not add template molecule N,O-Diacetylmuramidase.
Embodiment 2:
Be dissolved in the Tris-HCl buffered soln of 30mL pH 9 by 18mg N,O-Diacetylmuramidase, add 300mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h; Add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 100mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15 minutes; Add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15 minutes, then normal temperature magnetic agitation 48h under anaerobic.By solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH 9 to wash 5 times to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, obtains carbon nano tube surface lysozyme molecular imprinting polymkeric substance.The synthesis of blank molecularly imprinted polymer is identical therewith with elution process, just in building-up process, does not add template molecule N,O-Diacetylmuramidase.
Embodiment 3:
Be dissolved in the Tris-HCl buffered soln of 30mL pH 9 by 18mg N,O-Diacetylmuramidase, add 350mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h; Add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 100mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15 minutes; Add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15 minutes, then normal temperature magnetic agitation 48h under anaerobic.By solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH 9 to wash 5 times to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, obtains carbon nano tube surface lysozyme molecular imprinting polymkeric substance.The synthesis of blank molecularly imprinted polymer is identical therewith with elution process, just in building-up process, does not add template molecule N,O-Diacetylmuramidase.
Embodiment 4:
Be dissolved in the Tris-HCl buffered soln of 30mL pH 9 by 18mg N,O-Diacetylmuramidase, add 400mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h; Add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 100mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15 minutes; Add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15 minutes, then normal temperature magnetic agitation 48h under anaerobic.By solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH 9 to wash 5 times to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, obtains carbon nano tube surface lysozyme molecular imprinting polymkeric substance.The synthesis of blank molecularly imprinted polymer is identical therewith with elution process, just in building-up process, does not add template molecule N,O-Diacetylmuramidase.
Embodiment 5:
Be dissolved in the Tris-HCl buffered soln of 30mL pH 9 by 18mg N,O-Diacetylmuramidase, add 400mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h; Add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 60mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15 minutes; Add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15 minutes, then normal temperature magnetic agitation 48h under anaerobic.By solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH 9 to wash 5 times to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, obtains carbon nano tube surface lysozyme molecular imprinting polymkeric substance.The synthesis of blank molecularly imprinted polymer is identical therewith with elution process, just in building-up process, does not add template molecule N,O-Diacetylmuramidase.
Embodiment 6:
Be dissolved in the Tris-HCl buffered soln of 30mL pH 9 by 18mg N,O-Diacetylmuramidase, add 400mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h; Add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 80mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15 minutes; Add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15 minutes, then normal temperature magnetic agitation 48h under anaerobic.By solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH 9 to wash 5 times to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, obtains carbon nano tube surface lysozyme molecular imprinting polymkeric substance.The synthesis of blank molecularly imprinted polymer is identical therewith with elution process, just in building-up process, does not add template molecule N,O-Diacetylmuramidase.
Embodiment 7:
Be dissolved in the Tris-HCl buffered soln of 30mL pH 9 by 18mg N,O-Diacetylmuramidase, add 400mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h; Add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 150mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15 minutes; Add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15 minutes, then normal temperature magnetic agitation 48h under anaerobic.By solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH 9 to wash 5 times to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, obtains carbon nano tube surface lysozyme molecular imprinting polymkeric substance.The synthesis of blank molecularly imprinted polymer is identical therewith with elution process, just in building-up process, does not add template molecule N,O-Diacetylmuramidase.
Embodiment 8:
Be dissolved in the Tris-HCl buffered soln of 30mL pH 9 by 18mg N,O-Diacetylmuramidase, add 400mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h; Add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 200mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15 minutes; Add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15 minutes, then normal temperature magnetic agitation 48h under anaerobic.By solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH 9 to wash 5 times to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, obtains carbon nano tube surface lysozyme molecular imprinting polymkeric substance.The synthesis of blank molecularly imprinted polymer is identical therewith with elution process, just in building-up process, does not add template molecule N,O-Diacetylmuramidase.
Embodiment 9:
Be dissolved in the Tris-HCl buffered soln of 30mL pH 9 by 18mg N,O-Diacetylmuramidase, add 400mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h; Add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 250mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15 minutes; Add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15 minutes, then normal temperature magnetic agitation 48h under anaerobic.By solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH 9 to wash 5 times to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, obtains carbon nano tube surface lysozyme molecular imprinting polymkeric substance.The synthesis of blank molecularly imprinted polymer is identical therewith with elution process, just in building-up process, does not add template molecule N,O-Diacetylmuramidase.
Embodiment 10:
Be dissolved in the Tris-HCl buffered soln of 30mL pH 9 by 18mg N,O-Diacetylmuramidase, add 400mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h; Add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 300mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15 minutes; Add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15 minutes, then normal temperature magnetic agitation 48h under anaerobic.By solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH 9 to wash 5 times to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, obtains carbon nano tube surface lysozyme molecular imprinting polymkeric substance.The synthesis of blank molecularly imprinted polymer is identical therewith with elution process, just in building-up process, does not add template molecule N,O-Diacetylmuramidase.
Embodiment 11:
Be dissolved in the Tris-HCl buffered soln of 30mL pH 9 by 18mg N,O-Diacetylmuramidase, add 400mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h; Add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 350mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15 minutes; Add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15 minutes, then normal temperature magnetic agitation 48h under anaerobic.By solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH 9 to wash 5 times to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, obtains carbon nano tube surface lysozyme molecular imprinting polymkeric substance.The synthesis of blank molecularly imprinted polymer is identical therewith with elution process, just in building-up process, does not add template molecule N,O-Diacetylmuramidase.
Embodiment 12:
Be dissolved in the Tris-HCl buffered soln of 30mL pH 9 by 18mg N,O-Diacetylmuramidase, add 35mg N tert butyl acrylamide (being dissolved in 1mL ethanol) and 320mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h; Add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 100mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15 minutes; Add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15 minutes, then normal temperature magnetic agitation 48h under anaerobic.By solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH 9 to wash 5 times to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, obtains carbon nano tube surface lysozyme molecular imprinting polymkeric substance.The synthesis of blank molecularly imprinted polymer is identical therewith with elution process, just in building-up process, does not add template molecule N,O-Diacetylmuramidase.
Embodiment 13:
Be dissolved in the Tris-HCl buffered soln of 30mL pH 9 by 18mg N,O-Diacetylmuramidase, add 70mg N tert butyl acrylamide (being dissolved in 1mL ethanol) and 240mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h; Add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 100mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15 minutes; Add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15 minutes, then normal temperature magnetic agitation 48h under anaerobic.By solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH 9 to wash 5 times to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, obtains carbon nano tube surface lysozyme molecular imprinting polymkeric substance.The synthesis of blank molecularly imprinted polymer is identical therewith with elution process, just in building-up process, does not add template molecule N,O-Diacetylmuramidase.
Embodiment 14:
Be dissolved in the Tris-HCl buffered soln of 30mL pH 9 by 18mg N,O-Diacetylmuramidase, add 87.5mg N tert butyl acrylamide (being dissolved in 1mL ethanol) and 200mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h; Add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 100mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15 minutes; Add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15 minutes, then normal temperature magnetic agitation 48h under anaerobic.By solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH 9 to wash 5 times to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, obtains carbon nano tube surface lysozyme molecular imprinting polymkeric substance.The synthesis of blank molecularly imprinted polymer is identical therewith with elution process, just in building-up process, does not add template molecule N,O-Diacetylmuramidase.
Embodiment 15:
Be dissolved in the Tris-HCl buffered soln of 30mL pH 9 by 18mg N,O-Diacetylmuramidase, add 105mg N tert butyl acrylamide (being dissolved in 1mL ethanol) and 160mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h; Add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 100mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15 minutes; Add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15 minutes, then normal temperature magnetic agitation 48h under anaerobic.By solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH 9 to wash 5 times to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, obtains carbon nano tube surface lysozyme molecular imprinting polymkeric substance.The synthesis of blank molecularly imprinted polymer is identical therewith with elution process, just in building-up process, does not add template molecule N,O-Diacetylmuramidase.
Embodiment 16:
18mg N,O-Diacetylmuramidase is dissolved in the Tris-HCl buffered soln of 30mL pH 9, add 87.5mg N tert butyl acrylamide (being dissolved in 1mL ethanol) and 200mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h; Add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 60mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15 minutes; Add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15 minutes, then normal temperature magnetic agitation 48h under anaerobic.By solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH 9 to wash 5 times to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, obtains carbon nano tube surface lysozyme molecular imprinting polymkeric substance.The synthesis of blank molecularly imprinted polymer is identical therewith with elution process, just in building-up process, does not add template molecule N,O-Diacetylmuramidase.
Embodiment 17:
18mg N,O-Diacetylmuramidase is dissolved in the Tris-HCl buffered soln of 30mL pH 9, add 87.5mg N tert butyl acrylamide (being dissolved in 1mL ethanol) and 200mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h; Add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 80mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15 minutes; Add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15 minutes, then normal temperature magnetic agitation 48h under anaerobic.By solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH 9 to wash 5 times to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, obtains carbon nano tube surface lysozyme molecular imprinting polymkeric substance.The synthesis of blank molecularly imprinted polymer is identical therewith with elution process, just in building-up process, does not add template molecule N,O-Diacetylmuramidase.
Embodiment 18:
Be dissolved in the Tris-HCl buffered soln of 30mL pH 9 by 18mg N,O-Diacetylmuramidase, add 87.5mg N tert butyl acrylamide (being dissolved in 1mL ethanol) and 200mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h; Add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 150mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15 minutes; Add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15 minutes, then normal temperature magnetic agitation 48h under anaerobic.By solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH 9 to wash 5 times to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, obtains carbon nano tube surface lysozyme molecular imprinting polymkeric substance.The synthesis of blank molecularly imprinted polymer is identical therewith with elution process, just in building-up process, does not add template molecule N,O-Diacetylmuramidase.
Embodiment 19:
Be dissolved in the Tris-HCl buffered soln of 30mL pH 9 by 18mg N,O-Diacetylmuramidase, add 87.5mg N tert butyl acrylamide (being dissolved in 1mL ethanol) and 200mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h; Add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 200mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15 minutes; Add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15 minutes, then normal temperature magnetic agitation 48h under anaerobic.By solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH 9 to wash 5 times to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, obtains carbon nano tube surface lysozyme molecular imprinting polymkeric substance.The synthesis of blank molecularly imprinted polymer is identical therewith with elution process, just in building-up process, does not add template molecule N,O-Diacetylmuramidase.
Fig. 1 is above-described embodiment 1-11 carbon nano tube surface lysozyme molecular imprinting Macroscopic single crystal schematic diagram.First, N,O-Diacetylmuramidase and function monomer 1-allyl group-3-Methylimidazole phosphofluoric acid form mixture by electrostatic, hydrogen bond and pi-pi accumulation effect; There is free radical polymerization thing between function monomer, linking agent and acrylamide are carbon nano-tube modified to react, thus N,O-Diacetylmuramidase/function monomer mixture is wrapped in carbon nano tube surface; Elute template molecule, carbon nano tube surface leaves imprinted sites N,O-Diacetylmuramidase to specific adsorption ability.
Fig. 2 is above-described embodiment 12-19 carbon nano tube surface lysozyme molecular imprinting Macroscopic single crystal schematic diagram.Compared with embodiment 1-12, except adding the 1-allyl group-3-Methylimidazole phosphofluoric acid of polarity as except function monomer in embodiment 13-20, also add hydrophobic N tert butyl acrylamide as mixing functions monomer.Therefore N,O-Diacetylmuramidase with these two kinds of function monomer compound tenses, action site is more, and reactive force is stronger, and the binding performance of molecular imprinted polymers obtained and imprinting effect are likely stronger.
Fig. 3 be above-described embodiment 1-4 add different content 1-allyl group-3-Methylimidazole phosphofluoric acid function monomer synthesis molecularly imprinted polymer and blank molecularly imprinted polymer to the adsorptive capacity of N,O-Diacetylmuramidase.As seen from the figure, along with the increase of ionic liquid, the adsorptive capacity of blank molecularly imprinted polymer to N,O-Diacetylmuramidase significantly declines, and molecularly imprinted polymer first declines and increases afterwards.When ionic liquid content is 200-350mg (function monomer/template molecule ratio is about 11-19), the adsorptive capacity of blank molecularly imprinted polymer to N,O-Diacetylmuramidase is greater than molecularly imprinted polymer, and when the content of ionic liquid reaches 400mg (function monomer/template molecule ratio is about 22), molecularly imprinted polymer to the adsorptive capacity of N,O-Diacetylmuramidase apparently higher than blank molecularly imprinted polymer, its imprinting factor can reach 2.1, and selective adsorption capacity strengthens.
Fig. 4 is that above-described embodiment 1-2 adds 200 and the molecularly imprinted polymer of 300mg 1-allyl group-3-Methylimidazole phosphofluoric acid function monomer synthesis and the scanning electron microscope (SEM) photograph of blank molecularly imprinted polymer.As can be seen from the figure, there is very big-difference in the surface topography of molecularly imprinted polymer and blank molecularly imprinted polymer, for molecularly imprinted polymer, N,O-Diacetylmuramidase add the dispersiveness improving carbon nanotube, the polymeric coating that carbon nano tube surface is formed is smooth, in obvious reticulated structure, and blank molecularly imprinted polymer, because dispersiveness is not so good, the polymkeric substance that carbon nano tube surface is formed has the phenomenon that hardens.
Fig. 5 is that above-described embodiment 4-11 adds different content N, and the molecularly imprinted polymer of N '-methylene-bisacrylamide linking agent synthesis and blank molecularly imprinted polymer are to the adsorptive capacity of N,O-Diacetylmuramidase.As can be seen from the figure, when the content of linking agent is 60mg, imprinting effect is not had; When the content of linking agent is 80-250mg, molecularly imprinted polymer is to the adsorptive capacity of N,O-Diacetylmuramidase apparently higher than blank molecularly imprinted polymer, and imprinting effect is remarkable; And when linking agent is increased to 300-350mg, imprinting effect is lost.The function of linking agent is fixed up by template molecule/function monomer mixture by radical polymerization, after removing template, forms stable space structure.The amount of linking agent can affect snappiness and the rigidity of molecularly imprinted polymer space structure, when the amount of linking agent is too much, abundant traces site is embedded in the inside of polymkeric substance, and when the amount of linking agent is less, because the flexibility of molecularly imprinted polymer is too large, the imprinted sites formed on its surface is very limited.Therefore, rational cross-linked dosage can make molecularly imprinted polymer have best snappiness, make template protein can smoothly from trace hole wash-out out, rapid diffusion can enter trace hole again, reduce non-specific binding simultaneously, obtain best imprinting effect.In the present invention, best dosage of crosslinking agent is 100mg, and its imprinting factor can reach 4.5.
Fig. 6 is above-described embodiment 6, and 4,7 add 80, the molecularly imprinted polymer of 100 and 150mg N, N '-methylene-bisacrylamide linking agent synthesis and the scanning electron microscope (SEM) photograph of blank molecularly imprinted polymer.As can be seen from the figure, along with the increase of linking agent, the polymeric coating of carbon nano tube surface progressively thickens, when the amount of linking agent reaches 150mg, because the polymeric coating generated is thicker, do not see obvious carbon nanotube pattern, declined to some extent on the contrary compared with when therefore imprinting effect is 100mg with linking agent.
Fig. 7 be above-described embodiment 4,12-15 add different mol ratio N tert butyl acrylamide and 1-allyl group-3-Methylimidazole phosphofluoric acid function monomer synthesis molecularly imprinted polymer and blank molecularly imprinted polymer to the adsorptive capacity of N,O-Diacetylmuramidase.As seen from the figure, when the mol ratio of N tert butyl acrylamide and 1-allyl group-3-Methylimidazole phosphofluoric acid is 50%:50%, the molecularly imprinted polymer imprinting effect of synthesis is best, and imprinting factor is 1.6.In addition, compared with the molecularly imprinted polymer only synthesize as function monomer with ionic liquid, adding of N tert butyl acrylamide, add the interaction in N,O-Diacetylmuramidase between hydrophobic amino acid and function monomer, therefore total adsorptive capacity increase.
Fig. 8 is that above-described embodiment 12-14 adds N tert butyl acrylamide and 1-allyl group-3-Methylimidazole phosphofluoric acid mol ratio is the molecularly imprinted polymer of 20%:80%, 40%:60% and 50%:50% synthesis and the scanning electron microscope (SEM) photograph of blank molecularly imprinted polymer.As we know from the figure, increase the ratio of N tert butyl acrylamide in mixing functions monomer, the molecularly imprinted polymer pattern that carbon nano tube surface is formed obtains larger improvement, when N tert butyl acrylamide accounts for 20%, coatingsurface has certain phenomenon that hardens, when N tert butyl acrylamide is increased to 40% and 50%, present the website structure of porous.On the contrary, the increase of N tert butyl acrylamide can not improve the surface topography of blank molecularly imprinted polymer, and because the dispersiveness of carbon nanotube is bad, the polymer sheet junction phenomena of synthesis is still serious.
Fig. 9 is that above-described embodiment 14,16-19 adds different content N, and the molecularly imprinted polymer of N '-methylene-bisacrylamide linking agent synthesis and blank molecularly imprinted polymer are to the adsorptive capacity of N,O-Diacetylmuramidase.As we know from the figure, when the content of linking agent is 150mg, molecularly imprinted polymer is to the adsorptive capacity of N,O-Diacetylmuramidase apparently higher than blank molecularly imprinted polymer, and imprinting factor is about 2.3.
Figure 10 is that above-described embodiment 14,18-19 adds 100, the molecularly imprinted polymer of 150 and 200mg N, N '-methylene-bisacrylamide linking agent synthesis and the scanning electron microscope (SEM) photograph of blank molecularly imprinted polymer.As we know from the figure, the content of linking agent on the pattern impact of molecularly imprinted polymer significantly, when linking agent is 100, during 150mg, the polymkeric substance that carbon nano tube surface is formed is the reticulated structure of porous, and when linking agent is increased to 200mg, because degree of crosslinking is too large, the polymkeric substance that carbon nano tube surface generates is too thick, can't see the tubular structure of carbon nanotube.When linking agent is 150mg, the distribution of carbon nano tube surface coating is the most even, and reticulated structure is best, and therefore imprinting effect is also best.
Table 2 compares different content N, the molecular imprinting of N '-methylene-bisacrylamide linking agent synthesis and the specific surface area of blank imprinted polymer and the adsorptive capacity to N,O-Diacetylmuramidase thereof.As can be seen from the table, for molecularly imprinted polymer, along with the increase of linking agent, its specific surface area is from 44.48m
2/ g is increased to 105.68m
2/ g, it is the trend of first increases and then decreases to the adsorptive capacity of N,O-Diacetylmuramidase, and on the one hand, when linking agent is less, prepared polymer rigid is inadequate, and can not produce abundant imprinted sites, therefore the imprinting effect of material is not obvious; On the other hand, when linking agent is too much, because the too thick imprinted sites that causes of polymeric coating formed in function monomer/template protein composite surface embedded deeply, reduce the specific adsorption ability of molecularly imprinted polymer on the contrary, the result presented is that absorption total amount and imprinting effect all decline.For blank imprinted polymer, along with the increase of linking agent, its specific surface area is from 48.88m
2/ g is increased to 117.43m
2/ g, it is increase tendency to the adsorptive capacity of N,O-Diacetylmuramidase on the whole, when linking agent is increased to 100-200mg, the specific surface area of molecularly imprinted polymer is less than blank imprinted polymer, but in this is interval molecularly imprinted polymer to the adsorptive capacity of N,O-Diacetylmuramidase higher than blank imprinted polymer, particularly when linking agent is 150mg, imprinting factor reaches 2.3, and imprinting effect is obvious.These results all show that molecular imprinting polymerization is determined by specific adsorption and non-specific adsorption jointly to the adsorptive capacity size of N,O-Diacetylmuramidase, and blank imprinted polymer then depends primarily on nonspecific physical adsorption to the adsorptive capacity of N,O-Diacetylmuramidase.
Specific surface area and the adsorptive capacity of the molecular imprinting that table 2 different content NNMBA linking agent synthesizes and blank imprinted polymer compare
Figure 11 is that the molecularly imprinted polymer that synthesizes of above-described embodiment 4 and embodiment 18 and blank molecularly imprinted polymer are to the adsorption isothermal curve of N,O-Diacetylmuramidase.As we know from the figure, along with Lysozyme in Solution concentration increases, molecularly imprinted polymer and the adsorptive capacity of blank molecularly imprinted polymer to N,O-Diacetylmuramidase also increase thereupon, and molecularly imprinted polymer to the avidity of N,O-Diacetylmuramidase apparently higher than blank molecularly imprinted polymer, this mainly owing to carbon nano tube surface formed imprinted sites N,O-Diacetylmuramidase to specific recognition capability.
Langmuir matching is carried out to the isotherm adsorption data accorded of Figure 11, the results are shown in Table 3.As seen from the table, compared with only having the system of 1-allyl group-3-Methylimidazole phosphofluoric acid function monomer, larger with the loading capacity of the molecularly imprinted polymer of N tert butyl acrylamide and 1-allyl group-3-Methylimidazole phosphofluoric acid mixing functions monomer synthesize, imprinting effect is better.This is mainly because there is multiple action site in the N,O-Diacetylmuramidase surface as imprinted templates, when itself and function monomer compound tense, electronegative, polarity and containing aromatic base amino acid can with 1-allyl group-3-Methylimidazole phosphofluoric acid generation electrostatic, hydrogen bond and pi-pi accumulation effect, hydrophobic amino acid can with N tert butyl acrylamide generation hydrophobic interaction, therefore the reactive force between template protein and function monomer is stronger, the molecularly imprinted polymer loading capacity obtained and imprinting effect better.
The molecularly imprinted polymer of table 3 the present invention synthesis and the Langmuir fitting parameter of blank molecularly imprinted polymer isotherm adsorption data accorded
Figure 12 compares above-described embodiment 18 with the selective adsorption capacity of 1-allyl group-3-Methylimidazole phosphofluoric acid and the N tert butyl acrylamide molecularly imprinted polymer that is mixing functions monomer synthesize to N,O-Diacetylmuramidase and cytochrome C.Result shows, and the imprinting factor of carbon nano tube surface lysozyme molecular imprinting polymkeric substance to N,O-Diacetylmuramidase, cytochrome C of synthesis is respectively 1.73 and 1.27, is 1.36 to the selectivity factor of cytochrome C.Also certain imprinting effect is there is to cytochrome C in molecular imprinting to polymkeric substance, this mainly because the molecular weight of cytochrome C and N,O-Diacetylmuramidase and iso-electric point closely, in the trace hole stayed after causing a small amount of cytochrome C can enter N,O-Diacetylmuramidase template removal.These results show, the molecularly imprinted polymer of the present invention's synthesis has stronger selective adsorption capacity to N,O-Diacetylmuramidase, if when there is the mutual interference albumen such as cytochrome C in absorption system, molecularly imprinted polymer can preferentially selective sorption N,O-Diacetylmuramidase.
Figure 13 compares function monomer, linking agent and the carbon nano tube surface lysozyme molecular imprinting of synthesis and the infrared spectrogram of blank imprinted polymer.As can be seen from the figure, the diffuse reflectance infrared spectroscopy peak of molecular imprinting (d) and blank molecularly imprinted polymer (e) is almost overlap, and the chemical constitution of both explanations is consistent, and N,O-Diacetylmuramidase wash-out is complete.Similarly, it can also be seen that from figure, the infrared spectra of the molecularly imprinted polymer synthesized with ionic liquid (d) or mixing functions monomer (f) is also closely similar, and this can be explained as follows: the characteristic peak of function monomer 1-allyl group-3-Methylimidazole phosphofluoric acid (c) imidazole ring is at 1000 ~ 1750cm
-1with N tert butyl acrylamide (a) and N, N in scope '-methylene-bisacrylamide (b) overlaps, and therefore when participation radical polymerization, in its polymkeric substance, the characteristic peak of imidazole ring is covered.827cm
-1for the characteristic peak of ionic liquid phosphofluoric acid, all not obvious in the molecular imprinting and blank molecularly imprinted polymer of synthesis, this may be because phosphofluoric acid negatively charged ion still retains in the solution after ionic liquid participation polyreaction.
Figure 14 compares the carbon nano tube surface lysozyme molecular imprinting of function monomer and synthesis and the thermogravimetric analysis figure of blank imprinted polymer.As we know from the figure, the weightlessness of 1-allyl group-3-Methylimidazole phosphofluoric acid mainly occurs between 300 ~ 450 DEG C, and the weightlessness of N tert butyl acrylamide mainly occurs between 50 ~ 120 DEG C.Adopt the molecularly imprinted polymer of same procedure synthesis substantially identical with the weightless trend of blank imprinted polymer, illustrate that the polymer thickness that carbon nano tube surface is formed is consistent, molecular imprinting and blank imprinted polymer can not owing to coat-thicknesses to the Recognition Different of N,O-Diacetylmuramidase, and should owing to the generation in trace hole with specific recognition capability.For for the molecular imprinting of ionic liquid or mixing functions monomer synthesize and blank molecularly imprinted polymer, first time is there is weightless when room temperature rises to 100 DEG C, mass loss is respectively 13.24% and about 13.59%, is mainly derived from the evaporation of moisture in polymkeric substance and the decomposition of part N tert butyl acrylamide; Second time occurs between 300 ~ 450 DEG C weightless, mass loss is respectively 48.63% and about 71%, mainly comes from polymkeric substance intermediate ion liquid and N, N ' decomposition of-methylene-bisacrylamide, last residuals mainly carbon nanotube.From figure, we can also learn, compared to the synthetic system taking ionic liquid as function monomer, when function monomer is 1-allyl group-3-Methylimidazole phosphofluoric acid and N, during N '-methylene-bisacrylamide mix monomer, the polymeric coating that carbon nano tube surface is formed is thicker, and this is also the reason that its loading capacity is larger.Generally speaking, the molecularly imprinted polymer of synthesis has good thermostability.
Claims (10)
1. a carbon nano tube surface lysozyme molecular imprinting polymkeric substance, it take N,O-Diacetylmuramidase as template molecule, 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid and N tert butyl acrylamide are mixing functions monomer, acrylamide is carbon nano-tube modified is matrix, the Tris-HCl buffered soln of pH9 is reaction system, molecular imprinted polymer on surface N,O-Diacetylmuramidase to Selective recognition ability obtained under linking agent, initiator and catalyst action.
2. carbon nano tube surface lysozyme molecular imprinting polymkeric substance as claimed in claim 1, is characterized in that: described acrylamide is carbon nano-tube modified, the weight ratio of N,O-Diacetylmuramidase, N tert butyl acrylamide, 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid is followed successively by (20 ~ 40): (15 ~ 25): (30 ~ 120): (160 ~ 400).
3. carbon nano tube surface lysozyme molecular imprinting polymkeric substance as claimed in claim 1, is characterized in that: described acrylamide is carbon nano-tube modified, the weight ratio of N,O-Diacetylmuramidase, N tert butyl acrylamide, 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid is followed successively by 30: 18: (80 ~ 100): (180 ~ 220).
4. carbon nano tube surface lysozyme molecular imprinting polymkeric substance as claimed in claim 1, is characterized in that: described linking agent is N, N '-methylene-bisacrylamide.
5. carbon nano tube surface lysozyme molecular imprinting polymkeric substance as claimed in claim 4, is characterized in that: described linking agent and the carbon nano-tube modified weight ratio of acrylamide are 3.3 ~ 5: 1.
6. carbon nano tube surface lysozyme molecular imprinting polymkeric substance as claimed in claim 1, is characterized in that: described initiator is ammonium persulphate.
7. carbon nano tube surface lysozyme molecular imprinting polymkeric substance as claimed in claim 1, is characterized in that: described catalyzer is N, N, N', N'-Tetramethyl Ethylene Diamine.
8. prepare a method for carbon nano tube surface lysozyme molecular imprinting polymkeric substance, it is characterized in that comprising the following steps:
1) be dissolved in the Tris-HCl buffered soln of 30mL pH9 by 15 ~ 25mg N,O-Diacetylmuramidase, add 30 ~ 120mg N tert butyl acrylamide and 160 ~ 400mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h;
2) add 20 ~ 40mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 60 ~ 200mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15-20 minute;
3) add 20-40mg ammonium persulphate, 50-100 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15-20 minute, then normal temperature magnetic agitation 12-48h under anaerobic;
4) by solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH9 to wash 3-5 time to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, to obtain final product.
9. prepare the method for carbon nano tube surface lysozyme molecular imprinting polymkeric substance as claimed in claim 8, it is characterized in that comprising the following steps:
1) be dissolved in the Tris-HCl buffered soln of 30mL pH9 by 18mg N,O-Diacetylmuramidase, add 80 ~ 100mg N tert butyl acrylamide and 180 ~ 220mg 1-allyl group-3-Methylimidazole Hexfluorophosphate ionic liquid, normal temperature lower magnetic force stirs 2h;
2) add 30mg acrylamide carbon nano-tube modified, ultrasonic disperse is even, adds 100 ~ 150mg N, N '-methylene-bisacrylamide, logical nitrogen deoxygenation 15-20 minute;
3) add 40mg ammonium persulphate, 50 μ L N, N, N', N'-Tetramethyl Ethylene Diamine, continue logical nitrogen deoxygenation 15-20 minute, then normal temperature magnetic agitation 12-48h under anaerobic;
4) by solid-liquid two-phase laminated flow, adopt the Tris-HCl buffered soln of pH9 to wash 3-5 time to solid product, then be eluted to without protein residues with ultrapure water, lyophilize, to obtain final product.
10. prepare the method for carbon nano tube surface lysozyme molecular imprinting polymkeric substance as claimed in claim 8, it is characterized in that: described acrylamide is carbon nano-tube modified to carry out surface chemical modification to carboxylic carbon nano-tube and obtain, caliber≤the 8nm of described carboxylic carbon nano-tube, length is 20 ~ 30 μm, carboxyl-content is 3.86wt%, purity >95%, the method of surface chemical modification is: take 0.6g carboxylic carbon nano-tube, add 60mL thionyl chloride and 10 dimethyl formamides, 70 DEG C of backflow 24h, after solvent evaporate to dryness, continue to add 3g acrylamide and 50mL dimethyl formamide, ultrasonic dissolution 10min, in 45 DEG C of backflow 24h, product ultrapure water is washed till neutrality, 60 DEG C of oven dry.
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