CN102114416B - Method for preparing protein molecularly imprinted polymer particles - Google Patents
Method for preparing protein molecularly imprinted polymer particles Download PDFInfo
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- CN102114416B CN102114416B CN 200910248928 CN200910248928A CN102114416B CN 102114416 B CN102114416 B CN 102114416B CN 200910248928 CN200910248928 CN 200910248928 CN 200910248928 A CN200910248928 A CN 200910248928A CN 102114416 B CN102114416 B CN 102114416B
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Abstract
The invention relates to preparation of protein molecularly imprinted polymer particles, in particular relates to a particulate organic polymer synthesized by a protein molecularly imprinted technology. The protein molecularly imprinted polymer particles are prepared through the following steps: preparing a reacting solution; adsorbing a protein solution in the ducts of porous silica gel by oscillation or filtration and other methods; pumping the reacting solution into the ducts of the silica gel through a vacuum pump or other methods; performing a polymerization or agglomeration reaction in the ducts of the silica gel; and corroding silicon spheres after the polymerization or agglomeration reaction is completed so as to obtain the protein molecularly imprinted polymer particles. The invention has the advantages that the preparation method is simple and convenient, and the polymer particles have regular particle diameters and large adsorption capacity to template protein. Compared with other molecularly imprinted polymer particle preparation methods, the method provided by the invention has few preparation steps, and the prepared polymer particles have the characteristics of high adsorption and desorption speed on the template protein as well as good specificity identification to the template protein.
Description
Technical field
The present invention relates to the preparation of protein molecule engram polymer beads, specifically prepare a kind of granular molecularly imprinted polymer, realize specific identification and combination to the template albumen in the solution.
Background technology
At nature, a lot of life processes all relate to molecular recognition, touch reaction etc. such as immune response, ligand-receptor effect and enzyme.Therefore, molecular recognition plays a part important and special in the life evolution process.Molecular imprinting (MIT) then is the manual simulation to this process.Contain the molecularly imprinted polymer (MIP) in manual identified site by preparation, realize the high selectivity identification to a certain specific target molecule.Because the final goal of MIT is realize artificial antibody's molecule synthetic, therefore relates to numerous subjects such as polymer chemistry, material science, biochemistry, host-guest chemistry and supramolecular chemistry, is to have challenging research field.Traditional molecular engram material is prepared into first integral material mostly, sieves by grinding again, obtains particle diameter large (5-100 μ m) and particle in irregular shape.Therefore not only as chromatograph packing material the time post effect lower, and for large molecule, during such as Western blotting, sorption and desorption speed is all very slow.For addressing the above problem, the polymer beads engram technology has obtained people's concern in recent years, the imprinted polymer particle of regular shape, and the mass transfer velocity of energy Effective Raise sample, and can be used as SPE and chromatograph packing material.Recently, the employing microemulsion polymerization methods such as Tong have synthesized the protein molecule engram magnetic-particle, have larger adsorption capacity (Tan, C.J.; Tong, Y.W.Anal.Chem.2007,79,299-306), but preparation process is loaded down with trivial details, and prepared imprinted polymer is selectively relatively poor.Bossi etc. are take the silicon ball as matrix, having prepared HAS is the trace particle of template albumen, the sorption and desorption speed, but need first template albumen covalency to be fixed on silicon ball surface, remove with oxalic acid again after forming imprinted sites, complex steps, and the trace silicon ball adsorption capacity of preparation is lower, selectively also undesirable.(Bonini，F.；Piletsky，S.；Turner，A.P.F.；Speghini，A.；Bossi，A.Biosens.Bioelectron.2007，22，2322-2328)。
Summary of the invention
The object of the present invention is to provide a kind of preparation method of protein-imprinted polymer particle, in the hope of solving template protein wash-out difficulty when preparing protein-imprinted polymer, resulting imprinted material adsorption-desorption speed is slow, and adsorption capacity is little, the shortcoming of poor selectivity.
For achieving the above object, the technical solution used in the present invention is:
Take the organic polymer reaction solution as example: use Methacrylamide, methacrylic acid is function monomer, and the piperazine bisacrylamide is crosslinking agent, and ammonium sulfate and polysorbas20 are auxiliary reagent, and phosphate buffer is that solvent is mixed with reaction solution.Protein solution is adsorbed in the duct of Bio-sil by methods such as vibration or suction filtrations.After in reaction solution, adding initator, in the duct of vavuum pump with reaction solution suction silica gel.Starvation room temperature reaction 24 hours, erode the silicon ball after, obtain the protein molecule engram polymer beads.
Be specially:
1). elute for fear of protein denaturation with the albumen that adsorbs in the silica gel duct, the proposal reactions system is water, and reacting solution pH value is 4-11 (isoelectric point according to protein is determined);
Contained organic solution can not make protein denaturation, volumetric concentration 0-20%; Such as acetonitrile, dimethyl sulfoxide (DMSO); The inorganic salt concentration 0.01-1M of pore-foaming agent.Because kinds of protein is numerous, character is complicated, can be adjusted according to actual conditions.If reaction system is nonaqueous phase, selection principle does not elute the protein that is adsorbed on first Silica Surface again for must guarantee that the reactant liquor solvent can not make protein denaturation simultaneously.The reaction solution viscosity is decided on pore size and reperfusion mode, must guarantee that reaction solution can enter in the silica gel duct.
The pH value of used phosphate buffer is 5-6.5 in this example.The final weight concentration of each component of adding in the phosphate buffer is: template albumen 0.2～1.6%, function monomer 3.0～6.0%, crosslinking agent 5.0～12.0%, auxiliary reagent 2.0～5.0%, the ratio of phosphate buffer and acetonitrile volume is 1: 4～1: 6.
2). can be take the Bio-sil particle of different-grain diameter as matrix, in order to be conducive to protein and reaction solution transporting in the duct, advise channel diameter minimum be 5 times of protein molecule diameters, be preferably protein molecule diameter more than 10 times.The template protein solution is adsorbed in the duct of Bio-sil by methods such as vibration or suction filtrations, the mass ratio of protein and silica gel is 1: 10～1: 20, in reaction solution, add initator, weight concentration is 0.08～0.2%, reaction solution is pumped in the duct of silica gel starvation reaction 24 hours by vavuum pump.Polymerisation finishes post-etching and falls the silicon ball, obtains the protein molecule engram polymer beads.
3). the protein molecule engram polymer beads for preparing washed 24 hours with containing 10% dodecyl sodium sulfate (mass/volume concentration) and the acetic acid mixed solution of 10% (volumetric concentration), repeatedly wash with distilled water at last, until efflux pH value is about 7.Dry preservation under the room temperature.
Advantage of the present invention is: (1) this method preparation method is easy, and step is few; (2) the polymer beads particle diameter of preparation rule; (3) the Western blotting block polymer of preparation has faster mass transfer rate, higher adsorption capacity and better molecular recognition characteristic; (4) the protein molecule engram particulate polymers for preparing can be used as sorbing material and joins in the solution, perhaps comes template albumen in the specific binding solution as the filler of solid-phase extraction column.
Description of drawings
Fig. 1 is the scanning electron microscope (SEM) photograph (* 20000) of the protein molecule engram polymer beads of preparation
Fig. 2 is the HPLC chromatogram of four protein mixed solutions, from left to right is respectively ribonuclease B, cromoci, the chromatographic peak of PSA and beta lactoglobulin.
Fig. 3 is the chromatogram after the mixed solution of four albumen passes through protein molecule engram polymer beads adsorption treatment.Can see that the template albumen (PSA) in the solution is removed by specificity after processing, and very little to the absorption of other protein.This shows that the protein molecule engram polymer beads has preferably selective recognition capability to template molecule.
The specific embodiment
Prepare in accordance with the following steps a kind of protein-imprinted polymer particle.
Embodiment 1
The final weight concentration of each component of adding in the phosphate buffer is: template albumen 0.2～1.6%, function monomer 3.0～6.0%, crosslinking agent 5.0～12.0%, auxiliary reagent 2.0～5.0%, the ratio of phosphate buffer and acetonitrile volume is 1: 4～1: 6.The pH value of phosphate buffer is the interior adjusting of the scope of template albumen isoelectric point ± 2.
The template protein solution is adsorbed in the duct of Bio-sil by methods such as vibration or suction filtrations, the mass ratio of protein and silica gel is 1: 8-1: 20, in reaction solution, add initator and rate accelerating material, weight concentration is 0.08～0.2%, reaction solution is pumped in the duct of silica gel by vavuum pump, starvation reaction 24 hours, heat cause the control temperature below 60 ℃.Polymerisation is used hydrogen fluoride after finishing, and ammonium acid fluoride or sodium hydroxide solution erode the silicon ball, obtain the protein molecule engram polymer beads.
The protein molecule engram polymer beads for preparing washed 24 hours with containing 10% dodecyl sodium sulfate (mass/volume concentration) and the acetic acid mixed solution of 10% (volumetric concentration), repeatedly wash with distilled water at last, until efflux pH value is about 7.Dry preservation under the room temperature.
Template albumen is PSA (PSA).
The synthesis condition of polymer beads is as shown in the table:
Application examples
The protein molecule engram particulate polymers for preparing can be used as sorbing material and joins in the solution, perhaps comes template albumen in the specific binding solution as the filler of solid-phase extraction column.
Fig. 2 is the HPLC chromatogram of four protein mixed solutions, from left to right is respectively ribonuclease B, cromoci, the chromatographic peak of PSA and beta lactoglobulin.
Fig. 3 is the chromatogram after the mixed solution of four albumen passes through protein molecule engram polymer beads adsorption treatment.Can see that the template albumen (PSA) in the solution is removed by specificity after processing, and very little to the absorption of other protein.This shows that the protein molecule engram polymer beads has preferably selective recognition capability to template molecule.
Claims (3)
1. the preparation method of a protein molecule engram polymer beads is characterized in that:
First with protein adsorption in the hole of Bio-sil, then pour into reaction solution, and react, carry out at last the corrosion of silica gel, obtain the protein molecule engram polymer beads;
Described reaction solution is organic polymer soln, and reaction system is water, and reacting solution pH value is 4-11;
The reaction that reaction solution occurs in the silica gel duct is polymerisation or aggregation; Reaction result is to be full of continuous integral material in the silica gel duct;
The Bio-sil that the polymer beads of preparation different-grain diameter is selected different-grain diameter and aperture is as matrix, and the duct of Bio-sil is interconnected; Silica gel channel diameter 〉=5 times protein molecule diameter;
Described protein is the protein solution that any or multiple protein is made into, and protein solution concentration 0.1-5mg/mL, the mass ratio of protein and silica gel are 1: 8-1: 20;
Described protein solution is the phosphate buffer of protein, and the pH value is 4-11, and the protein final weight concentration of adding in the phosphate buffer is 0.2～1.6%;
Described reaction solution adopts the phosphate buffer preparation, and the final weight concentration of each component is: function monomer concentration is 3.0～6.0%, and crosslinking agent piperazine bisacrylamide concentration is 5.0～12.0%,
Function monomer is methacrylic acid and Methacrylamide, and their mass ratio is 1: 15-21; Initator is that the ammonium persulfate weight concentration is 0.01-0.04%, and rate accelerating material is N, N, and N`, N`-tetramethylethylenediamine weight concentration is 0.05-0.2%; Dispersant Tween-20 or acetonitrile weight concentration are 0-20%; The pore-foaming agent ammonium sulfate concentrations is 0.01-1M;
The mol ratio of function monomer and protein consumption is 2000-4000: 1.
2. according to preparation method claimed in claim 1, it is characterized in that: reaction temperature 23-27 ℃, time 14-24 hour.
3. according to preparation method claimed in claim 1, it is characterized in that:
The protein molecule engram polymer beads for preparing washed 24 hours with the acetic acid mixed solution of the dodecyl sodium sulfate that contains mass/volume concentration 10% and volumetric concentration 10%, repeatedly wash with distilled water at last, until efflux pH value is 7, dry preservation under the room temperature;
The protein molecule engram polymer beads for preparing has the ability of with template protein-specific being combined similar with antibody.
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| CN103585979B (en) * | 2013-09-26 | 2016-08-17 | 湖南中烟工业有限责任公司 | A kind of cigarette filter rod polymeric sorbent and its preparation method and application |
| CN104403043B (en) * | 2014-12-12 | 2016-06-22 | 南开大学 | The molecular engram microsphere of discrete group albumen H4-K16 acetylation labeling polypeptide |
| CN108079957B (en) * | 2016-11-21 | 2020-08-11 | 中国科学院大连化学物理研究所 | N-phosphorylated peptide fragment and protein enrichment material, and preparation and application thereof |
| CN111068624B (en) * | 2019-12-27 | 2023-07-21 | 西北工业大学 | Preparation method of molecular imprinting mesoporous material with enlarged aperture |
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| CN101463105A (en) * | 2007-12-21 | 2009-06-24 | 中国科学院大连化学物理研究所 | Protein imprinted material and use thereof for removing albumin from human blood serum |
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Non-Patent Citations (2)
| Title |
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| M.E. Brown et al..Protein binding to peptide-imprinted porous silica scaffolds.《Chemical Engineering Journal》.2008,第137卷参见第98页第2节. * |
| 刘秋叶等.硅胶修饰-表面分子印迹牛血红蛋白及其识别性能的研究.《化学学报》.2008,第66卷(第1期),第57页第1.2节. * |
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