Surface Modification Of Magnetic Affinity Material By Mpeg For Rapid Separation Of Mixed Peptides

In recent years, metal ion affinity chromatography (IMAC) has been widely used because of its advantages such as rapid separation, mild separation conditions, low cost. In order to weaken the interference of high molecular weight protein in the actual system, a new media named restricted-access media has certain advantages for improve the separation of the active small molecules. The main character of restricted-access media was that modification with a layer of semi permeability barrier formation in the surface of media for size exclusion, and combined with the IMAC affinity binding selectivity for the target protein. Therefore, research for preparation with a high load capacity and high selectivity of restricted-access affinity adsorption medium. Details of the experimental results were as follows:(1) Chitosan-microspheres(M-CS) were prepared by emulsion crosslinking method. Microspheres particle size was 25 microns. Activation condition was optimized by series of experiments. When the swelling time was 4h, the activation temperature was 50 ℃, activation time was 6 h, epoxy volume was 50%, concentration of NaOH was 0.4M.The density of epoxy could reach 260μmol·g-1.The M-CS@Cu2+ were preparation through the amino-oxalic acid as a spacer which was cheated with copper ions (Cu2+). Bovine serum albumin(BSA) were choose as molecular protein model. With BSA adsorption quantity as an index, series of experiments were carried out. When buffer ion concentration was 0.02 M, pH= 7, adsorption time was 60 min, ligands density was 140μmol·g-1.The adsorption of BSA (1 mg·mL-1) could reached 117.6 mg·g-1.(2) Polyethylene glycol (mPEG1900) were used for the surface modification, preparation condition was optimized. When using boron trifluoride as catalyst, catalyst dosage (mole ratio) was 1:0.08, reaction time was 6h, reaction temperature was 50 ℃, mPEG medium surface grafting rate could reached 26.46%. Infrared spectrum (FT-IR), scanning electron microscope (SEM), differential calorimeter (DSC) and thermo-gravimetric analyzer (TG) were used for the characterization, proved successful medium surface grafting mPEG1900. Study the limit in the grafting rate of metal ion affinity medium blocking effect of BSA adsorption, When the mPEG1900 grafting rate increase the adsorption of BSA were decrease. When the legend density was 140umol·g-1, mPEG surface grafting rate was 26.46%, rejection rate of BSA reached 50.68%.(3) In order to further explore the application of M-CS@Cu2+ @mPEG 1900.Simulation system and the casein digestion system were researched for applied to the M-CS@Cu2+@mPEG1900. Val-Ser- Leu-Pro-Glu-Try (VW-6) were choose as the model of small molecular peptides, BSA were choose as macromolecular protein molecular model, the simulation system and the casein digestion system were established. Compared with the before and the after modification in hybrid system and casein enzyme hydrolysis system. Results show that the M-CS@Cu2+ @mPEG1900 was improved the adsorption effect of VW-6 obviously than M-CS@Cu2+. Explanation of magnetic limited into the enrichment of small molecular peptides affinity medium could effectively reduce the nonspecific adsorption of large molecules, and improve the efficiency of the separation of active small molecules which provides a feasible method for improve the separation efficiency of small molecules.