Study On Separation And Enrichment Of Proteins With Magnetic Molecularly Imprinted Nanoparticles

The molecular imprinting technology,combined with magnetic nanoparticles is significant for further research and theoretical study for the separation and purification of proteins.Fe₃O₄@SiO₂-AA was prepared in this study by modifying the groups of O-Si-O and-COOH on the surface of Fe₃O₄ nanoparticles.Bovine hemoglobin?BHb?was chose as the template to synthesized molecularly imprinted magnetic nanoparticles?MIP?on the basis of Fe₃O₄@SiO₂-AA.The MIP was characterized and its adsorption of BHb and the elution properties of BHb were also studied.First of all,the method of chemical precipitation was used to prepare the Fe₃O₄nanoparticles,and the Fe₃O₄ particles were modified by the groups of O-Si-O and-COOH.With methylacrylic acid and itaconic acid as functional monomers,and N'N-MBA as crosslinking agent,using molecular imprinting technology,the best ratio of substrates for the MIPs was obtained when 0.3%Fe₃O₄@SiO₂-AA,0.35%methylacrylic acid,0.125%itaconic acid,and 0.08%N'N-MBA were adopted to synthesize MIPs.Secondly,the magnetic particles were characterized in this study.As shown by the result of Fourier Transform Infrared Spectroscopy?FTIR?,the groups of O-Si-O and-COOH and and the molecularly imprinted polymer layer were successfully modified on the surface of magnetic particles.Transmission electron microscope?TEM?characterization showed that the size of MIP particles is about 20 nm,and the dispersity of MIPs is good.According to thermogravimetric analysis?TGA?,the magnetic content of MIPs is about 84%,with high magnetic induction and good stability.Elemental analysis found that the C content of MIP was 4.06%,and the template molecules in MIP particles were completely eluded.The isoelectric point of MIP,which is about 4.5 was analyzed by Zeta potential.In addition,the influence of different conditions on the process of MIP to absorb BHb was researched in this paper,as well as the mechanism.The kinetic adsorption found that after12 h,the adsorption of MIPs to BHb reached equilibrium,and the maximum adsorption capacity was 116.69 mg/g.The kinetic adsorption process was in accordance with the pseudo-second-order kinetic model.Isothermal adsorption study has found that when the initial concentration of BHb increased to 1.0 mg/mL,the adsorption quantity of MIPs tended to balance,which meant the concentration of 1.0 mg/mL was the best initial concentration of BHb.The isothermal adsorption process of MIPs to BHb can be well described by the Langmuir adsorption model.According to the study of BHb adsorption capacity of MIP particles under different solution conditions,the adsorption capacity of MIPs can reach169.29 mg/g MIPs at p H 6.2,due to the promoting effect of electrostatic attraction.The adsorption capacity of MIPs to BHb was found to be the large when NaCl concentration was low because the structure of proteins was stable in low ionic strength,while the adsorption was inhibited when the NaCl concentration increased because of charge shielding effects and the structural change of BHb.From the results of absorption in urea solutions,hydrogen bonds were proved to be contributing.In SDS solution and urea solution with high concentractions,the absorption amount decreased when the structure of BHb changed.Through the research of the selective and competitive adsorption of BHb by MIPs,it can be found that BHb was the only protein that can be specially absorbed by MIPs while the absorption of other proteins was few.What's more,the eluting effect of different eluent on the BHb from MIPs was studied in this paper.After elution of 12 h,the deionized water showed higher elution rate of 64.23%to BHb.While using NaCl solution with different concentrations,PBS solution of different pH,urea solution of different concentrations as the eluent of BHb,the elution rates were all smaller than that of deionized water.Using SDS solution as eluent,it can be found that when the concentration of SDS reached 1%,the elution rate of BHb molecules on MIPs was close to 100%.From the ultraviolet-visible scans of different eluents,it can be found that in deionized water,NaCl eluents,and PBS eluents,the molecular structure of the eluted BHb was intact.While in the urea solution with high concentrations and SDS eluents,the blue shift of the characteristic absorption peak of BHb happened,which meant the structure of BHb eluted was changed.Last but not least,it was found that after repeated adsorption and elution experiments for five times,the adsorption capacity of MIPs to BHb molecules could still reach more than 110mg/g,which showed excellent reusability and material stability of MIPs.