Study On Protein Macromolecularly Imprinted Agarose-based Polymer Microspheres

Protein macromolecularly imprinted agarose-based polymer microspheres (P-Agr MIPMs) were prepared by reversed phase suspension gelation, using salad oil as medium, using egg albumin (EA), bovine serum albumin (BSA) and lysozyme (Lyz) as templates respectively, the conditions of the preparation process including amount of dispersants, ratio of oil to water, agarose concentration, and stirring rate were optimized. The removal solution for removing templates was also optimized. The rebinding performance and its influencing factors of EA-Agr MIPMs were studied. Further more, the rebinding dynamics and the rebinding capacity and the imprinting efficiency (IE) of the P-Agr MIPMs made by different types of protein to objective molecules respectively were compared, then the rebinding dynamics of Lyz-Agr MIPMs in different concentration solution was observed, at last the rebinding competition kinetics of EA-Agr MIPMs and BSA-Agr MIPMs to relevant target molecules was studied. The morphology of the MIPMs was observed by optics microscope. The rebinding properties were tested by a Hitachi UV-1800 spectrophotometer and a DDSJ-308A conductivity meter.The results of experiments showed that the ratio of oil to water, dispersant, agarose concentration and stirring rate were mainly factors influencing the particle size and its distribution of MIPMs. When the ratio of oil to water was 4/1, agarose concentration was 2.5%w/v, and stirring rate was 420 rpm, the average size was 240m and their particle distribution index (PDI) was 0.24. The optimal selection of removal solution was the buffer solution of 0.001mol/L sodium dodecyl sulfonate (SDS) and 0.001mol/L NaCl. All MIPMs with different types of protein had a imprint effect, which was different as the templates was different. In addition, factors such as the type of electrolyte in rebinding solution and temperature could have an influence on IE and rebinding capacity of MIPMs.According to the contrast experiments on rebinding capacity and rebinding dynamics of P-Agr MIPMs with three types of protein imprinted to objective molecules respectively, rebinding competition kinetics of EA-Agr MIPMs and BSA-Agr MIPMs to different relevant target molecules and the results of rebinding thermodynamics of P-Agr MIPMs, we discussed the characteristics of agarose acting as the macromolecular imprinted polymeric matrix and concluded that imprinted cavities played a leading role during the process of rebinding. Further more, according to the analysis of selective rebinding experiments, both EA-Agr MIPMs and BSA-Agr MIPMs exhibited an effect of selective rebinding capacity, for which the separation factors (β) were 1.19 and 1.34 respectively.