Functional Membranes And Magnetic Microspheres For Selective Separation And Enrichment Of Biomolecules

Adsorbents play significant roles in the separation and enrichment of target molecules. Among the various matrixs for the preparation of adsorbents, magnetic nano-adsorbents are the hot areas of research due to their high specific surface area, good biocompatibility and fast adsorption equilibrium as well as the convenient operation of magnetic solid-phase extraction. Membrane separation is an effective method for the large scale purification of bio-macromolecules because the membrane adsorbents possess fast mass transfer, allowing to dealing with huge amount of samples in a short time. However, these two kinds of materials have relatively low adsorption capacities at present, and the selectivity also needs to be improved for different targets. Therefore, in this paper, the newly designed surface modification strategies were applied to anchor high density of functional groups on the surface of the magnetic microspheres and regenerated cellulose membranes to enhance the adsorption capacity and selectivity. The work has made some foundations for the efficient purification and enrichment of active micromolecules and proteins from the biological samples. Mainly including:?1? Selective enrichment and determination of monoamine neurotransmitters ?MNTs? by Cu²⁺ immobilized magnetic solid-phase extraction ?MSPE? coupled with high-performance liquid chromatography-fluorescence detection:Cu²⁺was immobilized on the Fe₃O₄@SiO₂ by iminodiacetic acids as chelators and the selective adsorption of MNTs at pH 5.0 was achieved via the specific coordination interaction between the amino groups of MNTs and the immobilized Cu²⁺. The employed weak acidic extraction condition avoided the oxidation of MNTs, thus facilitated operation and ensured higher recoveries. Under optimal conditions, the MSPE coupled with HPLC/FD for the determination of MNTs in rabbit plasma exhibited high selectivity, sensitivity and re-productivity. Also, the utilization of coordination interaction to improve the selectivity might open another way to selectively enrich small alkaloids from complex samples.?2? Magnetic microspheres with multi-Iigands boronate affinity system mediated by the assembly of polyhedral oligomeric silsesquioxanes ?POSS? and a di-nuclear phenylbronic acid ?d-PBA? for the adsorption of glycoprotein with enhanced capacity and affinity:Magnetic microspheres with multi-ligands boronate affinity system ?Fe₃O₄@SiO₂@p-dPBA? were prepared by the assembly of POSS-8NH₂ and an intra-molecular d-PBA ligand. The Fe₃O₄@SiO₂@p-dPBA exhibited high adsorption capacity for glycoproteins and nucleosides due to the high density of phenylbronic acid groups ?PBA?. More interestingly, the octahedral structure of POSS-8NH₂ makes the PBA molecules "stretch" in the three-dimensional space and each direction has two "neighboring" PBA molecules originated from the intra-molecular d-PBA ligand. The specially designed multi-ligands boronate affinity system benefits the glycoprotein interacts with multiple PBA simultaneously. As a result, the affinity strength towards glycoproteins was greatly enhanced. Finally, the Fe₃O₄@SiO₂@p-dPBA was used to purity glycoproteins from egg white and a high purity was obtained, suggesting that the purposed method could be a potential alternative solution for glycoproteomics.?3? Preparation of boronate affinity magnetic microspheres with high adsorption capacity via surface-initiated atom transfer radical polymerization ?SI-ATRP? for the enrichment of nucleoside in human urine:To further increase the adsorption capacity, SI-ATRP, a living-controlled polymerization technique, was adopted to graft polymer brushes containing PBA from Fe₃O₄@SiO₂, obtaining Fe₃O₄@SiO₂@pPBA microspheres. Owing to the high density of PBA containing polymer brushes, a much higher adsorption capacity was obtained. Therefore, SI-ATRP is a more effective method to improve the adsorption capacity of materials. The Fe₃O₄@SiO₂@pPBA microspheres can effectively enrich nucleosides from human urine with high purity. Therefore, the MSPE coupled with HPLC/UV for the determination of nucleosides in human urine achieved high selectivity, sensitivity and recovery.?4? Preparation of by high-capacity weak cation-exchange ?WCX? membrane via SI-ATRP for the adsorption and purification of Lysozyme:A WCX membrane with high adsorption capacity ?Q_m? was prepared via "post-polymerization modification" method based on the application of SI-ATRP. By varying the duration of SI-ATRP, the Q_m and permeability of the WCX membrane optimized. The new parameters, the utilization percentage of carboxyl and the stoichiometric displacement parameter, were introduced to theoretically investigate the adsorption behavior of Lysozyme on the three dimensional polymer brushes for the first time. At last, the WCX membranes were applied to purify Lysozyme from egg white with high recovery, which depends significantly on the duration of SI-ATRP. Therefore, SI-ATRP is an effective method to improve the purification recovery of protein by adjusting the polymerization time.?6? Preparation of a Zr⁴⁺-immobilized metal affinity membrane ?IMAM? for selective adsorption and purification of phosphoprotein:A novel phosphate-Zr⁴⁺IMAM was prepared based on SI-ATRP, and the Q_m and selectivity were evaluated by comparatively adsorption of standard phosphoproteins and non-phosphoproteins. The results demonstrated that the phosphate-Zr⁴⁺ IMAM had higher Q_m and affinity for phosphoproteins over non-phosphoproteins owing to the application of SI-ATRP in the preparation process and the specific coordination interaction between the phosphate groups on the surface of phosphoprotein and the immobilized Zr+ during adsorption. Finally, the phosphate-Zr⁴⁺ IMAM was applied to purity phosphoprotein from real sample with high purity. Therefore, the as-prepared phosphate-Zr⁴⁺ IMAM could be a promising affinity material for the efficient purification of phosphoprotein from complex bio-samples in the phosphate proteomics research.