Synthesis Of Functionalized Polystyrene Nanospheres For Isolation And Purification Of Proteins

As an important biological macromolecule,proteins have been widely applied in many fields such as food,biomedicine and bio-pharmacy due to their excellent biochemical characteristics.Protein separation and purification technology has attracted much attention because it is the premise and basis of protein research.However,the traditional methods for protein separation and purification can only be used to isolate crude protein products,which cannot meet the requirements of modern industrial production for high-purity proteins.In this thesis,styrene was used as a raw material to prepare polystyrene?PS?nanospheres by emulsion polymerization,which was further employed as a platform to synthesize sulfonated polystyrene?SPS?nanospheres,carboxylated polystyrene?PS-COOH?nanospheres,metal chelated polystyrene nanospheres(PS-ANTA-M²⁺)and metal chelated magnetic polystyrene nanospheres(Fe₃O₄@PS-ANTA-M²⁺).These modified PS nanospheres were used to study the separation and purification properties of proteins.It aims to develop a cost-effective and reusable protein separation and purification medium and achieve simple and efficient protein separation and purification process.The main content of this thesis are listed as follows:?1?Synthesis of SPS nanospheres for proteins separation.PS nanospheres were prepared by emulsion polymerization using styrene as a monomer.SPS nanospheres were obtained by the sulfonation of PS nanospheres.SPS nanospheres have uniform particle sizes and homogeneous dispersion.The degree of sulfonation was 2.36±0.08 mmol/g.SPS nanospheres have efficient and fast adsorption properties for proteins.Adsorption kinetics was well discribed by Pseudo-second-order while the adsorption isotherms can be fitted by the Langmuir model.The maximum adsorption capacities of bovine hemoglobin?BHb?protein was 2831.4 mg/g.?2?Synthesis of PS-COOH nanospheres.PS-COOH nanospheres were prepared by emulsion polymerization with styrene and acrylic acid as functional monomers.The prepared PS-COOH nanospheres have uniform particle size and homogeneous dispersion.During the polymerization process,the volume ratio of acrylic acid to styrene was 3:10 m L/m L,the content of carboxyl group on the surface of the nanospheres was determined to be 0.43±0.03 mmol/g.The resulting PS-COOH nanospheres can be well dispersed in aqueous solutions.?3?Synthesis of PS-ANTA-M²⁺nanospheres for separation and purification of proteins.PS-ANTA-M²⁺?M=Ni,Cu,Zn,Co?nanospheres was obtained by using PS-COOH nanospheres as immobilized carrier,grafted with N?,N?-Bis?carboxymethyl?-L-lysine hydrate?ANTA?and chelating different metal ions.PS-ANTA-M²⁺nanospheres have uniform particle size and single dispersion.Histidine?His?-tagged proteins can be specifically isolated from complex biological systems by PS-ANTA-M²⁺nanospheres,and have an excellent specific separation performance within 3 times of recycling.?4?Synthesis of Fe₃O₄@PS-ANTA-M²⁺nanospheres for separation and purification of proteins.Fe₃O₄ magnetic nanoparticles were synthesized by co-precipitation,and added to the emulsion polymerization system to prepare Fe₃O₄@PS-COOH nanospheres,then ANTA was used to chelate different metal ions on the surface of the nanospheres.Fe₃O₄@PS-ANTA-M²⁺nanospheres with Fe₃O₄ nanoparticles core and PS-ANTA-M²⁺shell were obtained.The prepared Fe₃O₄@PS-ANTA-M²⁺nanospheres possess an outstanding magnetic response and re-dispersibility,which is conducive to rapid and efficient separations in the application of protein purification.The adsorption process of BHb can reach the adsorption equilibrium within 24 h,and adsorption kinetics was well discribed by Pseudo-second-order while the adsorption isotherms can be fitted by the Langmuir model.Fe₃O₄@PS-ANTA-M²⁺nanospheres can selectively isolate BHb,but has no adsorption capacity on bovine serum?BSA?.His-tagged proteins can be specifically isolated by Fe₃O₄@PS-ANTA-M²⁺nanospheres from complex biological systems,and the purity of isolated His-tagged proteins was better than that of commercial NTA-Ni column.