中空多孔SiO2纳米微球的自刻蚀制备及其亲和分离蛋白质研究

Affinity precipitation is one of the most effective methods of purifying proteins, enzymes and other biomolecules. In view of the problem of small ratio surface, limited amount of binding ligand and low adsorption capacity existed in the affinity support of inorganic micro/nano particles, this project using hollow porous SiO2 nanospheres (HPSNSs) as a novel support for affinity precipitation is proposed to improve the separating efficiency of target proteins. It aims to study systematically how SiO2 nanospheres with different structures are converted via hydrothermal treatment into hollow structure, improve the self-etching theory and experimental technique, and tailor the structure and property of HPSNSs..It is also intended to activate the obtained HPSNSs by grafting thiol, amino and carboxyl groups or hydrolyze directly silane coupling agents to produce HPSNSs with active groups, study the orientation and distribution of active groups on the surface of HPSNSs, and tune the kind and concentration of active groups. It would utilize spacer molecules to tune the binding mode and spatial structure of affinity ligands and bond chemically affinity ligands or immobilize metal ions on the surface of HPSNSs. It would study fully the specific adsorption behavior of target proteins on the HPSNSs, relate their structure, surface feature and binding mode of ligands to adsorption capacity, optimize the structure of HPSNSs, and offer fundamental data and the basic of the theory for HPSNSs used as a novel support for affinity precipitation.

亲和沉淀是纯化蛋白质、酶等生物大分子最有效的方法之一。针对当前无机微/纳米微粒亲和载体比表面小、配体结合量有限和吸附容量低等缺点，本项目拟利用中空多孔SiO2纳米微球为亲和沉淀的载体，以提高其对目标蛋白的亲和分离效率。通过研究不同结构SiO2纳米微球的自刻蚀行为，总结自刻蚀理论与实验技术，实现中空多孔SiO2纳米微球结构与性能的有效调控。.在中空多孔SiO2纳米微球表面接枝巯基、氨基、羧基等基团或利用硅烷偶联剂水解制备功能化中空多孔微球，研究表面活性基团的取向与分布规律，调控表面活性基团的种类和浓度；通过引入间臂分子，调节配体结合方式及其空间结构，实现不同亲和配体的有效结合或在微球表面络合上金属离子；研究中空多孔SiO2纳米微球对目标蛋白的亲和吸附行为；建立微球结构、表面特性、配体结合方式与吸附容量的构效关系，优化结构，为中空多孔SiO2纳米微球用作亲和沉淀的载体提供基础数据和理论依据。

本项目以SiO2纳米微球为起始物，利用自刻蚀技术实现了纳米微球中空化转变，完善了自刻蚀制备理论; 通过调控微球结构与表面特性，实现了其对目标蛋白亲和纯化和药物靶向。研究表明，反应条件对所制备SiO2的形貌有很大影响，其中SiO2-GSH微球可用于谷胱甘肽（GST）及GST融合蛋白的分离纯化；水热处理硅烷化SiO2纳米微球可得到中空微球，带有双螯合基团SiO2微球对二价金属离子具有很高的络合能力，且SiO2-SH/IDA-Ni对不同分子量组氨酸(His)融合蛋白均表现出很高的亲和特性；在二价金属离子存在下处理SiO2纳米微球可得到相应的中空MSiO3纳米微球、纳米管及纳米纤维；中空MSiO3纳米微球对His融合蛋白质表现出良好的特异性，且具有很好的再生能力；水热处理具有核/壳结构SiO2纳米微球，可以分别得到摇铃结构和中空结构复合纳米微球，这些微球可实现对His，GST和麦芽糖(MPB)融合蛋白质亲和纯化, 其中Fe3O4/NiSiO3 和Fe3O4/SiO2微球在外磁场作用下可实现目标蛋白的快速分离。多孔羟基磷灰石基吸附剂对His融合蛋白表现出高的亲和特性和普适性。利用乳液与水热相结合的方法，可一步制备中空SiO2纳米微球, 调节溶剂组成和反应温度可以实现中空微球结构从介孔、树枝状孔、双孔结构到中空结构的调控；通过表面功能化可实现微球对目标蛋白亲和纯化、药物响应释放和载体靶向识别。