多孔聚合物酶反应器及其基于手性毛细管电泳的酶反应动力学研究

In this work, new polymerization technique for obtaining block copolymers will be studied. Meanwhile, enzyme kinetics study based on porous stiuli-resposive polymer enzyme reactor and chiral capillary electrophoresis protocol will be realized with amino acids enantiomers as the substrates. The investigation will include: (1) Controlled/living radical polymerization technique (RAFT and ATRP) will be utilized for preparation of unique block copolymers and stimuli-responsive intelligent polymers; (2) Porous polymer membrane will be formed by breath figure method and spin coating method. The intelligent polymer chains and enzymes will be modified onto the surface of the porous membrane to form micelle. The micelle nanopores will be employed to improve the enzyme efficiency; (3) New chiral ligand exchange capillary electrophoresis system will be constructed for chiral separation with peptides as chiral liagnds and for enzyme kinetics study with amino acids enantiomers as the substrats; (4) Enzyme reaction of real amino acids samples under normal physiological and pathophysiological conditions will be investigated by using the porous polymer based enzyme reactor and chiral capillary electrophoresis assay. This exploited work will provide good performance with the intelligent stimuli-responsive porous polymers based enzyme reactor for deep understanding of life science. More over, this study will expand the application fields of stimuli-responsive polymers and chiral capillary electrophoresis technique in analytical chemistry.

开展新型聚合物的合成制备及基于手性分析的酶反应动力学的研究是目前分析化学的研究热点，但刺激响应智能聚合物基质酶反应器的构建、氨基酸的手性分析及其酶解应用研究急待突破。本申请的研究内容包括：（1）采用可控/活性自由基聚合法制备新型嵌段聚合物及刺激响应聚合物；（2）以静态呼吸图法/旋涂法制备多孔聚合物；将刺激响应聚合物链键合到其表面并修饰氨基酸相关酶，构建新型多孔聚合物基质酶反应器；（3）开展以手性肽为配体的手性配体交换毛细管电泳新方法研究；（4）以氨基酸对映体为酶底物，开展酶反应研究并探索微纳孔限域效应提升酶解效率的机理；进一步开展生理病理条件下氨基酸的酶解应用研究。本项目具有较强的探索性，预期研究若能取得一定进展，必将为手性毛细管电泳技术的深入研究提供新思路，同时也将拓展多孔智能聚合物新材料在生命分析化学及疾病诊疗相关领域中的应用范围。

发展了智能嵌段聚合物的合成制备方法，构建了系列多孔膜型酶反应器；构建了基于寡肽的手性配体交换毛细管电泳（CLE-CE）分析系统，实现了氨基酸对映体的良好分离，探讨了其分离机理；结合CLE-CE分析技术，探索了基于聚合物刺激响应特性的纳米限域效应对氨基酸底物酶解效率的影响；开展了酶反应器的活体分析应用研究并发展了氨基酸酶的抑制剂筛选的CLE-CE新方法，促进了智能聚合物与CE研究领域的交叉发展。发表SCI论文16 篇（11篇IF>5），获授权专利1项，参加国内外学术会议并作邀请报告10次。.具体创新点：① 采用活性可控自由基聚合法，实现了刺激-响应聚合物的可控制备；② 结合酶的化学键合技术，构建了刺激-响应多孔聚合物膜型酶反应器；③ 设计合成了数十种寡肽，发展了基于寡肽配体的CLE-CE分析系统并探索了氨基酸对映体手性分离CLE-CE机理；④ 结合所发展的CLE-CE分析方法及所构建的聚合物多孔膜型酶反应器，通过改变环境条件，实现了对氨基酸底物高效酶解的调控；揭示了纳米孔限域效应是氨基酸酶的酶解效率大幅提升的关键；⑤ 发展了氨基酸酶的抑制剂筛选的CLE-CE新方法，探索了手性氨基酸、氨基酸酶与相关疾病的关系及在活体分析的应用潜力。