核酸适配体-分子印迹协同识别磁性固相微萃取技术及其在复杂生物样品前处理中的应用研究

分子印迹与核酸适配体是国内外广泛研究及应用的分子识别技术，但均存在短板。本项目拟有机结合核酸适配体与分子印迹技术，在Fe3O4磁性纳米粒子表面包裹SiO2涂层，表面改性后将核酸适配体固载于微球表面，加入模板分子与功能单体孵育形成适配体-模板-单体复合物，在水相中自由基聚合制备适配体-分子印迹聚合物@SiO2@Fe3O4磁性微球。分子印迹聚合中引入核酸适配体单元，可产生适配体-功能单体协同识别效应，实现一加一大于二的高选择性；适配体通过端基酰胺反应键合于磁性微球表面，同时其碱基序列中引入胸腺嘧啶核苷不饱和键可参与自由基聚合，实现聚合物骨架结构中多点固载适配体，解决表面固载模式下适配体易酶解导致的稳定性差、寿命短问题，实现两种分子识别技术优势互补，建立核酸适配体-分子印迹协同识别磁性固相微萃取技术，结合液相色谱，应用于复杂样品中赭曲霉素、孔雀石绿等物质高选择性分离富集与检测。

Molecular imprinting and nucleic acid aptamer are extensively studied and applied as the molecule recognition technique at home and abroad, but they all have evident drawbacks. In this project, nucleic acid aptamer and molecular imprinting technique are tightly integrated. The Fe3O4 nanoparticles were synthesized and coated with SiO2 in the surface. Subsequently, the SiO2 coating was chemically modified, and then the aptamer was immobilized chemically onto the surface of SiO2@Fe3O4 nanoparticles. After the incubation with template molecule and functional monomer, an aptamer-template-monomer complex would be obtained. When mixed with cross linker in aqueous solution, the free radical polymerization was initiated to obtain aptamer-molecularly imprinted polymer@SiO2@Fe3O4 magnetic particles. When aptamer was introduced into the molecularly imprinted polymerization, high selectivity of "1+1>2" could be realized through aptamer-monomer synergistic recognition effect. The aptamer was immobilized onto the surface of magnetic microsphere through amide interaction on terminal group, and simultaneously the unsaturated bond of thymidine in aptamer base sequence could participate in the free radical polymerization. As a result, the aptamer could be fixed into the polymer framework through multipoint crosslink immobilization, to solve the weak stability and short lifetime problems of aptamer caused by easy enzymolysis in conventional surface immobilization mode. Finally, a nucleic acid aptamer-molecularly imprinted polymer synergistic recognition based magnetic solid-phase microextraction technique would be established through the complementary advantages integration of two molecule recognition method. When combined with liquid chromatography technology, the technique could be applied for highly selective separation, enrichment and detection of ochracin and malachite green in complicated samples.