基于核酸适配体/纳米金粒子修饰石英毛细管的在线循环流动式管内固相微萃取技术及其应用研究

In this project, nucleic acid aptamer with high affinity and specificity would be adopted in sample pretreatment technique. A long hollow quartz capillary tube is modified with gold nanoparticles on the inner wall, and then aptamer is coated on the gold nanoparticle surface through Au-S chemical bonding to enhance the bonding strength and density. Consequently, the amount of bonded aptamer and the extraction capacity would be improved. Subsequently, the tube would be connected with a peristaltic pump, a sample cell and a closed circuit to perform the circulation flow in-tube solid-phase microextraction, and the sample extraction efficiency could be enhanced remarkably. Simultaneously, an on-line in-tube solid-phase microextraction device is set up with six-way valves and linked with liquid chromatography, so on-line extraction, desorption, sample injection and washing could be achieved, and fast analysis is realized and sample pretreatment efficiency could be enhanced. The on-line circulation flow in-tube solid-phase microextraction technique, based on nucleic acid aptamer/gold nanoparticle modified quartz capillary tube, would be established and applied to the fast, highly efficient and selective separation, enrichment, and detection of trace or ultra-trace nucleotides such as adenosine and adenosine triphosphatase and alkaloids such as theophylline in complex biological samples.

本项目拟将亲合力高、特异性强的核酸适配体生物识别体系应用于样品前处理技术，在一段长的空心石英毛细管内壁修饰纳米金粒子，通过巯金键将核酸适配体键合至纳米金粒子表面，增强键合强度及密度，提高适配体键合量及萃取容量；该毛细管连接蠕动泵、样品池及闭合管路实现循环流动式管内固相微萃取，提高样品萃取效率；同时通过六通阀搭建管内固相微萃取在线样品前处理装置，与高效液相色谱联用，实现在线萃取、解吸、进样及清洗，加快分析速度，提高工作效率，从而建立基于核酸适配体/纳米金粒子修饰石英毛细管的在线循环流动式管内固相微萃取技术，并应用于复杂生物样品中痕量、超痕量腺苷、三磷酸腺苷、茶碱等核苷酸或生物碱类物质快速、高效、高选择性分离富集与检测。

样品前处理是样品分析过程中的关键步骤。对于复杂样品如血浆、尿液、食品等而言，由于基体干扰的多样性和复杂性，痕量或超痕量待测组分的分离、富集需要高亲和力、高选择性的样品前处理技术。本项目将核酸适配体高亲合力、强选择性的特性与固相微萃取技术结合在一起，开发基于核酸适配体-配体生物识别系统及空心毛细管柱、毛细管整体柱及磁性微球为介质的新型高选择性固相微萃取技术。采用多次化学键合方法将纳米金粒子修饰至石英毛细管内壁，将巯基核酸适配体通过化学键合修饰于纳米金粒子表面，建立基于核酸适配体/纳米金修饰石英毛细管的新型管内固相微萃取技术，实现血浆、尿液中痕量腺苷高效、高选择性分离富集，检出限可达到0.45 ng/mL。通过溶胶凝胶法和点击化学方法在在空心毛细管中搭建多孔硅胶基质和多孔聚合物基质骨架结构，通过化学修饰固载核酸适配体，建立基于核酸适配体/硅胶整体柱及核酸适配体/聚合物整体柱的新型管内固相萃取技术，实现食品中赭曲霉毒素A及环境水样中双酚A高选择性分离富集，赭曲霉素A检出限低至6.7 ng/L，双酚A检出限低至0.50 ng/L。采用四氧化铁磁性纳米粒子作为载体,通过溶胶-凝胶法及反相乳液法分别在磁性粒子表面包裹二氧化硅及壳聚糖壳层，表面化学修饰核酸适配体后，得到核壳结构的SiO2/Fe3O4磁性微球，建立基于适配体-SiO2/Fe3O4磁性微球及适配体修饰壳聚糖/Fe3O4磁性微球的新型固相微萃取技术，实现血浆、尿液中腺苷高选择性分离富集，检出限分别为0.31 ng/mL及0.48 ng/mL。项目研究成果对于复杂生物样品分析及样品前处理技术的发展具有重要意义。