新型适配体功能化电纺丝增强固相微萃取技术及其应用研究

Solid-phase microextraction (SPME) is a very important, simple, efficient and solventless sample preparation method. The project is proposed to develop a novel aptamer-functionalized electrospun nanofiber-based SPME, and to study the ultra-trace, high sensitive and rapid method for separation and analysis of trace disease-related proteins from biological samples. The main research works are focused on as follows: (1) to synthesize polymers with multiple -COOH groups and then to prepare the corresponding SPME probes and membranes by electrospinning technique; (2) to select and produce the corresponding aptamers for recognition of heat shock protein 90α (Hsp90α), myoglobin, lysozyme and thrombin; (3) to prepare aptamer-functionalized electrospun nanofiber-based SPME probes and membranes, and to study their corresponding specific characteristics; (4) to develop novel aptamer-functionalized electrospun nanofiber-based SPME-LC-MS method for rapid and accurate determination trace Hsp90, myoglobin，thrombin and lysozyme in biological samples; (5) to develop on-line mutiple-aptamer founctionalized SPME-LC-MS method for simultaneous analysis of multiple analytes, and to investigate the corresponding thermodynamic and kinetic mathematical model of the proposed SPME method; (6) to separate and analyze trace disease-related proteins in biological samples by using the proposed methods and to monitor some new disease-related biomarkers. The object of the project is to develop aptamer functionalized electrospun nanofiber based SPME methods for providing novel analytical plateforms to analyze trace proteins in biological samples.

固相微萃取技术（SPME）是一项重要的样品前处理技术。本项目拟发展适配体功能化电纺丝增强SPME新技术，研究其超微量、高灵敏分析生物样品中痕量疾病相关蛋白的新方法。主要研究内容有：1)多羧基聚合物的合成及电纺丝法制备SPME纳米纤维丝涂层；2)热休克蛋白90a、肌红蛋白、溶菌酶和凝血酶等痕量疾病相关蛋白所对应的适配体筛选及合成；3)适配体功能化电纺丝增强SPME探针和薄膜的制备及特性研究；4）适配体功能化电纺丝增强SPME-LC-MS联用分析生物样品中痕量蛋白质的新方法；5）建立多组分同时分析的适配体功能化电纺丝增强SPME-LC-MS在线联用新方法，构建新型SPME萃取蛋白质所对应的热力学和动力学数学模型；6）采用构建的新方法分析临床生物样品，检测痕量疾病相关蛋白，发现新的疾病相关蛋白标志物。总体目标是构建适配体功能化电纺丝增强SPME新技术，为研究生物样品中痕量蛋白质组提供新技术平台。

固相微萃取技术（SPME）是一项重要的样品前处理技术。本项目按照研究计划构建了适配体功能化电纺丝增强SPME新技术，研究其超微量、高灵敏分析生物样品中痕量疾病相关蛋白的新方法。主要研究内容有：1）建立了凝血酶前体蛋白、凝血酶、血红蛋白、血清白蛋白、细胞色素C等蛋白质LC-MS/MS分析方法；2）合成了聚马来酸酸-丙烯腈、聚丙烯腈-甲基丙烯酸缩水甘油酯-氨基葡萄糖聚合物、氧化石墨烯接枝棉纤维丝、氧化石墨烯掺杂聚马来酸酸-丙烯腈薄膜和氧化石墨烯掺杂高内相乳液聚合物等多羧基聚合物，进而制备成适配体功能化SPME萃取材料；3）研究了适配体功能化电纺丝增强SPME探针和薄膜的制备及特性研究；4）构建了适配体功能化电纺丝增强SPME-LC-MS/MS联用分析生物样品中痕量蛋白质的新方法；5）构建了适配体功能化石墨烯掺杂电纺丝薄膜SPME-LC-MS/MS联用分析生物样品中痕量蛋白质的新方法；6）构建了适配体功能化石墨烯掺杂高内相乳液聚合物SPME-LC-MS/MS联用分析生物样品中痕量蛋白质的新方法； 7）采用构建的新方法分析临床生物样品，检测痕量疾病相关蛋白，实现相关疾病的检测。总体目标是构建适配体功能化电纺丝增强SPME新技术，为研究生物样品中痕量蛋白质组提供新技术平台。