基于量子点/NBD荧光比率的高灵敏、高通量功能寡糖筛选和构效关系评估体系构建及其应用

项目名称： 基于量子点/NBD荧光比率的高灵敏、高通量功能寡糖筛选和构效关系评估体系构建及其应用

项目编号： No.31201384

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 食品科学、农学基础与作物学

项目作者： 张洪涛

作者单位： 江南大学

项目金额： 25万元

中文摘要： 如何实现功能寡糖的高通量筛选及构效关系评估一直是整个寡糖产业链的核心和难题，目前尚无有效方法。为解决该问题，本课题拟以申请人已掌握的高通量分析"糖- 蛋白"作用关系的拟糖脂糖芯片体系为基础，针对目前因无法实现芯片上糖探针的实时测定以及AF647 做为靶蛋白检测标记时灵敏度相对较低，而使得糖芯片尚不能用于"糖-蛋白"构效关系评估的缺陷，利用已有的葡聚寡糖库和CR3 靶标蛋白，首先将灵敏的NBD荧光标记引入到葡聚寡糖探针，通过分析荧光强度实现芯片上寡糖探针的软测定；然后用检测灵敏度比AF647 高100 倍的量子点作为检测标记来实现CR3 蛋白的高灵敏实时检测；最后通过分析量子点/NBD 荧光值比率从而建立高灵敏、高通量的功能寡糖筛选和构效关系评估体系，同时实现CR3 与葡聚寡糖之间的构效关系评估。本研究对加快我国功能糖的筛选与开发、提升我国在功能寡糖领域的研发水平将起重要促进作用。

中文关键词： 寡糖；结构解析；ESI-CID-MSMS；DC-SIGN；构效关系

英文摘要： Achieving high-throughput screening of the functional oligosaccharides and assessment of their structure-activity relationship are the most important and difficult in the oligosaccharides industry. There is still no effective method to address these problems. In this proposal, a high-throughput analysis of oligosaccharides- protein relationship by the proposed glycolipid sugar chips was used to solve this dilemma. Since the amount of the oligosaccharides on the chip cannot be determined and the sensitivity of AlexFluo 647 towards a target protein is low, we introduce the chemical NBD fluorescent label to the oligosaccharide probes from gluco-oligosaccharides library and CR3 target protein. A soft determination was achieved through fluorescence intensity assay. Due to 100 times higher sensitivity than AlexFluo 647, quantum dots are used to achieve a better detection of the CR3 target protein. Finally, a system for high sensitivity and high-throughput screening of functional oligosaccharides and structure-activity relationship evaluation was established through analyzing the ratio of quantum dot and NBD fluorescence intensity. In addition, the structure-activity relation between CR3 and gluco-oligosaccharides was assessed. This study will play an important role in accelerating the screening and development of fu

英文关键词： Oligosaccharides；Sequence define；ESI-CID-MSMS；DC-SIGN；The relationship of structure and function