基于核壳量子点-金纳米颗粒FRET探针的高灵敏度食源性肉毒毒素分型检测新技术研究

项目名称： 基于核壳量子点-金纳米颗粒FRET探针的高灵敏度食源性肉毒毒素分型检测新技术研究

项目编号： No.31201352

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 食品化学残留与控制

项目作者： 王琴

作者单位： 中国人民解放军军事医学科学院

项目金额： 22万元

中文摘要： 肉毒毒素是自然界已知毒性最强的物质，食入毒素污染的食品是肉毒中毒的最主要途径，死亡率高达40%，列食物中毒之首。我国至少有21个省区市曾发生A、B、E型肉毒中毒事件。痕量肉毒毒素即可致人中毒，而现有技术不能满足食源性肉毒毒素痕量检测和快速分型甄别的技术需求。本研究依据毒素内肽酶对底物高度专一性切割和荧光共振能量转移（FRET）的原理，针对传统荧光融合蛋白探针CFP-SNAPtides-YFP检测灵敏度低的缺欠，通过引入核/壳量子点-Au 纳米颗粒替代荧光蛋白，同时将线性底物SNAPtides改进为构象型底物BoNTtides, 创新性地构建新型FRET探针CdSe/ZnS-BoNTtides-AuNPs。利用型特异的增敏FRET探针，通过捕获毒素特异切割底物引起的荧光信号变化，建立快速分型检测技术，实现对食品或临床样品中痕量A、B、E型肉毒毒素的高灵敏度检测。

中文关键词： 肉毒毒素；荧光共振能量转移；荧光报告分子；构象型底物；切割

英文摘要： Botulinum neurotoxins(BoNTs), produced by the bacteria of the genus Clostridium, are the most lethal substances known. Foodborne and clinical botulism accidents had happened at more than 21 cities in China. The "gold-standard" mouse-lethality test is currently the only widely accepted method of confirming BoNT exposure.The majority of the remaining BoNT detection approaches involve either immunological or endopeptidase activity assays, or some combination of the two. However, the mouse bioassay has its own disadvantages for lots of mice killed, long experimental time and low-throughput. While immunoassays and MS detections have demonstrated low pictogram limits of detection(LOD). So Botulism Technologies providing rapid, sensitive, and specific detection of BoNT exposure remain essential for providing timely diagnosis and effective treatments. At previous research, we monitored changes in Fluorescence resonance energy transfer(FRET) upon peptide cleavage. However, the photophysical properties of the organic dyes commonly utilized in most FRET-based assays continue to be problematic. These fluorophores can suffer from pH-sensitivity, low quantum yields, photo bleaching, and chemical degradation which significantly complicate FRET formats and subsequent data analysis. The growing utility of semiconductor nanocryst

英文关键词： botulinum toxin；Fluorescence Resonance Energy Transfer；fluorescent report molecular；comformational substrate；cleavage