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

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 nanocrystals or quantum dots(QDs) can help address many of these issues. As FRET donors and acceptors, QDs are physio-chemically robust and manifest unique photophysical properties which are cumulatively unavailable to conventional organic dyes in the same role. Except for the new QDs selected, the commercial lined SNAPtides will be changed into the comformational BoNTtides to improve the Km value of enzyme-substrate. Based on QDs and BoNTtides, a new high-sensitivity FRET probe will be designed and constructed. This novel probe-based FRET technology`may reached the attogram limits of detection(LOD) for foodborne botulinum toxins.

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。

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%。然而，现有检测技术在灵敏度、高通量和应急检测等方面存在一些缺欠。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，建立快速分型检测技术。通过优化检测方法中底物浓度、酶浓度、动态检测间隔时间、检测持续时间等系统条件，找到最适反应条件组合以提高检测系统的灵敏度、特异性和稳定性，并利用实验室模拟的食品和临床样本对建立的新型肉毒毒素检测方法进行评价。我们原核表达和纯化了肉毒毒素检测系统中的5种关键蛋白，分别为A/ E型和B型毒素底物蛋白SNAP-25和VAMP-2，以及针对A/ E型和B型毒素底物专一性切割的内肽酶ALc和BLc，并利用基因重组技术设计和构建了一个适用于多型肉毒毒素分析的底物融合蛋白SNVP。利用内肽酶ALc和BLc分别对报告分子CYA和CYB进行活性鉴定，结果显示CYA能被ALc酶解，而不能被BLc酶解；CYB能被BLc酶解，而不能被ALc酶解。将发射波长比(528/485)标准化为CYA的减少与SDS-PAGE分析CYA的直接减少作图，两种方法得到的结果呈线性相关(R2=0.96)。对FRET探针报告分子的一系列浓度检测得到CYA合适范围为0.5-32uM，CYB合适范围为0.2-32uM，合适的动态监测间隔时间和持续时间分别为2min和30-120min。CYA在内肽酶ALc作用下随时间变化与发射波长比率(528/485)作图比值最大时在0.9，最小在0.5，CYB最大时在1.5，最小在0.5。测定ALc切割底物CYA的酶动力学参数Km和kcat分别为2.33±0.21uM和5±0.4s-1，BLc切割底物CYB的Km和kcat值分别为17.6±2.6uM和4.16±0.28s-1。本研究建立的FRET探针检测灵敏度高（LOD为0.1LD50(小鼠)/ml(相当于0.4pg/ml)，LOQ为1.6 LD50(小鼠)/ml(相当于6.4pg/ml)），满足高通量检测要求 (96孔板检测)，对梯度稀释的A、B型肉毒毒素标准品，进行3次以上的重复检测，发现重复性良好(CV<5%)。由于毒素对底物的选择性切割效应，其检测的特异性为100％。该FRET探针不仅可尝试用于食品中痕量肉毒毒素的分型检测，还可用于肉毒毒素抑制剂的体外筛选。