β-环糊精协同过渡金属对黄曲霉毒素的超分子组装和识别研究

The current maximum levels of aflatoxins (AFs) set by developed countries are more and more strict while the events of exceeding standard happened frequently. Development of the high sensitivity, rapid and quantitative detection techniques and kits are effective ways to get rid of technical barriers to trade (TBT) and aflatoxins contamination. The molecular assembly and recognition between cyclodextrins and aflatoxins, include to AFB1, AFB2, AFG1, AFG2 and AFM1, will be researched and discussed in this project while the coordination was confirmed by aflatoxins and the transition metal ion in advance. The inclusion compounds will be characterized by means of fluorescence spectrum, NMR, IR spectroscopy and so on. Based on the chemical shife and other variations of host and guest molecules before and after the inclusion process, the molecular recognition of cyclodextrin will be investigated in the case of molecular size mutual match or approximate match, the recognition rule of cyclodextrin to aflatoxins will be elucidated. The effect of metal ion on the inclusion system stability will be explored. The object of this project is to enhance the fluorenscence intension of AFs in a very short time while ultra-high sensitivity（0.05μg/Kg-0.5μg/Kg）, quantitative（R>0.99）, and rapid detection for aflatoxin can be achieved together. The contradiction of sensitivity and quantitative and rapid detection will be solved. The project will explore and provide foundation data and theory for developing fast test technology and products for aflatoxins quantitative detection.

随着黄曲霉毒素限量标准的不断严格和各种安全事故频发，提高其检测灵敏度，建立快速简易的高灵敏度定量检测技术成为控制AFs的有效途径。本项目拟采用环糊精作为主体分子，引入过渡金属与黄曲霉毒素形成双重客体分子，对AFB1、AFB2、AFG1、AFG2、AFM1进行分子组装和识别研究，并用荧光光谱、核磁共振波谱和红外光谱等手段对所形成的糊精-金属-黄曲霉毒素超分子组装体系进行表征。根据主客体分子在包合前后的化学位移等构象变化，探索了在分子尺寸匹配或近似匹配的情况下环糊精的分子识别特点，总结环糊精对黄曲霉毒素的识别规律，发现过渡金属与三元体系稳定性的关系，实现快速荧光增强，达到高灵敏度快速荧光定量检测。解决复杂食品基质和水相基质中真菌毒素荧光分析灵敏度较低以及灵敏度与准确定量不能兼顾的瓶颈问题，发展快速简易且高灵敏度的黄曲霉毒素定量检测技术，为后期开发高灵敏度快速定量检测技术和产品打下坚实基础。

黄曲霉毒素污染是影响农产品质量与食品安全的最重大问题之一，随着黄曲霉毒素限量标准的不断严格和各种安全事故频发，提高其检测灵敏度，建立快速简易的高灵敏度定量检测技术成为控制AFs的有效途径。本项目采用环糊精作为主体分子，引入过渡金属Hg2+与黄曲霉毒素形成双重客体分子，对AFB1、AFB2、AFG1、AFG2、AFM1进行分子组装和识别研究，分别组装出环糊精-过渡金属-AFs（AFB1、AFG1、AFM1等）三元超分子包合物M-β-CD-Hg-AFB1、HE-β-CD-Hg-AFG1、M-β-CD-Hg-AFM1，并用荧光光谱、紫外吸收光谱、热力学等手段对所形成的糊精-金属-黄曲霉毒素超分子组装体系进行表征。根据主客体分子在包合前后的化学位移等构象变化，环糊精、蛋白等大分子主体分子与小分子毒素客体分子之间的相互作用位点、作用力和热力学性质等，探索了在分子尺寸匹配或近似匹配的情况下超分子体系分子识别特点并进行分子模拟和对接，总结出环糊精等大分子对黄曲霉毒素-Hg二重客体分子的识别规律为黄曲霉毒素和Hg先进行配位反应形成八面体配位化合物，然后1：1与主体分子包合，形成三元超分子化合物，识别过程中配位反应与三元体系稳定性密切相关，且黄曲霉毒素荧光响应值显著增加，而大分子会发生能量共振转移及静态荧光猝灭，荧光增敏程度为AFB1>AFG1>AFM1，双呋喃环上的双键对超分子体系荧光响应值的变化以及黄曲霉毒素与大分子蛋白荧光基团反应的距离起着主要的影响作用。利用超分子识别和荧光增强现象，结合快速分离技术，项目建立了不同的快速简易且高灵敏度的黄曲霉毒素定量检测技术用于检测粮油和乳品中的黄曲霉毒素。AFB1浓度在0.1~40μg/L范围内与体系荧光强度呈线性关系，相关系数R为0.9998，检出限为0.08μg/L，回收率为90%~100%；与国标方法及速测方法比对分析，检测准确度均无显著性差异。AFM1浓度在0.01～2.00µg/L范围内，相关系数R为0.9996，检出限为0.0026μg/L。解决了复杂食品基质和水相基质中真菌毒素荧光分析灵敏度较低以及灵敏度与准确定量不能兼顾的瓶颈问题，为后期开发高灵敏度快速定量检测技术和产品打下坚实基础。