识别霉菌毒素的催化活性纳米材料辅助核酸适配体传感器阵列研究

apid, sensitive and accurate detection of mycotoxins is an important prerequisite to safeguard the security of food and feedstuffs. The existing methods for mycotoxins detection and monitoring suffer from the problem of high cost, poor sensitivity, complicated operation and limited targets. Therefore, it is urgent to develop an ultra-sensitive, cost-effective, and high throughput method for rapid detection of multiple mycotoxins. The project has proposed a novel sensor array based on catalytically active nanomaterials assisted aptamers for recognition and detection of mycotoxins in food. A series of aptamers with different length and sequence will be employed as recognition units, while nanomaterials featuring intrinsic catalytical activity will be ultilized as signal transduction units and signal amplifiers in the sensor array. The sensitivity and discrimination ability of the sensor array would be greatly improved by coupling the selectivity of aptamers and the catalytic ability of the nanomaterials. The interaction between aptamers and mycotoxins will be systematically investigated to screen a series of aptamers, which have moderated binding affinities with different mycotoxins. The catalytic activity of the nanomaterials can be effectively modulated by aptamers through changing the aggregation state of the nanoparticles. The recognition events of aptamers on mycotoxins will be effectively correlated with the modification of catalytic activity of the nanomaterials. Characteristic fingerprints for different mycotoxins will be obtained and applied to patten recognition to achieve detection of mycotoxins with high accuracy and sensitivity. The completion of this project will provide a new way to solve the problems of limited specific types of mycotoxin aptamers and screening difficulty. It is expected to be another emerging means of detection of mycotoxins in the field of food security analysis and might be extented to other potential applications.

食品及饲料中霉菌毒素的快速、灵敏且准确的检测是保障其安全的重要前提。现有的霉菌毒素快速检测方法存在成本高、灵敏度较差、检测霉菌毒素种类有限、操作繁琐等问题，急需发展高灵敏、低成本、可实现多种霉菌毒素快速检测的方法。本课题提出一种催化活性纳米材料辅助的核酸适配体传感器阵列，用于食品及饲料中多种霉菌毒素的识别检测。利用一系列具有不同长度不同序列的核酸适配体作为识别单元，具有催化活性的纳米材料作为信号转导单元，通过耦合适配体的分子识别功能与催化纳米材料的高效信号转换和放大功能，提高传感器的选择性和灵敏度。通过核酸适配体可控调节纳米材料的催化活性，建立识别反应与催化活性之间的相关关系，得到不同霉菌毒素的特征指纹图谱，结合模式识别，实现对多种霉菌毒素的准确、高灵敏检测。本项目为解决高特异性霉菌毒素适配体种类有限和筛选困难的问题提供了一种新思路，有望成为霉菌毒素分析的另一种新兴的检测手段。

食品及饲料中霉菌毒素的快速、灵敏且准确的检测是保障其安全的重要前提。现有的霉菌毒素快速检测方法存在成本高、灵敏度较差、检测霉菌毒素种类有限、操作繁琐等问题，急需发展高灵敏、低成本、可实现多种霉菌毒素快速检测的方法。本项目主要研究内容包括四部分：①黄曲霉毒素M1核酸适配体序列优化、作用位点探索、传感器构建；②基于RT-qPCR的高灵敏黄曲霉毒素B1核酸适配体传感器研究；③基于荧光探针的黄曲霉毒素B1核酸适配体传感器研究；④基于 “血糖仪”的便携式赭曲霉毒素A核酸适配体传感器研究。利用霉菌毒素核酸适配体对霉菌毒素进行识别，利用实时定量PCR对霉菌毒素核酸适配体的互补DNA链进行扩增作为信号输出，适配体互补链作为PCR扩增的模板可以大幅度提高检测灵敏度，使得该方法对黄曲霉毒素B1和黄曲霉毒素M1检出限分别可达2.5×10-8 mg/kg和3.0 ×10-5 mg/kg，是迄今为止检测AFB1最灵敏的方法。由于AFM1核酸适配体序列碱基数较多（72 nt），通过分段设计，确定了AFM1适配体与AFM1之间的作用位点，将AFM1核酸适配体序列碱基数优化为36 nt。开发简单、快速的传感器利用带有荧光素的霉菌毒素核酸适配体与带有淬灭基团的互补DNA链构建荧光传感器，检测婴幼儿配方米粉中的黄曲霉毒素B1，方法检出限为1.6 ng/mL。利用磁珠将霉菌毒素适配体固定，并设计与适配体部分互补的DNA链并修饰蔗糖酶，当有霉菌毒素存在的时候，通过核酸适配体特异性识别霉菌毒素，释放生物传感器上结合的修饰有蔗糖酶的互补DNA链到溶液中，将蔗糖水解为葡萄糖，采用血糖仪对生成的葡萄糖进行测定，从而测定样品中霉菌毒素的浓度，该传感器对赭曲霉毒素A的线性范围是1×10-8 mol/L～4×10-6 mol/L，检出限可达6.7×10-9 mol/L (2.69 μg/kg)。本项目为霉菌毒素检测所面临的灵敏度、简便性和便携性的需求提供了解决方案，在检测婴幼儿配方食品中痕量霉菌毒素方面具有很好的应用前景，为保障食品安全提供有效支撑。