环境雌激素通用型传感器构建及非酶信号放大方法研究

Environmental estrogens (EEs) are a class of endocrine disrupting compounds (EDCs) that have been frequently found in rivers, soils, atmosphere, and agricultural products. Traditional techniques for EEs detection mainly focused on instrumental analysis methods, although they are very sensitive, the analytical procedure are rather complicated and therefore labor-intensive and time-consuming that are not suitable for rapid and on-site analysis of trace amounts of EEs in environmental samples. In this project, DNA aptamer will be employed as the molecular recognition element to construct a universal biosensor for rapid, simple, and ultrasensitive detection of the model target 17β-estradiol. Strand displacement reaction will be triggered once the specifically binding between 17β-estradiol and its aptamer occurs, which, in turn, releases a universal DNA to activate the enzyme-free signal amplification process. Using G-quadruplex as a signal reporter, experimental results can be observed by the naked eye. The proposed method involves simple operation steps with much improved sensitivity for EEs detection without instrumentation. Replacing the aptamer sequences, the proposed universal platform and modular signal amplification strategy can be used for other EEs detection, such as bisphenol A and diethylstilbestrol. Our designed sensor is an ideal candidate for the development of rapid EEs detection kits. This project is expected to provide a new approach for on-site visual detection of trace amounts of EEs and has important implications in the monitoring and control of EEs pollution.

环境雌激素(EEs)是一类广泛存在于河流、土壤、大气以及农产品的内分泌干扰物。传统的痕量EEs检测技术主要依赖仪器分析，需要繁琐的样品前处理，检测成本高、费时耗力，难以实现大批量样本的现场快速分析。为建立一种简单快速的通用型EEs检测方法，本项目以17β-雌二醇为例，采用核酸适体为分子识别元件，通过17β-雌二醇与核酸适体间的特异性结合发生链置换反应，激活通用型DNA序列，启动DNA自组装介导的非酶信号放大过程，构建高灵敏、通用型的EEs传感器。以G-四聚体为信号报告分子，建立可视化分析平台使检测结果肉眼可见。更换核酸适体序列，验证通用型平台和模式化信号放大方法在其它环境雌激素（双酚A、己烯雌酚等）的适用性。研制环境雌激素检测试剂盒系列产品，为痕量环境雌激素的现场分析提供新的思路和手段，对环境雌激素的监控和治理具有重要意义。

环境雌激素(EEs)是一类广泛存在于河流、土壤、大气以及农产品的内分泌干扰物。传统的痕量EEs检测技术主要依赖仪器分析，需要繁琐的样品前处理，检测成本高、费时耗力，难以实现大批量样本的现场快速分析。本项目建立了一类简单快速的通用型EEs检测方法。以17β-雌二醇为例，采用核酸适体为分子识别元件，通过17β-雌二醇与核酸适体间的特异性结合发生链置换反应，激活通用型DNA序列，启动DNA自组装介导的非酶信号放大过程，构建高灵敏、通用型的EEs传感器。以G-四聚体为信号报告分子，建立可视化分析平台使检测结果肉眼可见。更换核酸适体序列，验证通用型平台和模式化信号放大方法在其它环境雌激素（双酚A、己烯雌酚等）的适用性。构建的17β-雌二醇可视化传感器具有较高的灵敏度，其检测限为1 fM，线性范围为10 fM到10 nM，用于实际样品检测的回收率为88-107%，能满足实际样品检测的需求。同时我们也构建了一种双酚A荧光传感器，结合核酸外切酶III介导的信号扩增以及采用荧光染料SyBr Green I进行检测，可实现免标记操作，具有成本低廉、操作简单的优点。构建的BPA传感器具有很高的灵敏度，对BPA的检测限可达到5 fM，能满足实际样品检测的需求，当用于实际水样检测分析时，具有满意的回收率，为90-118%。我们对构建的通用型传感平台进行了扩展与延伸，构建了一种比色法快速检测新技术。同时对检测物进行了延伸，以黄曲霉酶毒素B1 （AFB1）为目标物，验证了通用型平台的普适性。通过使用胶体金进行显色反应，结合DNA支点介导的链置换反应以及循环DNA自组装技术，实现了AFB1的高灵敏分析，其检测限为2 pM，线性范围为10 pM至1 μM。用于大米样品中加标AFB1的回收率为90-112％。该项目研制的环境雌激素检测新技术和新方法为痕量环境雌激素的现场分析提供新了思路和手段，对环境雌激素的监控和治理具有重要意义。