农药分子识别调控的基于多孔脂质体释放体系的超灵敏适配体传感器研究

项目名称： 农药分子识别调控的基于多孔脂质体释放体系的超灵敏适配体传感器研究

项目编号： No.21507156

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 化学工业

项目作者： 王庆琳

作者单位： 中国科学院苏州纳米技术与纳米仿生研究所

项目金额： 22万元

中文摘要： 面对我国食品安全检测中农药残留引发的问题，本项目拟发展基于多孔脂质体的对靶分子高效率的“有效识别”而释放大量信号分子的信号放大新策略。将采用SELEX技术筛选农药分子噻虫嗪的适配体，与已报道的啶虫脒适配体分别构建基于信号放大新策略的适配体传感器，用于噻虫嗪和啶虫脒的超灵敏检测。基于DNA交联的抗菌肽和环糊精分子分别构建两种多孔脂质体，在孔洞处精确定位修饰含有靶分子适配体的识别体系，通过识别体系可以高效封闭孔洞，通过靶分子与其适配体相互作用来精确调控识别体系开启孔洞，使包埋的信号分子释放出来。本项目在多孔脂质体内装载三类不同的信号分子，实现信号的放大。这项研究有望深入认识以多孔脂质体为载体的高灵敏适配体传感器的构建规律，揭示基于靶分子-适配体识别体系精确调控的脂质体跨膜孔洞开启信号放大新策略的内在机理。这一全新的信号放大策略可成为食品安全中的农药或其它污染物高灵敏检测的通用普适方法。

中文关键词： 农药残留检测；适配体传感器；信号放大；多孔脂质体；适配体筛选技术

英文摘要： Pesticide residue is one of the prominent issues in food safety and environmental pollution, and a rapid, simple, and ultrasensitive detection method for pesticide residues is urgently needed. Acetamiprid and thiamethoxam are chosen as model target molecules for this project. An aptamer targeted to pesticide, acetamiprid, has recently been selected by applying the systematic evolution of ligands by exponential enrichment (SELEX) strategy. In this study, the aptamer targeted to thiamethoxam will be selected by SELEX method. Meanwhile, a new strategy of signal amplification based on target-aptamer recognition triggering the releasing of signal molecules from porous liposomes will be developed. Unlike the conventional porous carrier releasing system, the functionalized arm DNA1 and arm DNA2 can locate precisely on the edge of pores by grafting to the transmembrane molecules, such as peptide or cyclodextrin, through “Click” chemistry approach. The target aptamer will hybridize with arm DNA1 and arm DNA2 to form the sandwich-type DNA structure resulting in the blockage of pores and the inhibition of the release of signal molecules. In the presence of target, the aptamer will be released from the DNA structure due to the interaction of the aptamer and target, leaving the arm DNA1 and arm DNA2 on the surface of porous liposomes. The signal molecules can be released because arm DNA1 and arm DNA2 are flexible so that the pores of liposomes are open. Through this process, the release of signal molecules from this porous system can be triggered by the addition of target. As the proof-of-principle, three kinds of signal molecules for porous liposomes are selected. Calcein is selected as the first kind of signal molecule because of its properties of self-quenching in the liposome and fluorescing in solution. Thioflavine T (ThT) is selected as the second signal molecules to build a“light-up” strategy. ThT is released through triggering by the addition of target, then binds with G-quadruplex already present in solution. The horseradish peroxidase-mimicking DNAzyme was also used to amplify the detection signal. Here, hemin is selected as the third signal molecule to build “double signal amplification” system. Hemin is triggered by the addition of target, then forms DNAzyme by mixing with PS2.M already in solution. The DNAzyme formed by hemin and PS2.M reveals peroxidased-like activities, catalyzing the H2O2-mediated oxidation of ABTS2- to produce the colored ABTS•-. By detecting the absorption intensity of ABTS•-, the amplified detection of acetamiprid can be obtained. The general mechnism of successful design of such a signal amplication strategy based on porous liposome release system will been studied and discussed. The developed construction strategy for such aptasensors for ultrasensitive detection of acetamiprid can be an universal method for the detection of analytes in food safety and other analytical applications.

英文关键词： pesticide residues detection;aptasensors;signal amplification;porous liposomes;SELEX