为了实现食品中铅(Pb2+)污染的快速检测,本文构建了一种基于Pb2+ 依赖型脱氧核酶(DNAzyme)及荧光共振能量转移(Fluorescence resonance energy transfer,FRET)效应的 “Turn on” 型铅离子检测传感器。荧光猝灭基团BHQ1可以作为FRET受体猝灭EvaGreen的荧光。当Pb2+存在时,底物链的特异性切割反应可以减弱BHQ1对EvaGreen的猝灭效果,BHQ1随底物链的断裂而释放,远离供体EvaGreen,因此FRET效应减弱,供体EvaGreen的荧光得以保留。本文利用Pb2+诱导的FRET效应的减弱及EvaGreen荧光信号的增强可以对铅离子进行定性、定量分析。当脱氧核酶臂长为15 nt - 5 nt、EvaGreen浓度为1?、底物链与酶链的浓度分别为400 nM与100 nM时,探究了该方法的检出限及线性范围。该反应可在室温条件下3 min内完成,同时对铅离子具有高度的特异性,可以区分不同的金属离子,并且成功应用于新鲜鸡蛋及自来水中的铅离子定量分析,加标回收率为86.79 %-113.32 %。本文设计的 “Turn on” 型铅离子检测传感器为一管混的室温反应,无需扩增,反应迅速,为检测食品中的铅污染提供了一些思路。
To achieve rapid detection of lead (Pb2+) pollution in food samples, here, a simple “Turn on” Pb2+ sensor based on DNAzyme and Fluorescence Resonance Energy Transfer (FRET) was established. The quencher BHQ1 worked as the FRET acceptor and quenched the fluorescence of EvaGreen. In the presence of Pb2+, the specific cleavage of the substrates weakened the reaction between EvaGreen and BHQ1, the quencher BHQ1 released as the substrate cleaved and was far away from the FRET donor EvaGreen, so the FRET effect was weakened, the fluorescence of the donor EvaGreen was retained. The Pb2+-induced FRET effect weakening and fluorescence increase of EvaGreen could be used for qualitative and quantitative analysis of Pb2+. When the armlength of the DNAzyme was 15 nt-5 nt, the concentration of EvaGreen, Substrate (GR-B) and DNAzyme were 1 ?, 400 nM and 100 nM, respectively, the limit of detection and linear range were explored. The reaction could be finished within 3 minutes at room temperature and had a high specificity for Pb2+, which could distinguish different metal ions, and it has been successfully applied to the quantitative analysis of Pb2+ in fresh eggs and tap water samples, which showed a satisfactory recovery rate from 86.79 % to 113.32 %. In summary, the proposed “Turn on” sensor was a rapid one-tube test at room temperature, no amplification was required, which might provide some ideas for detecting Pb2+ in food.