基于拆分型核酸适配体和硫代黄素T荧光“turn-on”模式的腺苷三磷酸检测

A “turn-on” Fluorescent Strategy for the Detection of Adenosine Triphosphate Based on Split Aptamer and Thioflavin T

利用拆分型核酸适配体(Apt)和染料硫代黄素T(ThT),构建一种荧光“turn-on”模式的生物传感策略,用于腺苷三磷酸(ATP)的高灵敏检测。不存在ATP时,两段拆分的适配体Apt-1和Apt-2可分别与ThT结合,形成含G4片层较少的分子内G-四链体,嵌入的ThT分子少,体系的荧光很弱;当存在ATP时,由于Apt与ATP之间具有更高亲和力,可形成由Apt-1、Apt-2和ATP构成的含G4片层较多的分子间G-四链体,嵌入更多ThT分子,荧光显著增强。该方法简便、快速、成本低,具有较高的选择性和实用性。与缓冲溶液中ATP的分析相比,其在稀释血清样品中显示出更好的检测性能,灵敏度可以提高两个数量级。

A simple,rapid,and sensitive detection of adenosine triphosphate(ATP) in complicated systems has potential applications for chemical and biological sensing,bioassays,and food analysis.Herein,a new “turn on” fluorescent strategy was designed to detect ATP based on split aptamer and the fluorescent dye thioflavin T(ThT).Firstly,the intact aptamer of ATP was split into two fragments.Then the split aptamer was optimized by adding DNA sequence containing different number of repeated GGG.The split aptamer with two groups of GGG at the terminus of two aptamer fragments(Apt-1,Apt-2) shows the best performance.In the absence of ATP,intramolecular G-quadruplex containing few G4-quartets was formed between Apt-1(or Apt-2) and ThT,which emits weak fluorescence because few ThT molecules intercalate into the G-quadruplex.In the presence of ATP,intermolecular G-quadruplex containing more G4-quartets was formed between Apt-1,Apt-2 and ATP due to higher affinity and specificity between ATP and the split aptamer.More ThT molecules intercalate into the G-quadruplex and emit much stronger fluorescence.This is a simple,sensitive,label-free and low cost method for the detection of ATP.