Three-way DNA junction structure combined with enzyme-powered cascade amplification for ultrasensitive electrochemiluminescence detection of microRNA via smart DNA walker

In this work, a target-triggered three-way junction (3-WJ) structure was combined with enzyme-powered cascade amplification strategy for ultrasensitive electrochemiluminescence (ECL) detection of microRNA via free-running DNA walker. In the presence of target microRNA-21 (miRNA-21), a three-way junction structure was formed to trigger enzyme-aided multiple DNA amplification, and thus exponentially amplified triggers were generated. Then, the trigger DNA could hybridize with the blocking DNA on the electrode to release walker DNA, which specifically combined with the quantum dots (QDs)-DNA probe, and formed a recognition site for Nt.BbvCI. The movement of DNA walker is powered by the nicking endonuclease that cleaves specific QDs-DNA probes on the track. Thus one walker DNA can automatically cleave multiple QDs probes on the electrode during the movement, resulting in significantly amplified changes of ECL signal, which could be used for the highly sensitive detection of target miRNA-21. Under the optimal conditions, the ECL signal change is linear with the concentration of miRNA-21 in the range from 10(-14) M to 10(-7) M with a detection limit of 1.5 fM. This newly established strategy could achieve rapid, isothermal, and homogeneous signal amplification for specific nucleic acids in complicated biomatrix, which hold great potential for application in early clinical diagnosis.

在这项工作中，一种靶标触发的三向连接（3 - WJ）结构与酶驱动的级联放大策略相结合，通过自由运行的DNA walker实现对微小RNA（microRNA）的超灵敏电化学发光（ECL）检测。在靶标微小RNA - 21（miRNA - 21）存在的情况下，形成一个三向连接结构以触发酶辅助的多重DNA放大，从而产生呈指数增长的触发物。然后，触发DNA可与电极上的阻断DNA杂交以释放walker DNA，walker DNA与量子点（QDs） - DNA探针特异性结合，并为Nt.BbvCI形成一个识别位点。DNA walker的移动由切割轨道上特定QDs - DNA探针的切口内切酶提供动力。因此，一个walker DNA在移动过程中可自动切割电极上的多个QDs探针，导致ECL信号发生显著放大的变化，可用于对靶标miRNA - 21进行高灵敏度检测。在最佳条件下，ECL信号变化在10⁻¹⁴ M到10⁻⁷ M的miRNA - 21浓度范围内呈线性，检测限为1.5 fM。这种新建立的策略能够在复杂的生物基质中对特定核酸实现快速、等温且均一的信号放大，在早期临床诊断中具有巨大的应用潜力。