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

•Three-way junction structure was applied to highly selective detection of miRNA.•A novel free-running DNA walker was used to the amplified ECL detection of miRNA.•The cascade amplification strategy was applied for ultrasensitive miRNA detection.•A highly sensitive miRNA-21 detection with low limit of 1.5 fM was achieved. 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.