Ultrasensitive multiplexed miRNA detection based on a self-priming hairpin-triggered isothermal cascade reaction

We herein describe an ultrasensitive isothermal strategy to detect miRNAs in a multiplexed manner by utilizing a self-priming hairpin-triggered cascade reaction and the adsorption properties of graphene oxide (GO). In principle, a self-priming hairpin probe (SHP) was designed to be opened through binding to the target miRNA and rearranged to serve as a primer. The following extension displaced the target miRNA to be recycled for opening another SHP and produced a double-stranded (ds) SHP with a longer stem region. The nicking enzyme recognition site within the ds SHP was then subjected to continuous repeated nicking and extension reactions, consequently producing a large amount of the trigger sequence. In the second reaction phase, the trigger also transformed another single-stranded (ss) target template probe (TTP) into ds TTP and simultaneously produced numerous target mimic strands (Target') in the same manner, which could activate the first reaction phase, mimicking the target miRNA. Since the ss portions of the two probes were all transformed to the ds forms (ds SHP and ds TTP), they are resistant to the adsorption by graphene oxide (GO) and then emitted intense fluorescence after the application of GO while the ss forms of the two probes produced a negligible fluorescence signal without the target miRNAs. Based on this unique design principle, we were able to simultaneously identify multiple target miRNAs very sensitively down to attomolar levels (42.63 aM for miRNA let-7a, 13.08 aM for miRNA-141, and 10.14 aM for miRNA-98) within 30 min.