Hairpin-inserted cross-shaped DNA nanoprobe for ultrasensitive microRNA detection based on built-in target analogue cycle amplification

It remains technically challenging to develop a sensitive assay system to isothermally amplify the signal for miRNA detection because of its low abundance in tested sample, sequence similarities and existence in complex biological environments. In this study, using miRNA-21 as target model, a hairpin-inserted cross-shaped DNA nanoprobe (CP) with four functional arms is constructed for the ultrasensitive detection of miRNA via one-step built-in target analogue (BTA) cycle-mediated signal amplification. BTA is pre-locked in one arm of CP probe and inactive. In the presence of target miRNA, BTA can be unlocked and initiate an isothermal amplification process. Utilizing as-designed CP probe, miRNA-21 can be detected to down to 500 fM, and the linear response range spans over five orders of magnitude. The nonspecific signal is less than 1% upon nontarget miRNAs. CP probe exhibits ∼six times enhancement in resistance to nuclease degradation and no obvious degradation-induced fluorescence change is detected during the assay period. The recovery yield ranges from 98.2～105.5% in FBS solution. Because of the high sensitivity, desirable specificity, strong anti-interference ability and substantial increase in nuclease resistance, CP probe is a promising tool for the detection of miRNAs in a complex biological milieu.