Triple cycling integrating with primer exchange reaction (TCiPER) for ultra-sensitive and label-free detection of MicroRNA

Primer exchange reaction (PER), a novel and effective isothermal amplification method, is extensively employed in the development of microRNAs (miRNAs) detection methods. Nevertheless, the PER-based miRNA detection methods necessitate enhanced sensitivity and interference resistance. Here, we propose a fluorescence biosensor, referred to as Triple Cycling integrating with Primer Exchange Reaction (TCiPER), for miRNA detection with straightforward operation, reduced interferences, and significantly increased sensitivity. This method has a high sensitivity with a low limit of detection of 56 aM because the target miRNA recognizes the detection probe and initiates triple signal cycles. PER template (PT) is initially occupied by the hairpin probe (HP), which reduces the possibility of fault initiation of PER by the primer similar sequences, and ii) the produced G-quadruplex sequences combine with thioflavin T (ThT) to produce significant fluorescence, which is superior to traditional fluorescent methods that are developed on the basis of costly and carefully selected fluorescent probes. In conclusion, this strategy substantially enhanced the convenience of sensors fabricated using PER strategies and provided a new perspective for the future development of extensive and sensitive nucleic acid detection analysis tools.