Ultrasensitive detection of microRNAs based on cascade amplification strategy of RCA-PER and Cas12a

Since microRNAs (miRNAs) serve as markers for early cancer diagnosis, it is crucial to develop a novel biosensor to detect miRNAs quickly, sensitively and selectively. Hence, we developed a fluorescence biosensor based on target miRNA-initiated rolling circle amplification (RCA) to generate RCA products with multiple tandem catalytic hairpin DNA templates that trigger primer exchange reactions (PER) which extend short single-strand DNA (ssDNA) primers into long ssDNA. Subsequently, the long ssDNA activates the <i>trans</i>-cleavage activity of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system to cleave a fluorescent reporter chain, enabling ultrasensitive detection of miRNAs through the output fluorescence signal. The biosensor could quantify miRNA-141 concentrations from 10⁰ to 10⁵ pM, with a detection limit of 94 fM. Therefore, the biosensing strategy proposed in this study offers a robust technique for the clinical diagnosis of miRNA-141.