Loading scaffold-mediated multiple signal cycle for ultrasensitive microRNA analysis with low background signal

Abstract Alterations in microRNA (miRNA) expression profiles play a pivotal role in the initiation and progression of various diseases, including pediatric pneumonia. Consequently, the development of sensitive, specific, and precise methodologies for miRNA detection represents a promising tool for clinical diagnosis and therapeutic intervention. This study proposes a highly sensitive miRNA detection approach utilizing catalytic hairpin assembly (CHA)-mediated probe release through chain extension and cascade signal amplification. The method enables precise discrimination of target miRNAs from other RNA species via a dual-target recognition mechanism. Furthermore, the incorporation of four signal amplification cycles confers substantial amplification efficiency, permitting accurate detection of low-abundance miRNAs. This system employs cascade isothermal amplification, demonstrating a linear response across concentrations from 1 fM to 100 pM and achieving a detection limit of 0.54 fM. The methodology exhibits significant potential for practical application in complex biological samples, providing a novel platform for profiling miRNA expression patterns and elucidating their mechanistic roles in disease pathogenesis.