Cyclic reverse transcription cooperating self-priming cyclic amplification for sensitive and label-free non-small cell lung cancer related microRNA analysis

Non-small cell lung cancer (NSCLC) is a leading cause of global cancer mortality, with a poor five-year survival rate primarily attributed to late diagnosis and therapeutic resistance. Emerging evidence highlights the critical role of microRNAs (miRNAs) in NSCLC pathogenesis, progression, and prognosis. However, conventional polymerase chain reaction (PCR)-based miRNA detection methods often lack sufficient sensitivity and robustness. This study presents a novel, self-assembled bio-sensing platform for the highly sensitive analysis of circulating NSCLC-associated miRNAs. The platform integrates a target-primed hybridization chain reaction (THSP) with cooperative self-primed circular amplification, utilizing a Variable Hairpin Probe (VHP) biosensor coupled with the SYBR Green I fluorescent dye system. Upon specific recognition of target miRNA-21, the system initiates a cascade amplification, minimizing nonspecific background. The activated reaction generates abundant double-stranded DNA products, which bind SYBR Green I to produce a strong fluorescent signal without requiring expensive labeled probes. This straightforward method achieves a detection limit of 1.33 fM, significantly enhancing the sensitivity and practicality. It offers a promising strategy for developing highly specific nucleic acid analysis tools, advancing non-invasive miRNA detection toward precise and personalized NSCLC management.