Isothermal and protein-free cascade catalytic hairpin assembly induced-DNAzyme sensing strategy for sensitive miRNA analysis

Sequence-specific microRNAs (miRNAs) represent promising biomarkers for tumor diagnosis, while current detection methods are often limited by stringent temperature requirements and excessive protease dependency. Herein, we present an isothermal, protein-free cascaded amplification circuit for the sensitive analysis of miRNA-21. This strategy, termed CHA-DNAzyme, integrates a miRNA-activated catalytic hairpin assembly (CHA) reaction with a deoxyribozymes (DNAzyme) component. Specifically, the target miRNA-21 initiates the CHA process via a toehold-mediated strand displacement reaction. This reaction unveils a previously sequestered DNAzyme sequence embedded within a hairpin structure, forming an active double-stranded complex. Subsequently, in the presence of Mg²⁺ cofactors, the catalytically activated DNAzyme drives the continuous cleavage of fluorescent reporter hairpins, releasing a measurable fluorescent signal. Implemented in a one-pot format, the CHA-DNAzyme strategy demonstrates excellent sensitivity (with a limit of detection of 8.70 pM), high specificity (the capability to discern single-base mismatches), short sample-to-answer time (approximately 60 min), and robust performance in analyzing serum samples. This approach holds significant potential to facilitate the advancement of miRNA detection tools and support their application in tumor diagnosis and prognosis assessment.