Catalytic hairpin assembled tripods combining primer exchange reaction for ultra-sensitive lumbar degenerative disease related MicroRNA detection

Abstract MicroRNAs (miRNAs), a class of small and low-abundance biomarker molecules, present considerable challenges for detection due to their size and limited expression. Catalyzed hairpin assembly (CHA) and primer exchange reaction (PER) represent two advanced isothermal amplification strategies that have been widely employed in miRNA detection. However, current PER-based approaches for miRNA analysis still require improvements in sensitivity and interference resistance. This study introduces a fluorescent biosensor that integrates primer-initiated triple amplification via CHA for the specific detection of miRNA-30d, a biomarker associated with lumbar degenerative disorders. The method employs target-specific recognition to initiate a cascaded signal amplification process: a dumbbell-shaped probe initially blocks the PER template. Upon binding to miRNA-30d, the CHA circuit effectively suppresses non-specific primer binding and erroneous amplification. Subsequently, DNAzyme-mediated cleavage activates the PER dumbbell structure, initiating PER to produce numerous G-quadruplex structures. These structures bind to thioflavin T, yielding a strong fluorescence signal that outperforms conventional methods reliant on costly custom fluorescent probes. This approach is straightforward to implement and demonstrates high sensitivity, achieving a detection limit as low as 56 aM. It significantly enhances the performance and reliability of PER-based sensing platforms and offers a viable strategy for developing highly sensitive and selective nucleic acid detection tools.