Self-Sustaining miRNA Amplification Diagnostics via Catalytic Hairpin Assembly and Split Activator-Initiated...

To meet the growing demand for ultrasensitive diagnostics, representative hybrid platforms integrating nonenzymatic isothermal nucleic acid amplification such as catalytic hairpin assembly (CHA) with CRISPR/Cas systems have been developed. However, two major challenges remain: background leakage from spontaneous hairpin hybridization and inherent fluorescence from conventional ssDNA reporters. Here, we present a self-sustaining isothermal biosensing platform that addresses these limitations by combining CHA with a split activator-initiated CRISPR/Cas12a feedback circuit for the ultrasensitive detection of miRNA-155, a key biomarker of breast cancer. In our design, miRNA-155 initiates CHA to form a DNA duplex, which, along with the miRNA, acts as split activators to trigger CRISPR/Cas12a. Cas12a cleaves a ds-loop DNA reporter, releasing fluorescence and regenerating the target. This dual-recognition mechanism ensures strict target dependence, reduces background noise, and, with the reporter design, minimizes leakage. The released miRNA reactivates CHA, enabling continuous signal amplification through a self-sustaining feedback loop involving successive CHA and Cas12a trans-cleavage cycles, enhancing detection sensitivity. Via these features, the platform achieves attomolar sensitivity and excellent specificity, even distinguishing single-base miRNA variants. Direct detection of endogenous miRNA-155 in serum samples from breast cancer patients demonstrated clear differentiation from healthy controls. This strategy provides a robust molecular detection platform for the accurate and ultrasensitive detection of low-abundance miRNAs in biomedical studies.