An integrated pre-amplified DNA logic gate platform with cascaded amplification for catalytic fluorogenic sensing of prostate cancer miRNAs.

Prostate cancer is one of the most common malignancies worldwide and a leading cause of cancer-related death in men. MicroRNAs (miRNAs) have emerged as promising and highly specific biomarkers for its detection. However, conventional miRNA-based biosensors face challenges of limited specificity, high false positives in single-target assays, and inadequate sensitivity. Here we developed an integrated pre-amplified DNA logic gate platform with cascaded signal amplification (IPL-CAFS-PCmi) for catalytic fluorogenic sensing of prostate cancer miRNAs. The system uses an AND-gate architecture, combining rolling circle amplification (RCA) for pre amplification, hybridization chain reaction (HCR) for signal enhancement and HCR-assembled multi-component nucleic acid enzymes (MNAzymes) for final cleavage-based amplification. The platform achieved detection limits of 2.21 fM for miR-141 and 2.66 fM for miR-375, with excellent specificity in discriminating single-base mismatches. In diluted human serum, recovery rates ranged from 98.51 % to 99.16 %, demonstrating strong robustness. By integrating RCA with HCR-mediated MNAzyme assembly in a programmable DNA logic framework, this design provides multi-level cascade amplification at room temperature, significantly improving both sensitivity and specificity. The platform thus represents a versatile tool with strong potential for precision oncology, clinical diagnostics, and point-of-care testing.