Cascade Signal Amplification-Based Fluorescent Biosensor Utilizing Exonuclease III and Self-Locking DNAzyme Synergy for microRNA Detection

Abstract

MicroRNAs (miRNAs), as pivotal regulators of gene expression, are critically associated with the progression of cancer, neurodegenerative, and cardiovascular diseases. However, their detection remains challenging due to low sensitivity, background noise, and matrix interferences in complex biological samples, hindering clinical applications such as early tumor screening and liquid biopsy. Herein, we present a cascade-amplified fluorescence biosensor driven by the synergistic integration of exonuclease III (Exo III) and self-locking DNAzyme (SLD). This strategy exploits Exo III-mediated cyclic digestion of target-induced DNA/RNA heteroduplexes to release multiple signal conversion chains, while the SLD’s rigid conformational gate suppresses nonspecific catalysis until unlocked by target-specific molecular keys. The activated DNAzyme then drives secondary amplification through iterative cleavage of fluorophore-labeled reporters. This dual-amplification mechanism achieves label-free, isothermal detection of miRNA-155 with a linear range of 50 fM–10 pM and a limit of detection (LOD) of 4.96 fM. Notably, the sensor demonstrates robust specificity in serum matrices, effectively overcoming homologous miRNA cross-reactivity and complex biological interferences. By merging target recycling with conformation-gated catalysis, this platform offers a cost-effective, high-precision solution for miRNA profiling, holding significant promise for point-of-care diagnostics and liquid biopsy-based early cancer detection.