Sensitive detection of miRNA-141 using an endogenous dual-switch activated bipedal DNAzyme walker biosensor

In this study, a novel dual-switch fluorescent biosensor based on a bipedal DNAzyme walker was developed for the highly sensitive detection of the tumour biomarker miRNA-141. By exploiting the typically elevated expression levels of miRNA-141 and apurinic/apyrimidinic endonuclease 1 (APE1) in tumour cells, an endogenous dual-gating system was constructed. The presence of both targets was required to initiate an enzyme-free strand displacement reaction, enabling target recycling and the subsequent release of bipedal DNAzyme walker components (D1/D2), which were efficiently isolated using streptavidin-modified magnetic beads. In the presence of Mg²⁺, the activated DNAzyme walker specifically cleaved fluorophore-labelled substrate strands on gold nanoparticles (AuNPs). Through repeated "walking-cleavage" cycles, fluorescence signals were progressively restored and amplified. By integrating AuNPs as carriers and magnetic separation technology, a cascade dual-amplification system based on non-enzymatic strand displacement and bipedal DNAzyme walking was established, markedly enhancing the detection sensitivity. The biosensor exhibited dual-switch-specific activation, effectively suppressing interference from non-target RNAs and other nucleases, and maintained a low background signal. It demonstrated excellent discrimination between tumour cells and normal cells, and showed good anti-interference performance in spiked serum samples. This study provides a new strategy for the development of high-precision tumour biomarker detection, offering potential for trace analysis in complex biological samples.