A dual-switch fluorescence biosensor with entropy-driven and DNA walker cascade amplification circuit for sensitive microRNA detection

To achieve precise diagnosis of tumor cells, designing nucleic acid amplification circuits with intelligent multi-switch responsiveness for the specific and sensitive detection of miRNA within tumor cells is an important strategy. Here, we developed a dual-switch fluorescence biosensor that integrates a two-step cascade signal amplification circuit of an entropy-driven circuit (EDC) and DNA walkers onto gold nanoparticles for highly sensitive and quantitative detection of target miRNA in tumor cells. The dual switches that trigger the fluorescence signal are miRNA and the APE1 enzyme, both of which are upregulated in tumor cells. To be specific, the target miRNA21 triggers the upstream EDC, releasing strands that serve as walkers for the downstream circuit. Under the cleavage-driven action of the APE1 enzyme, the walker strands walk along the track strands on the surface of AuNPs, releasing a strong fluorescence signal and presenting good linearity in the target miRNA concentration range of 40 pM-100 nM. This biosensor presents good specificity, strong anti-interference ability against multiple RNAs and enzymes and can effectively distinguish cancer cells from normal cell lysates. Overall, this dual-switch fluorescence biosensor provides a precise recognition and efficient amplification strategy for miRNA detection within tumors, indicating its potential for clinical applications in disease diagnosis.