tFNA-boosted strand displacement amplification for sensitive and reliable MicroRNA detection in human serum and live...

MicroRNAs (miRNAs) are critical biomarkers for disease diagnosis, yet their detection remains challenging due to limitations in sensitivity and specificity. To address this, we developed a three-dimensional entropy-driven DNA signal amplifier (3D-EDSA), which integrates a DNA tetrahedron framework with entropy-driven strand displacement reaction (SDR). This design provides precisely organized reaction sites, enhances binding specificity, and leverages entropic gain to improve amplification efficiency and reversibility. Using miR-34a-5p, a key biomarker of non-alcoholic fatty liver disease (NAFLD), as a model target, 3D-EDSA achieved a detection limit of 10 pM and high specificity in clinical samples, effectively distinguishing NAFLD patients from healthy controls. The system exhibited a significantly improved signal-to-noise ratio over conventional SDR method. Furthermore, we applied 3D-EDSA for live-cell imaging, revealing spatial heterogeneity in miR-34a-5p expression between MIHA and LM3 cells, which was corroborated by RT-qPCR. Our work establishes a robust and modular platform for miRNA detection and demonstrates a new paradigm for nucleic acid nanotechnology in clinical diagnostics and live-cell analysis.