Entropy-drive DNA circuit coupled with no-promoter rolling circle transcription to synthesize fluorescent gold nanoclusters for label-free detection of breast cancer biomarkers.

MicroRNAs (miRNAs) serve as critical biomarkers for cancer diagnosis, yet their detection faces challenges by their extremely low absolute abundance in clinical samples. Existing cascade detection strategies based on rolling circle amplification are often limited by nonspecific hybridization caused by sequence interference, which compromises sensing performance. To address this, we developed a novel cascade amplification strategy by integrating an entropy-driven DNA circuit (EDC) with no promoter rolling circle transcription (NP-RCT) to achieve highly sensitive, label-free detection of miRNA. The core innovation of this approach is the design of a single-stranded DNA probe that self-folds into a dumbbell structure, functioning simultaneously as both the EDC product and the transcription template. This dual-role design prevents sequence crossover between the two reaction systems and eliminates promoter dependency. The thymine-rich sequence in the dumbbell loop improves transcription efficiency, and the poly-adenine RNA generated by transcription can directly guide the in-situ synthesis of fluorescent gold nanoclusters. The sensor demonstrates a detection limit of 9.6 pM for miRNA-21 with single-base mismatch specificity. Moreover, it exhibits favorable performance in detecting miRNA-21 within complex biological samples, effectively distinguishing cancer cells from normal cells through the analysis of total RNA extracts. This study provides a new strategy for achieving sensitive and specific detection of miRNAs.