A label-free fluorescent sensor for miR-21 based on sequentially coupled CHA-HCR dual-stage amplification and synergistic signal regulation by graphene oxide and SYBR Green I.

MicroRNAs (miRNAs) are promising biomarkers for cancer diagnosis, and their sensitive and accurate detection is important for early cancer diagnosis. However, achieving sensitive miRNA detection and logic analysis while maintaining a simple workflow remains challenging. In this study, catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) were sequentially coupled to construct a dual-stage enzyme-free amplification circuit and integrated with a label-free fluorescence reporting strategy based on graphene oxide and SYBR Green I for logical recognition and sensitive detection of miR-21. CHA provides specific target recognition and primary signal amplification, and its product triggers the downstream HCR to generate dsDNA nanowires. The resulting dsDNA provides abundant SYBR Green I binding sites and enables continuous signal enhancement. Molecular simulations further supported the feasibility and kinetic behavior of the sequential cascade pathway. The modular cascade also produced a three-input AND logic response, demonstrating controllable logic operation. Under isothermal, enzyme-free, and label-free conditions, the sensor achieved a limit of detection of 184 pM and distinguished highly similar miRNA sequences at the single-base level. This CHA-HCR system provides a simple and reliable approach to miRNA analysis and shows potential for the convenient, sensitive detection of trace biomarkers.