Cascaded and autocatalytic dumbbell probe for exponentially amplified and label-free highly-sensitive fluorescent biosensing of miRNA from cancer cells

• A cascaded and autocatalytic amplification strategy based on a dumbbell probe enables exponentially amplified and label-free fluorescent biosensing of miRNA-21. • The integration of HCR, DNAzyme cleavage, and RCA achieves ultrahigh sensitivity with a detection limit down to 0.155 fM. • Dual binding sites of the dumbbell probe drive exponential G-quadruplex generation, producing strong fluorescence without external labeling. • miRNA-21 has been shown to be monitored in human serum samples and cancer cell lysates. miRNAs are valuable biomarkers for cancer diagnostics, but their low abundance and miRNAs are valuable biomarkers for cancer diagnostics, but their low abundance and sequence similarity pose challenges for reliable detection. The hybridization chain reaction mediated by a DNAzyme has been combined with the rolling circle amplification (HCRD-RCA) to develop an exponentially amplified, label-free, and highly sensitive detection method for miRNA-21. Within this framework, the miRNA-21 target sets off a hybridization chain reaction to produce a DNAzyme that cleaves hairpins and releases S1. Subsequently, S1 induces rolling circle amplification on a dumbbell probe with dual binding sites, whilst regenerating the DNAzyme to establish an autocatalytic cycle. The dual-site dumbbell probe facilitates the exponential generation of G-quadruplex structures, which bind to thioflavin T, resulting in strong fluorescence without the need for labeling. This method achieves an ultralow detection limit of 0.155 fM, excellent mismatch discrimination, and reliable performance in complex matrices. Furthermore, it quantifies miRNA-21 in serum and clearly distinguishes expression differences between MCF-7 and HeLa cells. Overall, this cascaded and autocatalytic platform offers a robust tool for the early detection of cancer and precise analysis of miRNA in biological samples.