An amplification-free fluorescence turn-on sensing platform for in situ monitoring miRNA release from live cells

MicroRNAs (miRNAs) are key regulators of numerous physiological and pathological processes and hold substantial promise for disease diagnosis and therapeutic evaluation. Although a variety of quantitative miRNA assays have been reported, none are suitable for real-time monitoring of miRNA release from live cells due to prolonged assay times, insufficient sensitivity, or interference from complex sample matrices. Herein, we report a novel amplification-free assay based on hybridization of a single-stranded DNA (ssDNA) probe to the target miRNA and fluorescence readout of the resulting DNA–RNA heteroduplex using the RNA-selective intercalating dye SYBR Green II. The proposed assay enables quantitative detection of target miRNAs with a detection limit of 9.1pM and a linear fluorescence response across a concentration range of 25 to 200pM (R 2 = 0.999). By leveraging a label-free and amplification-free design, this platform facilitates rapid and sensitive quantification, enabling—for the first time—the in-situ monitoring of miRNA release from live cells. Application of the assay to miRNA-146a revealed distinct, lineage-dependent secretion profiles under chemotherapeutic stress: while malignant MDA-MB-231 cells maintained sustained extracellular miRNA levels, non-malignant HEK-293 cells did not. Furthermore, treatment with paclitaxel and imatinib significantly inhibited miRNA-146a release from MDA-MB-231 cells. These results demonstrate the assay’s potential as a robust tool for real-time, quantitative miRNA analysis in both fundamental biomedical research and clinical diagnostics.