A Novel Dual Signal Amplification Strategy Integrating APE1-Assisted DNA Walker with Rolling Circle Amplification for Highly Sensitive, Label-Free Exosomal microRNA Analysis

Highly sensitive analysis of exosomal microRNAs (miRNAs) holds significant implications for early cancer diagnosis and prognostic evaluation, yet the low abundance, high homology, and short sequence length of exosomal miRNAs present critical challenges for achieving both sensitivity and specificity in detection. Herein, a dual signal amplification (DSA) strategy integrating an APE1 enzyme-assisted DNA walker with rolling circle amplification (RCA) was developed for highly sensitive, specific, and label-free analysis of exosomal miRNAs. Using miR-10b as a breast cancer proof-of-concept target, the system integrated an APE1 enzyme-assisted DNA walker with rolling circle amplification (RCA). Target miR-10b triggered the APE1-assisted DNA walker immobilized on magnetic beads, generating abundant RCA primer probes. Following magnetic separation, the obtained RCA primers were incubated with the RCA reaction mixture to initiate the RCA reaction, yielding long single-stranded DNA products with copious G-quadruplex (G4). Thioflavin T (ThT) then selectively bound to G4 structures, forming fluorescent G4-ThT complexes that enabled a label-free fluorescence readout. This approach achieved a detection limit of 0.27 fM with exceptional specificity, demonstrating the capacity to discriminate single-nucleotide variants. To validate its applicability, the DSA strategy successfully distinguished miR-10b expression between breast cancer-cell-derived and normal cell-derived exosomes. Furthermore, clinical validation revealed marked differences in serum exosomal miR-10b between breast cancer patients and healthy controls, exhibiting remarkable diagnostic accuracy (AUC = 0.990). This innovative DSA strategy provides a robust platform for ultrasensitive miRNA detection and holds promise for advancing early cancer diagnosis and therapeutic development.