Enzyme-assisted nanopore sensor for sensitive and multiplexed detection of microRNAs

MicroRNAs (miRNAs) play crucial roles in the pathogenesis of diverse diseases; however, their extremely low abundance in complex biological systems makes highly sensitive and specific multiplexed detection particularly challenging. Here, we report an enzyme-assisted nanopore sensor that enables simultaneous and ultrasensitive detection of multiple miRNAs. Capture DNA (cDNA) immobilized on streptavidin-coated magnetic beads specifically hybridizes with target miRNAs, opening the cDNA hairpin and facilitating the binding of alkaline phosphatase (ALP)-labeled detection DNA to form stable complexes. ALP catalyzes the dephosphorylation of designed phosphorylated peptide probes, inducing conformational and charge changes that yield distinct current blockade signatures in the presence of cucurbit [7]uril within the nanopore. This strategy reduces background interference via magnetic separation and amplifies detection signals through enzymatic reactions, thereby markedly improving sensitivity for low-abundance miRNAs. The sensor achieves a broad dynamic range from 10.0 fM to 10.0 nM across three target miRNAs, with femtomolar-level limits of detection. By integrating molecular separation, enzymatic amplification, and nanopore sensing, this platform provides a robust approach for precise miRNA analysis, offering strong potential for early disease diagnosis.