Selective electrochemical discrimination of 3' isomiRs differing by two nucleotides

MicroRNAs and isomiRs are promising biomarkers for the detection of diseases such as cancers, but distinguishing the highly similar microRNA sequences between these remains challenging. We report a five-strand four-way junction (5S-4WJ) electrochemical biosensor that combines universal reporter strands with target-specific probes for a modular, low-cost design. The system discriminates miR-146b from its isomiR (R + 2, +2 nt at 3' end) by tuning the probe length, introducing mismatches, and controlling the hybridization kinetics. This strategy yields sevenfold higher sensitivity for R + 2 while maintaining specificity at 10-50 nM. Compared to existing electrochemical biosensors, which struggle to or do not test targets with extra nts at either 5' or 3', the 5S-4WJ sensor enables precise isomiR detection, advancing nucleic acid diagnostics.