Competitive magneto enzyme-linked miRNA assay for quantification of microRNAs in exosomes

MicroRNAs (miRNAs) are non-coding, short RNA sequences involved in gene regulation. Although intracellular molecules, miRNAs are also found in biological fluids, where they serve as promising diagnostic and prognostic biomarkers. In liquid biopsies, miRNAs are protected from RNase activity through association with proteins or as part of the exosomal cargo. Current methods for miRNA detection are technically demanding and resource intensive. Here, we present a competitive magneto enzyme-linked miRNA (ELmiRNA) assay for the direct quantification of miRNAs in exosomes. The assay is based on a DNA scaffold containing two miRNA templates, each hybridized to a complementary double-tagged probe and anchored via a poly(A) tail to oligo(dT)-magnetic particles. Upon incubation with miRNA-containing samples, displacement of the tagged probes occurs, enabling indirect quantification through enzymatic labeling. As proof of concept, miRNA-21 and miRNA-27a, both upregulated in breast cancer, were quantified in MCF-7 and MDA-MB-231 cells and their derived exosomes. The assay achieved limits of detection of 1.5 nM (9.6 pg μL -1 ) and 2.7 nM (16.2 pg μL -1 ) for miRNA-21 and miRNA-27a, respectively, and yielded 10–30 copies per exosome, in agreement with RT-qPCR validation. The ELmiRNA assay combines sensitivity and selectivity with high-throughput analysis using a routine laboratory filter-based microplate reader. This approach represents a simplified, amplification-free alternative to RT-qPCR for miRNA quantification in exosomes, suitable for routine implementation in standard clinical and research laboratories. • Amplification-free magneto enzyme-linked assay for exosomal miRNA quantification • miRNA-21 and miRNA-27a measured in breast cancer cell–derived exosomes • Competitive displacement format on oligo(dT)-magnetic particles using tagged DNA probes • Low-nanomolar limits of detection (1.5 nM miRNA-21; 2.7 nM miRNA-27a) • Simple assay format compatible with routine clinical laboratory workflows