Hydrogel-BasedIn Situ DNA Extension Assay for Multiplexedand Rapid Detection of MicroRNA

MicroRNAs (miRNAs) are important biomarkers for liquid biopsy, with extensive applicability to diverse diseases. Among diverse miRNA sensing platforms, graphically encoded hydrogel-based miRNA detection technology is a highly promising diagnostic tool, in terms of sensitivity, specificity, and multiplexing capability. However, the conventional hydrogel-based miRNA detection process suffers from a long assay time (more than 3 h) and redundant assay steps, limiting the practical applicability to actual clinical fields. In this study, we develop a hydrogel-based in situ DNA extension assay for rapid, simple, and multiplexed miRNA detection. Unlike typical hydrogel-based assays, the target hybridization and biotinylation for fluorophore labeling are integrated into a single step via target miRNA-primed DNA extension in hydrogel microparticles. Therefore, multiple microRNA targets can be quantitatively detected within 45 min by two assay steps composed of (1) target capture/biotinylation and (2) fluorophore labeling via streptavidin–biotin interaction. We validate robust sensitivities (down to the low picomolar level) and specificities (single-nucleotide level) by conducting singleplex assays for breast cancer-related miRNA markers (miR-16, miR-92a, and let-7a). Furthermore, multiplexed detection of these miRNA markers is conducted to validate robust multiplexing capacity with negligible nonspecific signal expression. Finally, multiple types of miRNAs in the lysate of breast cancer cells (MCF-7) are successfully detected using the developed assay. We expect the developed hydrogel-based assay can contribute to biomedical and omic fields, enabling high-throughput profiling of multiple miRNAs.