Streptavidin–biotin immobilization enhances functional miRNA hybridization compared with direct EDC/amine coupling on multiplex microbeads

Abstract MicroRNAs (miRNAs) are promising diagnostic and prognostic biomarkers, but direct, amplification-free detection remains challenging because of their short length, sequence similarity, and low abundance. Microbead-based suspension assays enable scalable multiplexing, but assay performance depends on how capture probes are immobilized and presented at the microbead surface. Here, we compared direct carbodiimide (EDC/amine) coupling of amine-modified DNA probes to carboxylated microbeads with streptavidin (SA)–biotin affinity immobilization, in which SA was first covalently attached to the poly(methyl methacrylate) (PMMA) microbeads surface and dual-biotinylated probes were subsequently immobilized by affinity binding. Both workflows were performed under matched hybridization conditions to compare the immobilization methods. Using let-7b and miR-124 as model targets on optically encoded PMMA microbeads, SA-biotin immobilization showed higher apparent probe loading, higher quenching-based hybridization response, more reproducible melting transitions, and slower post-reset fluorescence decay than direct EDC/amine coupling. At a 50 nM target concentration, SA-biotin immobilization achieved approximately 1.5-fold higher quenching-based hybridization response than direct EDC/amine coupling. Because apparent probe loading was not matched between immobilization methods, the relative contributions of probe density and other features of the immobilization strategies could not be fully separated. These results show that immobilization strategy affects functional miRNA hybridization under the tested experimental conditions and that SA-biotin immobilization showed higher functional performance associated with higher apparent probe loading and possibly improved surface accessibility.