Highly Sensitive Fluorescence Assay for miRNA Detection: Investigation of the DNA Spacer Effect on the DSN Enzyme Activity toward Magnetic-Bead-Tethered Probes

[Image: see text] Researchers have recently designed various biosensors combining magnetic beads (MBs) and duplex-specific nuclease (DSN) enzyme to detect miRNAs. Yet, the interfacial mechanisms for surface-based hybridization and DSN-assisted target recycling are relatively not well understood. Thus, herein, we developed a highly sensitive and selective fluorescent biosensor to study the phenomenon that occurs on the local microenvironment surrounding the MB-tethered DNA probe via detecting microRNA-21 as a model. Using the above strategy, we investigated the influence of different DNA spacers, base-pair orientations, and surface densities on DSN-assisted target recycling. As a result, we were able to detect as low as 170 aM of miR-21 under the optimized conditions. Moreover, this approach exhibits a high selectivity in a fully matched target compared to a single-base mismatch, allowing the detection of miRNAs in serum with improved recovery. These results are attributed to the synergetic effect between the DSN enzyme activity and the neutral DNA spacer (triethylene glycol: TEG) to improve the miRNA detection’s sensitivity. Finally, our strategy could create new paths for detecting microRNAs since it obliterates the enzyme-mediated cascade reaction used in previous studies, which is more expensive, more time-consuming, less sensitive, and requires double catalytic reactions.