Nanopore-Based Electrochemiluminescence for Detection of MicroRNAs via Duplex-Specific Nuclease-Assisted Target Recycling

In this study, we proposed a nanopore-based electrochemiluminescence (ECL) sensor combined with duplex-specific nuclease (DSN)-assisted target recycling amplification to detect microRNAs. Because of the synergetic effect of electrostatic repulsion and volume exclusion of gold nanoparticle-labeled DNA capture (DNA-Au NPs) to the negatively charged luminol anion probe, the DNA-Au NP-modified anodized aluminum oxide (AAO) nanopore electrode exhibited high ECL decline in comparison with the bare AAO electrode. Upon the introduction of DSN and target microRNA, the specific DNA–RNA binding and enzyme cleaving could trigger the detachment of capture DNA from the membrane surface, resulting in uncapping of AAO and an increased ECL signal. For comparison, positively charged Ru(bpy) 3 2+ was used as the ECL probe instead of luminol. Because the electrostatic attraction effect between DNA and Ru(bpy) 3 2+ is partially offset by the volume exclusion effect of Au NPs, the AAO electrode modified with only DNA capture is more suitable for the Ru(bpy) 3 2+ case. In our experiment, the case of negatively charged luminol combined with the synergetic effect of electrostatic repulsion and volume exclusion of DNA-Au NPs provides a quantitative readout proportional to the target microRNA concentration in the range of 1.0 fM to 1.0 nM, with a lower detection limit of 1 fM.