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

Abstract

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) was used as the ECL probe instead of luminol. Because the electrostatic attraction effect between DNA and Ru(bpy) 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) 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.

Electrochemical Biosensor Based on Tetrahedral DNA Nanostructures and G-Quadruplex–Hemin Conformation for the Ultrasensitive Detection of MicroRNA-21 in Serum

“…When incubated with miRNA-21, the TSPs were combined with the target miRNA and shaped DNA−RNA double strands, which could be specifically cleaved by DSN. The released miRNA could then participate in the next cycle, 15,40,41 resulting in significant current reduction and contributing to obtaining satisfactory sensitivity of the established biosensor. EIS measurements were also implemented to provide further information about the preparation of the biosensor.…”

DNA Technology-assisted Signal Amplification Strategies in Electrochemiluminescence Bioanalysis

“…A nanopore-based ECL sensor was also integrated with the DSN-assisted target recycling strategy for miR-107 assay. During the process, the specific target RNA-DNA binding and DSN cleaving detached the AuNPslabeled DNA capture, exposing the nanopore electrode for enhanced ECL signal [75].…”

Nanoporous Anodic Alumina Photonic Crystals for Optical Chemo- and Biosensing: Fundamentals, Advances, and Perspectives

“…SAMs of silanes with hydrophobic terminal groups increase the hydrophobicity of NAA [ 107 ], SAMs of perfluoroalkylchorosilanes reduce the effective pore diameter of NAA for molecular transport [ 108 ], poly(ethylene glycol)silane SAMs improve the biocompatibility of NAA for immunoisolation [ 109 ], and SAMs of mercaptosilanes have been used for sensing applications [ 110 , 111 , 112 , 113 ]. Similar to SAMs of alkanoic and phosphonic acids, SAMs of organosilanes can act as chemical linkers for further immobilization of biomolecules [ 114 , 115 , 116 ], polymers [ 116 ], and nanoparticles [ 117 ]. Furthermore, organosilanes containing different terminal functionalities (e.g., amine, thiol, carboxyl, etc.)…”