Duplex-Specific Nuclease-Amplified Detection of MicroRNA Using Compact Quantum Dot–DNA Conjugates

摘要: Advances in nanotechnology have provided new opportunities for the design of next-generation nucleic acid biosensors and diagnostics. Indeed, combining advances functional nanoparticles, DNA nanotechnology, nuclease-enzyme-based amplification can give rise to assays with advantageous properties. In this work, we developed a microRNA (miRNA) assay using bright fluorescent quantum dots (QDs), simple probes, enzyme duplex-specific nuclease. We employed an isothermal target-recycling mechanism, where single miRNA target triggers cleavage many signal probes. The incorporation DNA-functionalized QDs enabled quantitative readout, mediated by Forster resonance energy transfer (FRET)-based interaction Our approach splits reaction two, performing enzyme-mediated QD-based detection steps separately such that each could be optimized performance active components. Target recycling gave ca. 3 orders magnitude amplification, yielding highly sensitive limit 42 fM (or 1.2 amol) miR-148, excellent selectivity versus mismatched sequences other miRNAs. Furthermore, used alternative (miR-21) FRET pair direct absolute quantification miR-21 RNA extracts from human cancer normal cell lines.