期刊
ACS APPLIED MATERIALS & INTERFACES
卷 10, 期 34, 页码 28290-28300出版社
AMER CHEMICAL SOC
DOI: 10.1021/acsami.8b07250
关键词
biosensing; miRNA; quantum dots; FRET; target-recycling; isothermal amplification
资金
- i-sense EPSRC IRC in Early Warning Sensing Systems for Infectious Disease grant [EP/K031953/1]
- China Scholarship Council
- European Union's Horizon 2020 research and innovation program through the Individual Marie Sklodowska-Curie Fellowship Ampidots [701994]
- National Research Foundation of Korea (NRF) - Ministry of Education [2015R1A6A3A03018919]
- Whitaker International Program, Institute of International Education, United States
- ERC Consolidator grant Naturale CG [616417]
- EPSRC grant Bio-functionalised nanomaterials for ultrasensitive biosensing [EP/K020641/1]
- EPSRC [EP/K031953/1, EP/K020641/1] Funding Source: UKRI
Advances in nanotechnology have provided new opportunities for the design of next-generation nucleic acid biosensors and diagnostics. Indeed, combining advances in functional nanoparticles, DNA nanotechnology, and nuclease-enzyme-based amplification can give rise to new assays with advantageous properties. In this work, we developed a microRNA (miRNA) assay using bright fluorescent quantum dots (QDs), simple DNA probes, and the enzyme duplex-specific nuclease. We employed an isothermal target-recycling mechanism, where a single miRNA target triggers the cleavage of many DNA signal probes. The incorporation of DNA-functionalized QDs enabled a quantitative fluorescent readout, mediated by Forster resonance energy transfer (FRET)-based interaction with the DNA signal probes. Our approach splits the reaction in two, performing the enzyme-mediated amplification and QD-based detection steps separately such that each reaction could be optimized for performance of the active components. Target recycling gave ca. 3 orders of magnitude amplification, yielding highly sensitive detection with a limit of 42 fM (or 1.2 amol) of miR-148, with excellent selectivity versus mismatched sequences and other miRNAs. Furthermore, we used an alternative target (miR-21) and FRET pair for direct and absolute quantification of miR-21 in RNA extracts from human cancer and normal cell lines.
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