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Duplex specific nuclease-amplified detection of microRNA using compact quantum dot-DNA conjugates

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Title: Duplex specific nuclease-amplified detection of microRNA using compact quantum dot-DNA conjugates
Authors: Wang, Y
Howes, P
Kim, E
Spicer, C
Thomas, M
Lin, Y
Crowder, S
Pence, I
Stevens, MM
Item Type: Journal Article
Abstract: 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 Förster 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.
Issue Date: 29-Aug-2018
Date of Acceptance: 31-Jul-2018
URI: http://hdl.handle.net/10044/1/62689
DOI: https://dx.doi.org/10.1021/acsami.8b07250
ISSN: 1944-8244
Publisher: American Chemical Society
Start Page: 28290
End Page: 28300
Journal / Book Title: ACS Applied Materials and Interfaces
Volume: 10
Issue: 34
Copyright Statement: © 2018 American Chemical Society. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.
Sponsor/Funder: Engineering & Physical Science Research Council (E
Engineering & Physical Science Research Council (EPSRC)
Commission of the European Communities
Funder's Grant Number: EP/K031953/1
EP/K020641/1
ERC-2013-CoG-616417
Keywords: Science & Technology
Technology
Nanoscience & Nanotechnology
Materials Science, Multidisciplinary
Science & Technology - Other Topics
Materials Science
biosensing
miRNA
quantum dots
FRET
target-recycling
isothermal amplification
RESONANCE ENERGY-TRANSFER
SENSITIVE DETECTION
GOLD NANOPARTICLES
MOLECULAR BEACON
HUMAN CANCERS
LABEL-FREE
ONE-STEP
AMPLIFICATION
ASSAY
FLUORESCENCE
0904 Chemical Engineering
0303 Macromolecular And Materials Chemistry
0306 Physical Chemistry (Incl. Structural)
Publication Status: Published
Online Publication Date: 2018-08-16
Appears in Collections:Faculty of Engineering
Materials
Faculty of Natural Sciences



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