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

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Author(s)

  • Ye Wang
  • Philip D Howes
  • Eunjung Kim
  • Christopher David Spicer
  • Michael R Thomas
  • Yiyang Lin
  • Spencer W Crowder
  • Isaac J Pence
  • Molly M Stevens

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Publication details

JournalACS applied materials & interfaces
DateAccepted/In press - 31 Jul 2018
DatePublished (current) - 16 Aug 2018
Issue number34
Volume10
Number of pages11
Pages (from-to)28290-28300
Original languageEnglish

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.

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© 2018 American Chemical Society

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