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

TY - JOUR

T1 - Duplex-Specific Nuclease-Amplified Detection of MicroRNA Using Compact Quantum Dot–DNA Conjugates

AU - Wang, Ye

AU - Howes, Philip D

AU - Kim, Eunjung

AU - Spicer, Christopher David

AU - Thomas, Michael R

AU - Lin, Yiyang

AU - Crowder, Spencer W

AU - Pence, Isaac J

AU - Stevens, Molly M

N1 - © 2018 American Chemical Society

PY - 2018/8/29

Y1 - 2018/8/29

N2 - 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.

AB - 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.

KW - FRET

KW - biosensing

KW - isothermal amplification

KW - miRNA

KW - quantum dots

KW - target-recycling

UR - http://www.scopus.com/inward/record.url?scp=85052701471&partnerID=8YFLogxK

U2 - 10.1021/acsami.8b07250

DO - 10.1021/acsami.8b07250

M3 - Article

SN - 1944-8244

VL - 10

SP - 28290

EP - 28300

JO - ACS applied materials & interfaces

JF - ACS applied materials & interfaces

IS - 34

ER -