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Open AccessArticle

Single-Step FRET-Based Detection of Femtomoles DNA

Department of Chemistry, Virginia Commonwealth University, Richmond, VA 23284, USA
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Author to whom correspondence should be addressed.
Sensors 2019, 19(16), 3495; https://doi.org/10.3390/s19163495
Received: 19 July 2019 / Revised: 2 August 2019 / Accepted: 5 August 2019 / Published: 9 August 2019
(This article belongs to the Section Biosensors)
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Abstract

Sensitive detection of nucleic acids and identification of single nucleotide polymorphism (SNP) is crucial in diagnosis of genetic diseases. Many strategies have been developed for detection and analysis of DNA, including fluorescence, electrical, optical, and mechanical methods. Recent advances in fluorescence resonance energy transfer (FRET)-based sensing have provided a new avenue for sensitive and quantitative detection of various types of biomolecules in simple, rapid, and recyclable platforms. Here, we report single-step FRET-based DNA sensors designed to work via a toehold-mediated strand displacement (TMSD) process, leading to a distinct change in the FRET efficiency upon target binding. Using single-molecule FRET (smFRET), we show that these sensors can be regenerated in situ, and they allow detection of femtomoles DNA without the need for target amplification while still using a dramatically small sample size (fewer than three orders of magnitude compared to the typical sample size of bulk fluorescence). In addition, these single-molecule sensors exhibit a dynamic range of approximately two orders of magnitude. Using one of the sensors, we demonstrate that the single-base mismatch sequence can be discriminated from a fully matched DNA target, showing a high specificity of the method. These sensors with simple and recyclable design, sensitive detection of DNA, and the ability to discriminate single-base mismatch sequences may find applications in quantitative analysis of nucleic acid biomarkers. View Full-Text
Keywords: single molecule; FRET; DNA; sensing; single nucleotide polymorphism (SNP) single molecule; FRET; DNA; sensing; single nucleotide polymorphism (SNP)
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Sapkota, K.; Kaur, A.; Megalathan, A.; Donkoh-Moore, C.; Dhakal, S. Single-Step FRET-Based Detection of Femtomoles DNA. Sensors 2019, 19, 3495.

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