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Nucleic Acid Sandwich Hybridization Assay with Quantum Dot-Induced Fluorescence Resonance Energy Transfer for Pathogen Detection
Department of Life Science and Center for Nano Bio-Detection, National Chung-Cheng University, Chia-Yi 62102, Taiwan
* Author to whom correspondence should be addressed.
Received: 22 October 2012; in revised form: 23 November 2012 / Accepted: 30 November 2012 / Published: 4 December 2012
Abstract: This paper reports a nucleic acid sandwich hybridization assay with a quantum dot (QD)-induced fluorescence resonance energy transfer (FRET) reporter system. Two label-free hemagglutinin H5 sequences (60-mer DNA and 630-nt cDNA fragment) of avian influenza viruses were used as the targets in this work. Two oligonucleotides (16 mers and 18 mers) that specifically recognize two separate but neighboring regions of the H5 sequences were served as the capturing and reporter probes, respectively. The capturing probe was conjugated to QD655 (donor) in a molar ratio of 10:1 (probe-to-QD), and the reporter probe was labeled with Alexa Fluor 660 dye (acceptor) during synthesis. The sandwich hybridization assay was done in a 20 μL transparent, adhesive frame-confined microchamber on a disposable, temperature-adjustable indium tin oxide (ITO) glass slide. The FRET signal in response to the sandwich hybridization was monitored by a homemade optical sensor comprising a single 400 nm UV light-emitting diode (LED), optical fibers, and a miniature 16-bit spectrophotometer. The target with a concentration ranging from 0.5 nM to 1 μM was successfully correlated with both QD emission decrease at 653 nm and dye emission increase at 690 nm. To sum up, this work is beneficial for developing a portable QD-based nucleic acid sensor for on-site pathogen detection.
Keywords: Alexa Fluor 660; avian influenza virus H5N1; fluorescence resonance energy transfer (FRET); indium tin oxide (ITO); quantum dot 655 (QD655); sandwich hybridization
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Cite This Article
MDPI and ACS Style
Chou, C.-C.; Huang, Y.-H. Nucleic Acid Sandwich Hybridization Assay with Quantum Dot-Induced Fluorescence Resonance Energy Transfer for Pathogen Detection. Sensors 2012, 12, 16660-16672.
Chou C-C, Huang Y-H. Nucleic Acid Sandwich Hybridization Assay with Quantum Dot-Induced Fluorescence Resonance Energy Transfer for Pathogen Detection. Sensors. 2012; 12(12):16660-16672.
Chou, Cheng-Chung; Huang, Yi-Han. 2012. "Nucleic Acid Sandwich Hybridization Assay with Quantum Dot-Induced Fluorescence Resonance Energy Transfer for Pathogen Detection." Sensors 12, no. 12: 16660-16672.