Nucleic Acid Sandwich Hybridization Assay with Quantum Dot-Induced Fluorescence Resonance Energy Transfer for Pathogen Detection

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.

[1]  Yi Zhang,et al.  Quantum Dot Enabled Molecular Sensing and Diagnostics , 2012, Theranostics.

[2]  M. Perdue,et al.  Public Health Risk from Avian Influenza Viruses , 2005, Avian diseases.

[3]  Luke P. Lee,et al.  Innovations in optical microfluidic technologies for point-of-care diagnostics. , 2008, Lab on a chip.

[4]  J. Pasick.  Advances in the molecular based techniques for the diagnosis and characterization of avian influenza virus infections. , 2008, Transboundary and emerging diseases.

[5]  D J Alexander,et al.  A review of avian influenza in different bird species. , 2000, Veterinary microbiology.

[6]  Itamar Willner,et al.  Chemiluminescent and chemiluminescence resonance energy transfer (CRET) detection of DNA, metal ions, and aptamer-substrate complexes using hemin/G-quadruplexes and CdSe/ZnS quantum dots. , 2011, Journal of the American Chemical Society.

[7]  A. Malcolm,et al.  Nucleic acid analysis by sandwich hybridization , 1989, Journal of clinical laboratory analysis.

[8]  Chun-Yang Zhang,et al.  Single quantum dot-based nanosensor for multiple DNA detection. , 2010, Analytical chemistry.

[9]  V. Josserand,et al.  Effect of poly(ethylene glycol) length on the in vivo behavior of coated quantum dots. , 2009, Langmuir : the ACS journal of surfaces and colloids.

[10]  I. Willner,et al.  Semiconductor quantum dots for bioanalysis. , 2008, Angewandte Chemie.

[11]  Ian H Brown,et al.  Nucleic acid sequence-based amplification methods to detect avian influenza virus , 2003, Biochemical and Biophysical Research Communications.

[12]  Igor L. Medintz,et al.  Quantum dot-based resonance energy transfer and its growing application in biology. , 2009, Physical chemistry chemical physics : PCCP.

[13]  Erkki Ruoslahti,et al.  Targeted quantum dot conjugates for siRNA delivery. , 2007, Bioconjugate chemistry.

[14]  U. Krull,et al.  Interfacial transduction of nucleic acid hybridization using immobilized quantum dots as donors in fluorescence resonance energy transfer. , 2009, Langmuir : the ACS journal of surfaces and colloids.

[15]  Jae Jun Song,et al.  Design and Application of Highly Responsive Fluorescence Resonance Energy Transfer Biosensors for Detection of Sugar in Living Saccharomyces cerevisiae Cells , 2007, Applied and Environmental Microbiology.

[16]  Matthew C. Smith,et al.  An integrated portable hand-held analyser for real-time isothermal nucleic acid amplification. , 2007, Analytica chimica acta.

[17]  U. Krull,et al.  Multiplexed interfacial transduction of nucleic acid hybridization using a single color of immobilized quantum dot donor and two acceptors in fluorescence resonance energy transfer. , 2010, Analytical chemistry.

[18]  C. Pachucki.  Rapid tests for influenza , 2005, Current infectious disease reports.

[19]  J. Taubenberger,et al.  Methods for molecular surveillance of influenza , 2010, Expert review of anti-infective therapy.

[20]  Zhen-Li Huang,et al.  Purification of denatured bovine serum albumin coated CdTe quantum dots for sensitive detection of silver(I) ions , 2007, Analytical and bioanalytical chemistry.

[21]  Beate Crossley,et al.  Conventional and future diagnostics for avian influenza. , 2009, Comparative immunology, microbiology and infectious diseases.

[22]  E. Kobatake,et al.  Direct detection of RNA transcription by FRET imaging using fluorescent protein probe. , 2008, Journal of biotechnology.

[23]  W. Maret,et al.  Domain-specific fluorescence resonance energy transfer (FRET) sensors of metallothionein/thionein. , 2005, Protein engineering, design & selection : PEDS.

[24]  D. Ye,et al.  Molecular beacons: an optimal multifunctional biological probe. , 2008, Biochemical and biophysical research communications.

[25]  Fang Fang,et al.  A thermostat chip of indium tin oxide glass substrate for static polymerase chain reaction and in situ real time fluorescence monitoring. , 2008, Analytica chimica acta.

[26]  Anna Grazia Mignani,et al.  A portable fluorometer for the rapid screening of M1 aflatoxin , 2007 .

[27]  R. Nitschke,et al.  Quantum dots versus organic dyes as fluorescent labels , 2008, Nature Methods.

[28]  W. Stemmer,et al.  Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. , 1995, Gene.

[29]  U. Krull,et al.  Toward a multiplexed solid-phase nucleic acid hybridization assay using quantum dots as donors in fluorescence resonance energy transfer. , 2009, Analytical chemistry.

[30]  Angus Nicoll,et al.  Avian influenza A (H5N1) infection in humans. , 2005, The New England journal of medicine.

[31]  U. Krull,et al.  Towards multi-colour strategies for the detection of oligonucleotide hybridization using quantum dots as energy donors in fluorescence resonance energy transfer (FRET). , 2007, Analytica chimica acta.

[32]  Ning Zhang,et al.  Colorimetric detection of anthrax DNA with a Peptide nucleic acid sandwich-hybridization assay. , 2007, Journal of the American Chemical Society.

[33]  Sangwei Lu,et al.  Detection and subtyping of influenza A virus based on a short oligonucleotide microarray. , 2009, Diagnostic microbiology and infectious disease.

[34]  Ulrich Melcher,et al.  Molecular Detection and Identification of Influenza Viruses by Oligonucleotide Microarray Hybridization , 2003, Journal of Clinical Microbiology.