Nucleic Acid Sandwich Hybridization Assay with Quantum Dot-Induced Fluorescence Resonance Energy Transfer for Pathogen Detection
Identifieur interne : 001145 ( Pmc/Curation ); précédent : 001144; suivant : 001146Nucleic Acid Sandwich Hybridization Assay with Quantum Dot-Induced Fluorescence Resonance Energy Transfer for Pathogen Detection
Auteurs : Cheng-Chung Chou ; Yi-Han HuangSource :
- Sensors (Basel, Switzerland) [ 1424-8220 ] ; 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.
Url:
DOI: 10.3390/s121216660
PubMed: 23211753
PubMed Central: 3571803
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Nucleic Acid Sandwich Hybridization Assay with Quantum Dot-Induced Fluorescence Resonance Energy Transfer for Pathogen Detection</title><author><name sortKey="Chou, Cheng Chung" sort="Chou, Cheng Chung" uniqKey="Chou C" first="Cheng-Chung" last="Chou">Cheng-Chung Chou</name></author><author><name sortKey="Huang, Yi Han" sort="Huang, Yi Han" uniqKey="Huang Y" first="Yi-Han" last="Huang">Yi-Han Huang</name></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">23211753</idno><idno type="pmc">3571803</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3571803</idno><idno type="RBID">PMC:3571803</idno><idno type="doi">10.3390/s121216660</idno><date when="2012">2012</date><idno type="wicri:Area/Pmc/Corpus">001145</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">001145</idno><idno type="wicri:Area/Pmc/Curation">001145</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">001145</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Nucleic Acid Sandwich Hybridization Assay with Quantum Dot-Induced Fluorescence Resonance Energy Transfer for Pathogen Detection</title><author><name sortKey="Chou, Cheng Chung" sort="Chou, Cheng Chung" uniqKey="Chou C" first="Cheng-Chung" last="Chou">Cheng-Chung Chou</name></author><author><name sortKey="Huang, Yi Han" sort="Huang, Yi Han" uniqKey="Huang Y" first="Yi-Han" last="Huang">Yi-Han Huang</name></author></analytic><series><title level="j">Sensors (Basel, Switzerland)</title><idno type="eISSN">1424-8220</idno><imprint><date when="2012">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>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.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Alexander, D J" uniqKey="Alexander D">D.J. Alexander</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Beigel, J H" uniqKey="Beigel J">J.H. Beigel</name></author><author><name sortKey="Farrar, J" uniqKey="Farrar J">J. Farrar</name></author><author><name sortKey="Han, A M" uniqKey="Han A">A.M. Han</name></author><author><name sortKey="Hayden, F G" uniqKey="Hayden F">F.G. Hayden</name></author><author><name sortKey="Hyer, R" uniqKey="Hyer R">R. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Sensors (Basel)</journal-id><journal-id journal-id-type="iso-abbrev">Sensors (Basel)</journal-id><journal-title-group><journal-title>Sensors (Basel, Switzerland)</journal-title></journal-title-group><issn pub-type="epub">1424-8220</issn><publisher><publisher-name>Molecular Diversity Preservation International (MDPI)</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">23211753</article-id><article-id pub-id-type="pmc">3571803</article-id><article-id pub-id-type="doi">10.3390/s121216660</article-id><article-id pub-id-type="publisher-id">sensors-12-16660</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Nucleic Acid Sandwich Hybridization Assay with Quantum Dot-Induced Fluorescence Resonance Energy Transfer for Pathogen Detection</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Chou</surname><given-names>Cheng-Chung</given-names></name><xref ref-type="corresp" rid="c1-sensors-12-16660"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>Huang</surname><given-names>Yi-Han</given-names></name></contrib><aff id="af1-sensors-12-16660">Department of Life Science and Center for Nano Bio-Detection, National Chung-Cheng University, Chia-Yi 62102, Taiwan; E-Mail:<email>je091410show@hotmail.com</email></aff></contrib-group><author-notes><corresp id="c1-sensors-12-16660"><label>*</label>Author to whom correspondence should be addressed; E-Mail: <email>bioccc@ccu.edu.tw</email>; Tel.: +886-5-272-0411 (ext. 66506); Fax: +886-5-272-2871.</corresp></author-notes><pub-date pub-type="collection"><month>12</month><year>2012</year></pub-date><pub-date pub-type="epub"><day>04</day><month>12</month><year>2012</year></pub-date><volume>12</volume><issue>12</issue><fpage>16660</fpage><lpage>16672</lpage><history><date date-type="received"><day>22</day><month>10</month><year>2012</year></date><date date-type="rev-recd"><day>23</day><month>11</month><year>2012</year></date><date date-type="accepted"><day>30</day><month>11</month><year>2012</year></date></history><permissions><copyright-statement>© 2012 by the authors; licensee MDPI, Basel, Switzerland</copyright-statement><copyright-year>2012</copyright-year><license><license-p><pmc-comment>CREATIVE COMMONS</pmc-comment>This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">http://creativecommons.org/licenses/by/3.0/</ext-link>).</license-p></license></permissions><abstract><p>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.</p></abstract><kwd-group><kwd>Alexa Fluor 660</kwd><kwd>avian influenza virus H5N1</kwd><kwd>fluorescence resonance energy transfer (FRET)</kwd><kwd>indium tin oxide (ITO)</kwd><kwd>quantum dot 655 (QD655)</kwd><kwd>sandwich hybridization</kwd></kwd-group></article-meta></front><floats-group><fig id="f1-sensors-12-16660" position="float"><label>Figure 1.</label><caption><p>The homemade FRET detection system based on a single 400 nm UV LED, a disposable ITO glass slide, a miniature 16-bit CCD-array spectrophotometer, and optical fibers: (<bold>a</bold>) schematic diagram of the detection system setup; (<bold>b</bold>) picture of the detection device setup (the main body of the device was made of PMMA with dimensions of 8.4 × 4.5 × 4.3 cm<sup>3</sup>); (<bold>c</bold>) ITO with a transparent, 20 μL microchamber created by the Microarray Gene Frame<sup>®</sup>.</p></caption><graphic xlink:href="sensors-12-16660f1"></graphic></fig><fig id="f2-sensors-12-16660" position="float"><label>Figure 2.</label><caption><p>Mobility shift assay of QD655 conjugation with capturing probes. <bold>M</bold>: 1 kb DNA ladder, <bold>lane 1</bold>: QD655 particles only, <bold>lane 2</bold>: QD655 modified with sulfo-SMCC crosslinker, <bold>lane 3</bold>: sulfo-SMCC modified QD655 conjugated with capturing probes. The QD655 particles surrounded with positively charged amine-derivatized PEG do not migrate very quickly (<bold>lane 1</bold>). The binding of sulfo-SMCC on the QD655 increases the mobility of the conjugated particles since sulfo-SMCC possesses a negatively charged sulfonate group on its NHS ring (<bold>lane 2</bold>). The binding of capturing DNA probes to sulfo-SMCC modified QD655 particles further increases the mobility of the conjugates due to the additional negative charge introduced by the oligonucleotide probes (<bold>lane 3</bold>), verifying the conjugation of QD655 with capturing probes.</p></caption><graphic xlink:href="sensors-12-16660f2"></graphic></fig><fig id="f3-sensors-12-16660" position="float"><label>Figure 3.</label><caption><p>Comparison between the excitation and emission spectra of QD655 and Alexa Fluor 660. The emission maxima of QD655 and Alexa Fluor 660 occur at 653 and 690 nm, respectively. The excitation spectra were measured by the lab-grade spectrophotometer with quartz cuvettes; the emission spectra were collected by the homemade UV LED/ITO/CCD spectrometer optical sensor.</p></caption><graphic xlink:href="sensors-12-16660f3"></graphic></fig><fig id="f4-sensors-12-16660" position="float"><label>Figure 4.</label><caption><p>Schematic illustration of the sandwich hybridization assay with a QD-induced FRET reporter system for H5 target DNA detection: (<bold>a</bold>) sandwich hybridization with label-free H5 sequence (target) by the capturing probes conjugated on QD655 (FRET donor) and the reporter probes labeled with Alexa Fluor 660 (FRET acceptor); (<bold>b</bold>) FRET emission shift before and after the sandwich hybridization.</p></caption><graphic xlink:href="sensors-12-16660f4"></graphic></fig><fig id="f5-sensors-12-16660" position="float"><label>Figure 5.</label><caption><p>The emission spectra of the sandwich hybridization with a high FRET donor concentration ([QD] = 1 μM): (<bold>a</bold>) hybridization with different amounts of H5 target ([H5] = 0, 0.5, and 1 μM) and a fixed amount of FRET acceptor ([dye] = 1 μM) and a negative control study in which non-target 60-mer H1 sequence ([H1] = 1 μM) was hybridized with QD-dye FRET pair ([QD] = [dye] = 1 μM) to evaluate the detection specificity of the QD-dye FRET pair; (<bold>b</bold>) hybridization with a fixed amount of H5 target ([H5] = 1 μM) and different amounts of FRET acceptor ([dye] = 10, 20, and 30 μM).</p></caption><graphic xlink:href="sensors-12-16660f5"></graphic></fig><fig id="f6-sensors-12-16660" position="float"><label>Figure 6.</label><caption><p>FRET detection of different amounts of (<bold>a</bold>) 60-mer H5 target (0–50 nM) and (<bold>b</bold>) 630-nt H5 cDNA fragment target (0–50 nM) with 50 nM FRET donor (QD) and 1.5 μM FRET acceptor (dye). The insets of (<bold>a</bold>) and (<bold>b</bold>) display the dose response relationship between the average FRET value from triplicate experiments and the target concentration.</p></caption><graphic xlink:href="sensors-12-16660f6"></graphic></fig></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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