Journal of Fluorescence

, 16:259 | Cite as

Investigation of a Fluorescence Signal Amplification Mechanism Used for the Direct Molecular Detection of Nucleic Acids

  • Kim Doré
  • Mario Leclerc
  • Denis Boudreau
Original Paper


A fluorescence signal amplification mechanism allowing detection limits for DNA in the zeptomolar range was investigated. Photophysical properties of the molecular system were studied in order to better explain the signal amplification that is observed. We show that the confinement of a fluorescent DNA hybridization transducer in aggregates improves its quantum yield and photostability. Furthermore, we show that the combination of the resonance energy transfer occurring within the aggregates with the use of a conjugated polymer as the hybridization transducer and donor allows ultrafast and efficient energy coupling to the aggregates and can lead to the excitation of a large number of acceptors by only one donor.


Fluorescence signal amplification Fluorescence resonance energy transfer Ultrasensitive DNA detection Orientation and confinement in aggregates Ultrafast energy transfer 



The authors would like to thank Drs. B. Simard and S. Denommée at the Steacie Institute for Molecular Sciences, NRC, Ottawa, Canada, for the lifetime measurements and Dr. H.A. Ho (U. Laval) for the gift of the polymeric transducer and fruitful discussions. K.D. also acknowledges the Natural Sciences and Engineering Council of Canada for a scholarship.


  1. 1.
    Daar AS, Thorsteinsdóttir H, Martin DK, Smith AC, Nast S, Singer PA (2002) Top ten biotechnologies for improving health in developing countries. Nat Genet 32:229–232PubMedCrossRefGoogle Scholar
  2. 2.
    Fodor SPA, Read JL, Pirrung MC, Stryer L, Lu AT, Solas D (1991) Light-directed spatially addressable parallel chemical synthesis. Science 251:767–773PubMedCrossRefGoogle Scholar
  3. 3.
    Tyagi S, Kramer FR (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14:303–308PubMedCrossRefGoogle Scholar
  4. 4.
    McQuade DT, Pullen AE, Swager TM (2000) Conjugated polymer-based chemical sensors. Chem Rev 100:2537–2574PubMedCrossRefGoogle Scholar
  5. 5.
    Drummond TG, Hill MG, Barton JK (2003) Electrochemical DNA sensors. Nat Biotechnol 21:1192–1199PubMedCrossRefGoogle Scholar
  6. 6.
    Storhoff JJ, Lucas AD, Garimella V, Bao YP, Müller UR (2004) Homogeneous detection of unamplified genomic DNA sequences based on colorimetric scatter of gold nanoparticle probes. Nat Biotechnol 22:883–887PubMedCrossRefGoogle Scholar
  7. 7.
    Nam JM, Stoeva SI, Mirkin CA (2004) Bio-bar-code-based DNA detection with PCR-like sensitivity. J Am Chem Soc 126:5932–5933PubMedCrossRefGoogle Scholar
  8. 8.
    Liu RH, Yang J, Lenigk R, Bonanno J, Grodzinski P (2004) Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection. Anal Chem 76:1824–1831PubMedCrossRefGoogle Scholar
  9. 9.
    Liu B, Bazan GC (2004) Homogeneous fluorescence-based DNA detection with water-soluble conjugated polymers. Chem Mater 16:4467–4476CrossRefGoogle Scholar
  10. 10.
    Ho HA, Boissinot M, Bergeron MG, Corbeil G, Dore K, Boudreau D, Leclerc M (2002) Colorimetric and fluorometric detection of nucleic acids using cationic polythiophene derivatives. Angew Chem Int Ed 41:1548–1551CrossRefGoogle Scholar
  11. 11.
    Gaylord BS, Heeger AJ, Bazan GC (2002) DNA detection using water-soluble conjugated polymers and peptide nucleic acid probe. Proc Natl Acad Sci USA 99:10954–10957PubMedCrossRefGoogle Scholar
  12. 12.
    Nilsson KPR, Inganäs O (2003) Chip and solution detection of DNA hybridization using a luminescent zwitterionic polythiophene derivative. Nat Mater 2:419–424PubMedCrossRefGoogle Scholar
  13. 13.
    Doré K, Dubus S, Ho HA, Levesque I, Brunette M, Corbeil G, Boissinot M, Boivin G, Bergeron MG, Boudreau D, Leclerc M (2004) Fluorescent polymeric transducer for the rapid, simple, and specific detection of nucleic acids at the zeptomole level. J Am Chem Soc 126:4240–4244PubMedCrossRefGoogle Scholar
  14. 14.
    Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim NS (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis from diagnosis of sickle-cell anemia. Science 230:1350–1354PubMedCrossRefGoogle Scholar
  15. 15.
    Ho HA, Doré K, Boissinot M, Bergeron MG, Tanguay RM, Boudreau D, Leclerc M (2005) Direct molecular detection of nucleic acids by fluorescence signal amplification. J Am Chem Soc 127:12673–12676PubMedCrossRefGoogle Scholar
  16. 16.
    Beljonne D, Pourtois G, Silva C, Hennebicq E, Herz LM, Friend RH, Scholes GD, Müllen K, Brédas JL (2002) Interchain vs. intrachain energy transfer in acceptor-capped conjugated polymers. Proc Natl Acad Sci USA 99:10982–10987PubMedCrossRefGoogle Scholar
  17. 17.
    Klimov VI, McBranch DW, Barashkov NN, Ferraris JP (1997) Femtosecond dynamics of excitons in π-conjugated oligomers: the role of intrachain two-exciton states in the formation of interchain species. Chem Phys Lett 277:109–117CrossRefGoogle Scholar
  18. 18.
    Chen L, McBranch DW, Wang HL, Helgeson R, Wuld F, Whitten DG (1999) Highly sensitive biological and chemical sensors based on reversible fluorescence quenching in a conjugated polymer. Proc Natl Acad Sci USA 96:12287–12292PubMedCrossRefGoogle Scholar
  19. 19.
    Scholes GD (2003) Long-range resonance energy transfer in molecular systems. Ann Rev Phys Chem 54:57–87CrossRefGoogle Scholar
  20. 20.
    Vallotton P, Tairi AP, Wohland T, Freidrich-Bénet K, Pick H, Hovius R, Vogel H (2001) Mapping the antagonist binding site of the serotonin type 3 receptor by fluorescence resonance energy transfer. Biochemistry 40:12237–12242PubMedCrossRefGoogle Scholar
  21. 21.
    Jones GM, Wofsy C, Aurell C, Sklar LA (1999) Analysis of vertical fluorescence resonance energy transfer from the surface of a small-diameter sphere. Biophys J 76:517–527PubMedCrossRefGoogle Scholar
  22. 22.
    Sautter A, Kaletas BK, Schmid DG, Dobrawa R, Zimine M, Jung G, van Stokkum IHM, de Cola L, Williams RM, Würthner F (2005) Ultrafast energy-electron transfer cascade in a multichromophoric light-harvesting molecular square. J Am Chem Soc 127:6719–6729PubMedCrossRefGoogle Scholar
  23. 23.
    Lakowicz JR (1999) Principles of fluorescence spectroscopy, 2nd edn. Plenum, New YorkGoogle Scholar
  24. 24.
    Stewart WW (1981) Synthesis of 3,6-disulfonated 4-aminonap- thalimides. J Am Chem Soc 103:7615–7620CrossRefGoogle Scholar
  25. 25.
    Halperin A, Buhot A, Zhulina EB (2004) Sensitivity, specificity, and the hybridization isotherms of DNA chips. Biophys J 86:718–730PubMedGoogle Scholar
  26. 26.
    Dalgarno SJ, Tucker SA, Bassil DB, Atwood JL (2005) Fluorescent guest molecules report ordered inner phase of host capsules in solution. Science 309:2037–2039PubMedCrossRefGoogle Scholar
  27. 27.
    Nesterov EE, Zhengguo Z, Swager TM (2005) Conjugation enhancement of intramolecular exciton migration in poly(p-phenylene ethynylene)s. J Am Chem Soc 127:10083–10088PubMedCrossRefGoogle Scholar
  28. 28.
    ISS, Innovations in fluorescence. Website: rces/fluorophores.htmlGoogle Scholar
  29. 29.
    Maliwal BP, Gryczynski Z, Lakowicz J (2001) Long-wavelength long-lifetime luminophores. Anal Chem 73:4277–4285PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  1. 1.Chemistry Department and Centre d’optique, photonique et laser (COPL)Université LavalQuébecCanada
  2. 2.Chemistry Department and Centre de recherche en science et ingénierie des macromolécules (CERSIM)Université LavalQuébecCanada

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