DNA hybridization using surface plasmon-coupled emission.
We describe a new approach to measuring DNA hybridization using surface plasmon-coupled emission (SPCE). Excited fluorophores are known to couple with surface oscillations of electrons in thin metal films, typically 50 nm thick silver on a glass prism. These surface plasmons then radiate into the glass at a sharply defined angle determined by the emission wavelength and the optical properties of the glass and metal. This radiation has the same spectral profile as the emission spectrum of the fluorophores. We studied the emission due to Cy3-labeled DNA oligomers bound to complementary unlabeled oligomers which were themselves bound to the metal surface. Hybridization resulted in SPCE due to Cy3-DNA into the prism. Directional SPCE was observed whether the sample was illuminated from the sample side or through the glass substrate at the surface plasmon angle for the excitation wavelength. A large fraction of the total potential emission is coupled to the surface plasmons resulting in improved sensitivity. When illuminated through the prism at the surface plasmon angle, the sensitivity is increased due to the enhanced intensity of the resonance evanescent field. It is known that SPCE depends on proximity to the silver surface. As a result, changes in emission intensity are observed due to fluorophore localization even if hybridization does not affect the quantum yield of the fluorophore. The use of SPCE resulted in suppression of interfering emission from a noncomplementary Cy5-DNA oligomers due to weaker coupling of the more distant fluorophores with the surface plasmons. We expect SPCE to have numerous applications to nucleic acid analysis and for the measurement of bioaffinity reactions.
Malicka
Joanna
J
Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, University of Maryland at Baltimore, 725 West Lombard Street, Baltimore, Maryland 21201, USA.
Gryczynski
Ignacy
I
Gryczynski
Zygmunt
Z
Lakowicz
Joseph R
JR
D004247:DNA / Q000032:analysis*; D015342:DNA Probes / Q000032:analysis / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000032:analysis / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods*
D015342:DNA Probes; D009841:Oligonucleotides; D004247:DNA