DNA Hybridization Using Surface Plasmon-Coupled Emission

Abstract

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.

Controlling polarization twisting of light resulting from surface plasmon interactions with threefold symmetric nanostructures

“…1͑a͔͒, which has previously been used to examine other SPP re-emission polarization processes. [14][15][16] With the illumination angle set at the strong SPP excitation angle ͑ = 70°͒, and the illumination-polarization set to TM, we have recorded the outgoing intensity monitored by the photodiode, as a function of analyzer polarization. Such an examination would typically present a cos 2 function of the angle between the polarizer and the analyzer; and this is exactly what is observed for illumination angles off SPP resonance, and also for the symmetric SPP/nanostructure interaction orientations ͑N and S͒.…”

Minimization of detection volume by surface plasmon-coupled emission

“…In the number of previous reports, the exceptional background rejection in SPCE measurements have been reported [21,25,31]. This was crucial for enhanced assays [28,29] and ultra sensitive detection [29,33,34].…”

“…Recently, a phenomenon related to SPR, surface plasmon-coupled emission (SPCE) has been applied to DNA hybridization [21][22][23][24][25] and immunoassays [26][27][28][29][30][31]. In these measurements, the exceptional background rejection has been reported by various groups.…”

Toward the standing wave surface plasmon resonance fluorescence microscopy

“…For example, Malicka et al demonstrated the use of SPCE in DNA hybridization measurements [9] and Borejdo et al extended the fluorescence correlation spectroscopy with SPCE to improve signal-to-noise ratio [10].…”