Surface-plasmon field-enhanced fluorescence spectroscopy

We describe the combination of surface plasmon- and fluorescence spectroscopy for sensor applications. The resonant excitation of PSP modes at a metal/buffer-interface in a flow cell results in optical field intensities largely enhanced compared to the incoming laser light: a factor of 16, calculated for a Au/water interface by Fresnel formulas was experimentally confirmed. This field enhancement can be used to increase the sensitivity for monitoring binding reactions of an analyte from the aqueous phase to the recognition sites at a functionalized interface, provided this interfacial architecture ensures that the bound (fluorescently labeled) analyte molecules are still within the exponentially decaying evancescent held of the PSP mode, however, also keeping them sufficiently away from the (acceptor states of the) metal to avoid Forster quenching of the emitted fluorescence. A quantitative analysis is given for two examples: one is the binding of fluorescently-doped latex particles, (at sub-monolayer coverage), carrying in addition biotin-moieties at their surface for binding to a streptavidin layer at the Au/buffer interface. Here, a correlation between fluorescence intensity and layer thickness can be analyzed. A second example concerns a small biotinylated chromophore, the very dilute binding of which to the streptavidin layer results in only a minute angular shift of the PSP resonance curve, too small to be detected. The fluorescence intensity, however, is easily recorded and gives a rough estimate of the obtainable enhancement factor of ca. 1000. (C) 2000 Elsevier Science B.V. All rights reserved.