Immunoassays based on directional surface plasmon-coupled emission.
We described a new approach to immunoassays using surface plasmon-coupled emission (SPCE). Fluorescence is visually isotropic in space, so that the sensitivity is limited in part by the light collection efficiency. By the use of SPCE, we can efficiently collect the emission and convert it to a cone-like directional beam in a glass substrate. SPCE is the coupling of excited fluorophores with a thin metal film, resulting in radiation of surface plasmons into the higher refractive index media. We used SPCE to develop a model affinity assay using labeled goat anti-rabbit immunoglobulin G (IgG) antibodies against rabbit IgG bound to a 50-nm-thick silver film. Binding of labeled IgG to the surface resulted in increased intensity observed at an angle of 75 degrees from the normal in the glass substrate. The SPCE intensity depends on proximity of the fluorophore to the silver film and does not require a change in quantum yield upon binding. The use of SPCE is shown to provide background suppression because excited fluorophores distant from the silver film do not result in SPCE. Sensitivity and selectivity can be further increased by excitation under conditions of surface plasmon resonance (SPR) because the evanescent field is enhanced by the resonance interaction and excitation is limited to the region near the metal. We believe SPCE will provide a new technology for high sensitivity and selectivity in surface-bound assays and microfluidic systems.
Matveeva
Evgenia
E
Department of Biochemistry and Molecular Biology, Center for Fluorescence Spectroscopy, University of Maryland at Baltimore, 725 West Lombard Street, Baltimore, MD 21201, USA. eva@cfs.umbi.umd.edu
Gryczynski
Zygmunt
Z
Gryczynski
Ignacy
I
Lakowicz
Joseph R
JR
D000818:Animals; D005456:Fluorescent Dyes / Q000737:chemistry; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D007074:Immunoglobulin G / Q000032:analysis*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance / Q000379:methods* / Q000592:standards
D005456:Fluorescent Dyes; D007074:Immunoglobulin G