Multicolor fluoroimmunoassays based on surface plasmon-coupled emission

We present a new method for multi-color fluoroimmunoassays based on directional surface plasmon-coupled emission (SPCE). SPCE is coupling of excited fluorophores with a nearby thin metal film (silver) resulting in strongly directional emission into the underlying glass substrate. The angle at which the radiation propagates through the prism depends on emission wavelength and makes possible measurement of multiple analytes using multiple emission wavelengths. We demonstrated this possibility using two antibodies labeled with different fluorophores, binding to an antigen protein immobilized on the silver surface. We observed independent emission at a different angle on the glass prism, resulting of the surface binding of each antibody. This methodology can be readily extended to 3 or more fluorophores. This technology presents opportunity to develop highly sensitive multiplex assay format for biological agents' detection.