Time-Resolved Fluorometric Method for One-Step Immunoassays Using Plasmonic Nanostructures

The use of plasmonic nanostructures for the detection of interactions between biomolecules is currently a very active field of research. In this report, we demonstrate that localized surface plasmons of metallic particles provide the opportunity for sensitive detection of protein−protein interactions in one-step fluoroimmunoassays. Glass coated with silver island films was used for the construction of a bioactive sensing surface where binding results in fluorescence intensity amplification and highly decreased lifetime of the bound probes. The one-step fluoroimmunoassay is demonstrated for detection of TNF-α using a secondary antibody labeled with DY488 dye. Kinetic and end-point measurements were performed using intensity and lifetime (phase shift and modulation) measurements. The ability to vary the ratio of bound to unbound probes provides a mean to tune the assay sensitivity using lifetime measurement modalities. The estimated enhancement in sensitivity is about 100-fold compared to the standard glass surface assay because of intensity amplification of 10.4-fold and similar lifetime decrease. The discussion of experimental results is supported with theoretical calculations with specific parameters for TNF-α assay and DY488 fluorophore. The combined effects of plasmonic structures on intensity and lifetime of fluorophores and the use of phase-modulation fluorometry open the opportunity for bioassay design with high sensitivities and simplified biochemical procedures.