Improved fluoroimmunoassays using the dye Alexa Fluor 647 with the RAPTOR, a fiber optic biosensor

The performance of the fluorescent dye Alexa Fluor 647 (AF647) was explored as an alternative to Cy5 for immunoassays on the RAPTOR, a fiber optic biosensor. The RAPTOR performs sandwich fluoroimmunoassays on the surface of small polystyrene optical waveguides for analyte detection. Fluorescence and immunoassay data were examined at various dye-to-protein (D/P) ratios for both Cy5 and Alexa Fluor 647. Primarily, due to the self-quenching characteristics of Cy5, Alexa Fluor 647 is substantially more effective in fluoroimmunoassays, yielding over twice the signal for any given analyte concentration. Alexa Fluor 647 can be attached to antibodies at higher ratios, D/P=6, before self-quenching begins to limit the dye's effectiveness. Furthermore, while Alexa Fluor 647 becomes quenched at high dye-to-protein ratios, D/P=9, the net fluorescence yield reaches a maximum, as opposed to Cy5-labeled proteins, which become nearly nonfluorescent at high labeling ratios, D/P> or =6. The limitations of Cy5 were elucidated with an immunoassay for ricin, while the advantages of Alexa Fluor 647 were demonstrated in both direct binding assays as well as in a sandwich immunoassay for staphylococcal enterotoxin B.