New-donor-acceptor pair for fluorescent immunoassays by energy transfer

A novel Forster donor-acceptor dye pair for an immunoassay based on resonan ce energy transfer (RET) is characterized with respect to its photophysical properties. As donor and acceptor, we chose the long-wavelength excitable cyanine dyes Cy5 and Cy5.5, respectively. Due to the perfect spectral overl ap, an exceptionally high R-0 value of 68.7 Angstrom is obtained in solutio n. For biochemical applications, antibodies (IgG) are labeled with Cy5, whi le a tracer for competitive binding is synthesized by labeling bovine serum albumin (BSA) with an analyte derivative and Cy5.5. Binding the dyes to pr oteins at a low dye/protein ratio increases the fluorescence lifetimes and quantum yields, leading to an enhanced R-0 value of 85.2 Angstrom. At highe r dye/protein ratios, the formation of nonfluorescent dimeric species cause s a decrease in the fluorescence lifetime and quantum yield due to RET from monomeric dyes to dimers within one protein molecule. The Forster distance s could be calculated using the dimer absorption spectra to 83.9 and 83.6 A ngstrom for Cy5 and Cy5.5, respectively. Upon binding of the Cy5-labeled Ig G to the tracer, efficient quenching of Cy5 fluorescence is observed. Stead y-state and time-resolved measurements reveal that approximately 50% of the quenching results in Forster-type RET, while the residual quenching effect is caused by static quenching processes. The applicability of this dye pai r is demonstrated in a homogeneous competitive immunoassay for the pesticid e simazine.