New Donor−Acceptor Pair for Fluorescent Immunoassays by Energy Transfer

A novel Förster donor−acceptor dye pair for an immunoassay based on resonance 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...

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Bibliographic Details
Published in:Bioconjugate chemistry 1999-11, Vol.10 (6), p.1107-1114
Main Authors: Schobel, Uwe, Egelhaaf, Hans-Joachim, Brecht, Andreas, Oelkrug, Dieter, Gauglitz, Günter
Format: Article
Language:English
Subjects:
Binding, Competitive
Carbocyanines - chemistry
Chemical Phenomena
Chemistry, Physical
Dimerization
Energy Transfer
Fluorescence
Fluorescent Dyes - chemistry
Fluoroimmunoassay
Herbicides - analysis
Immunoglobulin G - chemistry
Photochemistry
Serum Albumin, Bovine - chemistry
Simazine - analysis
Spectrometry, Fluorescence
Spectrophotometry
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Summary:A novel Förster donor−acceptor dye pair for an immunoassay based on resonance 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 overlap, an exceptionally high R 0 value of 68.7 Å is obtained in solution. For biochemical applications, antibodies (IgG) are labeled with Cy5, while a tracer for competitive binding is synthesized by labeling bovine serum albumin (BSA) with an analyte derivative and Cy5.5. Binding the dyes to proteins at a low dye/protein ratio increases the fluorescence lifetimes and quantum yields, leading to an enhanced R 0 value of 85.2 Å. At higher dye/protein ratios, the formation of nonfluorescent dimeric species causes a decrease in the fluorescence lifetime and quantum yield due to RET from monomeric dyes to dimers within one protein molecule. The Förster distances could be calculated using the dimer absorption spectra to 83.9 and 83.6 Å for Cy5 and Cy5.5, respectively. Upon binding of the Cy5-labeled IgG to the tracer, efficient quenching of Cy5 fluorescence is observed. Steady-state and time-resolved measurements reveal that approximately 50% of the quenching results in Förster-type RET, while the residual quenching effect is caused by static quenching processes. The applicability of this dye pair is demonstrated in a homogeneous competitive immunoassay for the pesticide simazine.
ISSN:1043-1802
1520-4812
DOI:10.1021/bc990073b