New Donor−Acceptor Pair for Fluorescent Immunoassays by Energy Transfer

Abstract

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 A 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 A. 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 A 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.

Improvement of the Mutation-Discrimination Threshold for Rare Point Mutations by a Separation-Free Ligase Detection Reaction Assay Based on Fluorescence Resonance Energy Transfer

“…These properties are similar to those of the relatively new dye AF647, which has a maximum excitation wavelength of 650 nm, maximum emission wavelength of 670 nm (information from IDT's homepage), and an extinction coefficient of 203000 cm -1 M -1 . 20 According to the manufacturer, the reported fluorescence quantum yields of Cy5 and AF647 dissolved in water are 0.26 21 and 0.33, respectively. In view of these values, AF647 appears to be more fluorescent than Cy5.…”

Time-resolved Fluorescence Resonance Energy Transfer Assay for Point-of-Care Testing of Urinary Albumin

“…Furthermore, the europium chelate emits at 613ϳ620 nm, which allows the use of acceptors emitting in the red or near-infrared range. Cy5 has high molar absorptivity at the wavelength of the europium emission and emits red light at 665ϳ670 nm, which is different from the europium emission (28 ). The system thus permits the use of spectral resolution and temporal resolution for the detection of the sensitized acceptor signal.…”

“…This may be attributable to the formation of nonfluorescent Cy5 dimer. It was previously demonstrated that an increasing Cy5/protein ratio would lead to the formation of nonfluorescent Cy5 dimer within one protein molecule, causing a decrease of the quantum yield (28 ). The highest signal-to-background ratio (32.7) was obtained with albumin labeled with 1.2-1.6 Cy5 molecules/ molecule of albumin.…”

Resonant electronic excitation energy transfer by Dexter mechanism in the quantum dot system

“…In conclusions, the results are useful in the correct interpretation of experimental data taking into account both the direct Coulomb interaction of the donor and acceptor electrons and their exchange interaction. This is of particular impotence for measurements of small distance within the biomolecule [2][3][4].…”

“…The strong dependence of the energy transfer rate on the distance between the energy donor and the energy acceptor enables to detect the formation of antigenantibody complexes, ensyme-substrate complexes, the DNA hybridization and cleavage [1] and also to study the structure and dynamics of biomolecules, where measurements of small distance within the molecule are necessary [2][3][4]. Results of these investigations are of the great importance for diagnostics and therapy of diseases, among which are oncological ones [5], (see also the literature in [1]).…”