Development of homogeneous binding assays based on fluorescence resonance energy transfer between quantum dots and Alexa Fluor fluorophores.

We studied the fluorescence resonance energy transfer (FRET) between quantum dots emitting at 565, 605, and 655 nm as energy donors and Alexa Fluor fluorophores with absorbance maxima at 594, 633, 647, and 680 nm as energy acceptors. As a first step, we prepared covalent conjugates between all three types of quantum dots and each of the Alexa Fluor fluorophores that could act as an energy acceptor. All of these conjugates displayed efficient resonance energy transfer. Then we prepared covalent conjugates of these quantum dots with biotin, fluorescein, and cortisol and established that the binding of these conjugates to suitable Alexa Fluor-labeled antibodies and streptavidin (in the case of biotin) can be efficiently detected by measuring the resonance energy transfer in homogeneous solutions. Finally, based on these observations, competitive binding assays for these three small analytes were developed. The performance of these assays as a function of the degree of labeling of the quantum dots was evaluated. It was found that decreasing the degree of loading of the quantum dots leads to decreases of the limits of detection. The results show the great potential of this FRET system for the development of new homogeneous binding assays.