Probing molecular interactions with homogeneous techniques based on rare earth cryptates and fluorescence energy transfer

TLDR

Short‑range biomolecular interactions are crucial for both analytical applications and mechanistic studies in biology. The authors adapted a rare‑earth cryptate‑based homogeneous assay to model systems such as EGF receptor interactions, kinase activity, Jun/Fos protein binding, and DNA hybridization. The assay employs long‑lived europium fluorescence, spectral and temporal discrimination, a cage structure with ion pairing, and dual‑wavelength detection to shield the signal from media perturbations. The method produced results comparable to conventional assays and proved versatile across the tested molecular and cellular models.

Abstract

A homogeneous assay method based on the long-lived fluorescence of rare earth cryptates and amplification by nonradiative energy transfer has been developed for immunoassays. The principles of the assay allow a double discrimination of the emitted signal through spectral and temporal selectivity. The cage structure of the complex, ion pairing around europium, as well as double-wavelength detection, fully shield the assay from perturbations of media. Events based on short-range interactions involving biomolecules are of tremendous importance in many domains of biology, either for analytical purposes or for molecular mechanism studies. Therefore, the principles and the reagents used to devise this homogeneous assay were adapted to various models representative of molecular and cellular processes and were chosen from the signaling pathways involved in cellular communication and expression: epidermal growth factor (EGF) receptor-ligand interaction, EGF receptor kinase activity, Jun/Fos protein-protein interaction, and DNA hybridization. Evaluation of the homogeneous assays yielded results compatible with those from comparison assays and demonstrates the versatility and wide range of applicability of this methodology.