Thin solid europium(III) dye layers as donors in time-resolved fluorescence resonance energy transfer assays

Lanthanide chelates and lanthanide nanoparticle labels are attractive donors for separation-free time-resolved fluorescence resonance energy transfer (TR-FRET) assays. In fully dyed nanoparticles, the inner volume of nanoparticle labels in TR-FRET assays are incapable of participating to energy transfer due to large distances to acceptors on the surface. Our interest was to study surface-based TR-FRET and, therefore, various europium(III) (Eu) chelate layers were investigated for TR-FRET efficiency. Eu(III) chelates incorporated in a siloxane layer, Eu(III) chelate covalently coupled on silanized surface and Eu(III) labeled protein surface were prepared and compared to nanoparticle-based TR-FRET. Energy transfer between the solid-phase donors and Cy5-labeled protein were obtained with signal-to-background ratios ranging from 1.2 to 9.9. In this study, a thin layer prepared using Eu(III)-labeled protein gave the most efficient TR-FRET. This thin donor layer was tested in a competitive separation-free immunoassay of human albumin (hAlb). hAlb was measured in a clinically relevant concentrations from 0.05 to 10 mg l⁻¹ with the coefficient of variation ranging from 1.0% to 12.4%.