<title>Metal-ligand complexes as a new class of long-lived fluorophores for protein hydrodynamics and fluorescence polarization immunoassay</title>

Abstract

We describe the use of asymmetric Ru-ligand complexes as a new class of luminescent probes. These complexes are known to display luminescent lifetimes ranging from 10 to 4000 ns. We show that the asymmetric complex Ru(bpy)<SUB>2</SUB>(dcbpy) (PF<SUB>6</SUB>)<SUB>2</SUB> displays a high anisotropy value near 0.26 when excited in the long wavelength absorption band, and an intensity decay time near 400 ns. For covalent linkage to proteins, we synthesized the N- hydroxy succinimide ester. We measured the intensity and anisotropy decays of [Ru(bpy)<SUB>2</SUB>(dcbpy)] when covalently linked to proteins and in solutions of increasing viscosity. These data demonstrated that the probes can be used to measure rotational motions on the microsecond(s) timescale, which so far has been inaccessible using luminescence methods. We also used this probe in a fluorescence polarization immunoassay of HSA. We found that the steady-state polarization of labeled HSA was sensitive to binding of anti-HSA, resulting in a 200% increase in polarization. The labeled HSA was also used in a competitive format with unlabeled HSA as the antigen. The time-resolved anisotropy decays demonstrate increased correlation times for labeled HSA in the presence of anti-HSA, an effect which was partially reversed in the presence of unlabeled HSA. These results demonstrate the potential of the metal-ligand complexes to be used in the fluorescence polarization immunoassay of high molecular weight analytes. The use of such metal-ligand complexes enable fluorescence polarization immunoassays which bypass the usual limitation to low molecular weight antigens.