Synthesis and evaluation of Ru-complexes as anisotropy probes for protein hydrodynamics and immunoassays of high-molecular-weight antigens.

We investigated three unsymmetrical Ru-complexes, namely [Ru(bpy)2 (phen-ITC)]2+, [Ru(bpy)2(dcbpy)] and [Ru(bpy)2(mcbpy)]+ for use as probes for rotational diffusion and in immunoassays of high-molecular-weight antigens. For this purpose we synthesized reactive forms of these metal-ligand complexes and conjugated them to human serum albumin (HSA). The maximal anisotropies (r0) for the HSA-bound forms in frozen solution are 0.23, 0.17 and 0.14 for the (dcbpy), (mcbpy) and (phen-ITC) derivatives, respectively. The activated Ru metal-ligand complexes have either one or two NHS-esters or an isothiocyanate group as the reactive moiety. The usefulness of these complexes in immunoassays was determined by titration of the labeled HSA with polyclonal anti-HSA. The highest steady state anisotropy (r) values (0.190) were observed for the [Ru(bpy)2(dcbpy)]-labeled HSA on titration with polyclonal antibody. However, a relative increase in the steady state anisotropy (r/r0) on titration with polyclonal antibody was found for the phen-ITC probe (96%), as compared to the dcbpy (83%) or mcbpy (79%) derivatives. These findings were confirmed by time-resolved frequency-domain measurements. In particular the higher mean correlation times calculated for the phen-ITC derivative suggests reduced local probe motion for this probe when bound to HSA as compared to the (mcbpy) and (dcbpy) conjugates.