USE OF A LONG LIFETIME RE(I) COMPLEX IN FLUORESCENCE POLARIZATION IMMUNOASSAYS OF HIGH-MOLECULAR-WEIGHT ANALYTES

We describe a new class of fluorescence polarization immunoassays base d on the luminescence from a Re(I) metal-ligand complex, Re(I) complex es are extremely photostable and possess useful photophysical properti es including long lifetimes, high quantum yields, and high emission po larization in the absence of rotational diffusion, In the present stud y, a conjugatable, highly luminescent Re(I) metal-ligand complex, [Re( bcp)(CO)(3)(4-COOHPy)](ClO4), where bcp is 2,9-dimethyl-4,7-diphenyl-1 ,10-phenanthroline and 4-COOHPy is isonicotinic acid, has been evaluat ed for use in fluorescence polarization immunoassays (FPIs) with high- molecular-weight antigens. This Re(I) complex (Re) displays highly pol arized emission (with a maximum anisotropy near 0.3) in the absence of rotational diffusion and a long average lifetime (2.7 mu s) when boun d to human serum albumin (HSA) in oxygenated aqueous solution, The emi ssion polarization of the Re-HSA conjugate is sensitive to the binding of anti-HSA, resulting in a significant increase in anisotropy. The l abeled HSA was also used in a competition immunoassay where unlabeled HSA was also used as an antigen, These experimental results, combined with theoretical predictions, demonstrate the potential of this Re(I) metal-ligand complex as a luminescence probe in FPIs of high-molecular -weight analytes (10(5)-10(8) Da).