Use of a Long-Lifetime Re(I) Complex in Fluorescence Polarization Immunoassays of High-Molecular-Weight Analytes

Abstract

We describe a new class of fluorescence polarization immunoassays based on the luminescence from a Re(I) metal−ligand complex. Re(I) complexes are extremely photostable and possess useful photophysical properties including long lifetimes, high quantum yields, and high emission polarization in the absence of rotational diffusion. In the present study, 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 evaluated for use in fluorescence polarization immunoassays (FPIs) with high-molecular-weight antigens. This Re(I) complex (Re) displays highly polarized emission (with a maximum anisotropy near 0.3) in the absence of rotational diffusion and a long average lifetime (2.7 μs) when bound to human serum albumin (HSA) in oxygenated aqueous solution. The emission polarization of the Re−HSA conjugate is sensitive to the binding of anti-HSA, resulting in a significant increase in anisotropy. The labeled 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 (10⁵−10⁸ Da).