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 microseconds) 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 (10(5)-10(8) Da).

Figure 1.

Molecular-weight-dependent anisotropy for a protein-bound luminophore with luminescence lifetimes of 4, 40, 400, and 2700 ns. The curves are based on eqs 3 and 8 assuming an aqueous solution at 20 °C with a viscosity of 1 cP and $\overline{v}+h=1.9$.

Figure 2.

Molecular structure of [Re(bcp)(CO)₃(4-COOHPy)]⁺.

Figure 3.

Room temperature absorption and emission spectra of [Re(bcp)(CO)₃(4-COOHPy)]⁺ conjugated to HSA in 0.1 M PBS buffer, pH 7.0. The emission spectrum was obtained with 400 ± 4 nm excitation. The solid line shows the excitation anisotropy spectrum measured in 100% glycerol at −60 °C, with an emission wavelength of 550 ± 8 nm.

Figure 4.

Emission spectra of [Re(bcp)(CO)₃(4-COOHPy)]⁺ conjugated to HSA in 0.1 M PBS buffer, pH 7.0, equilibrated with argon or air at 20 °C. The excitation wavelength was 400 ± 4 nm.

Figure 5.

Frequency domain intensity decays of [Re(bcp)(CO)₃(4-COOHPy)]⁺ conjugated to HSA. Sample was the under same conditions as in Figure 4. The modulated excitation was centered at 390 nm (Panasonic blue LED) and a 500 nm long-pass filter was used to isolate the emission.

Figure 6.

Steady-state fluorescence polarization of Re–HSA at various concentrations of IgG specific for HSA (anti-HSA, ●) or nonspecific IgG (■) measured at 20 °C. Anisotropy is also displayed on this plot for comparative purposes. The excitation and observation wavelengths were 400 and 550 nm, respectively, with a bandpass of 8 nm.

Figure 7.

Steady-state fluorescence anisotropy of Re–HSA added to preincubated mixtures of anti-HSA with various concentrations of unlabeled HSA measured at 20 °C. The excitation and observation wavelengths were 400 and 550 nm, respectively, with a bandpass of 8 nm. Error bars represent the standard deviations of three anisotropy readings.

Scheme 1.

Intuitive Description of a Fluorescence Polarization Immunoassay (Re-L is [Re(bcp)(CO)₃(4-COOHPy)]⁺, and θ is the Rotational Correlation Time)