Novel Intramolecular Energy Transfer Probe for the Detection of Benzo[a]pyrene Metabolites in a Homogeneous Competitive Fluorescence Immunoassay

Abstract

The spectroscopic properties of a novel intramolecular energy transfer probe (ET probe)--consisting of 3-hydroxybenzo[a]pyrene (3OH-BaP) as the donor covalently linked to sulforhodamine B (SRB) as the acceptor--for the detection of polycyclic aromatic hydrocarbons (PAH) antibody binding were characterized. Absorption and fluorescence spectra as well as fluorescence decay curves were recorded in methanol and aqueous solution, respectively. For comparison, the parent chromophores 3OH-BaP and SRB were investigated as well. In the case of the ET probe, a very strong fluorescence quenching of the BaP-moiety-related emission due to an efficient energy transfer (energy transfer efficiency of about 0.95 for methanol) to the SRB moiety was observed. Upon addition of the PAH antibody, the fluorescence intensity and anisotropy of the BaP moiety was drastically increased. On the other hand, the fluorescence anisotropy of the SRB moiety did not change. The anisotropy results clearly indicate the binding of the antibody. On the basis of these findings, we concluded the following model: the BaP moiety is incorporated in the antibody binding site, whereas the SRB moiety sticks out from the binding site, restricting the motion of the BaP moiety, but leaving the SRB moiety uninfluenced. More important, this structure results in a disruption of the intramolecular energy transfer. The antibody-induced disruption of the intramolecular energy transfer is envisaged as a detection scheme in a future homogeneous competitive fluorescence immunoassay (FIA). This may provide a novel general detection principle for the immunodetection of low-molecular analytes (haptens) in a homogeneous competitive FIA format.