A Resonance Energy Transfer Immunoassay Based on a Thiol-Reactive Ruthenium Donor Dye and a Longwave-Emitting Acceptor

Abstract

A novel immunoassay is described that applies a thiol-reactive ruthenium metal–ligand complex as the donor dye in a luminescence energy transfer (LET) detection scheme. Unlike amine-reactive labels, the LET with a thiol label allows improved specificity and better reproducibility of labelling positions on proteins, because the number of reactive thiol groups of proteins is distinctly smaller. This helps to reduce the risk of over-labelling and self-quenching of the fluorophore. The synthesis of the thiol label was significantly improved, resulting in almost quantitative yields of pure product. The absorption and emission maxima of the ruthenium donor dye are at 460 nm and 600 nm, respectively, and a Stokes' shift of 140 nm warrants distinct separation of excitation and emission wavelengths even in turbid samples. A cyanine dye with an absorption maximum at 642 nm was chosen as the acceptor label because it has good overlap with the emission spectrum of the donor label. The emission of the acceptor peaks at 660 nm, thus further increasing the Stokes' shift (to an overall 200 nm). The quantification of anti-HSA with the LET immunoassay is possible with this new approach at concentrations as low as 220 pmol L⁻¹.