Time-Resolved Luminescence Energy Transfer Immunobinding Study Using a Ruthenium–Ligand Complex as a Donor Label

Abstract

A novel immunosystem is described that exploits the effect of luminescence energy transfer from a luminescently labeled antigen to a fluorescent antibody. A luminescent ruthenium–ligand complex (D-455) with absorption/emission maxima at 456/639 nm, respectively, was employed as the donor label, and a squaraine-type cyanine label (636/655 nm), as the fluorescent acceptor label. Specifically, the system human serum albumin (HSA)/anti-HSA was studied. HSA was labeled with the donor dye D-455, and anti-HSA was labeled with the acceptor dye A-631. On formation of the antigen–antibody complex, energy transfer occurs. The radiationless energy transfer affects both the decay time of D-455 and the intensities of the emissions of both D-455 and A-631. The decay time of around 500 ns of D-455 allows frequency–domain measurements in the low kilohertz range and therefore can be based on the use of conventional optoelectronics. This also suggests gated measurements to be performed. The major difference from existing HSA immunosystems is the use of a slow decaying ruthenium–ligand complex as the donor and of a long-wave emitting cyanine acceptor dye having a high quantum yield and a decay kinetics that is governed by the rate of energy transfer from the slow decaying donor.