Fluorescence Energy Transfer Immunoassay Based on a Long-Lifetime Luminescent Metal–Ligand Complex

Abstract

We describe an immunoassay based on fluorescence resonance energy transfer (FRET). The antigen was human serum albumin (HSA), which was labeled with a ruthenium-ligand complex, [Ru(bpy)2(phen-ITC)]2+. The antibody (IgG) to HSA was labeled with a nonfluorescent absorber, Reactive Blue 4. Association of the Ru-labeled HSA with the antibody was detected by three spectral parameters, a decreased quantum yield of Ru-HSA, a decrease in its fluorescence lifetime, and an increase in its fluorescence anisotropy. The steady-state anisotropy of Ru-HSA increased approximately eightfold upon binding to the antibody. These spectral effects were observed both in the direct association of the Ru-HSA with Reactive Blue 4-labeled antibody, and in a competitive assay format wherein unlabeled HSA competed with Ru-HSA for the binding sites on the antibody. Some nonspecific interactions of HSA may have occurred with Reactive Blue 4-labeled AHA, a difficulty which can be avoided with a different acceptor. The use of FRET provides a reliable means to alter the spectral properties upon antigen-antibody binding. The advantages of a ruthenium-ligand fluorophore include its long-wavelength absorption and emission, long fluorescence lifetime, and high photo-stability. Long wavelengths minimize problems of autofluorescence from biological samples, and long life-times allow off-gating of the prompt autofluorescence.

Electrochemiluminescent hybridization chip with electric field aided mismatch discrimination

“…Bis(2,2 -bipyridine)-5-amino-1,10-phenanthroline ruthenium(II) (Ru-1) was synthesized according to previously published procedures (Sprintschnik et al, 1977;GarciaFresnadillo and Orellana, 2001;Youn et al, 1995). Briefly, bipyridine and tri-chlororuthenium were heated and refluxed during 3 h in DMF.…”

Time-resolved Fluorescence Resonance Energy Transfer Assay for Point-of-Care Testing of Urinary Albumin

“…However, when an analyte is present in the circulation or in the body fluids at high concentrations, an insensitive assay is actually required. Homogeneous FRET assays with a competitive format have reportedly been used for the measurement of serum albumin (6,31 ), IgG (7 ), and thyroxine (32 ), which are all present at high concentrations in the circulation.…”

The quenching of electrochemiluminescence upon oligonucleotide hybridization

“…Ru(bpy) 3 2+ and other Ru-polypyridines have been extensively studied as sensitizers of photochemical electron (15)(16)(17)(18)(19) and energy transfer (20)(21)(22)(23)(24) processes. Long lifetimes of their excited states make them useful tools for studying electron and energy transfer mechanisms via quenching of their luminescence.…”

“…Ru(bpy) 3 2+ and its derivatives have been used as energy donors in several luminescence resonance energy transfer (LRET)-based bioassays (24)(25)(26)(27). A time-resolved immunoassay, in which Ru(bpy) 3 2+ -labelled human serum albumin was used as an energy donor and a squaranine-type cyanine label as an acceptor, has been described (25).…”