Journal of the American Chemical Society, Vol.126, No.50, 16451-16455, 2004
Time gating improves sensitivity in energy transfer assays with terbium chelate/dark quencher oligonucleotide probes
Lanthanides are attractive as biolabels because their long luminescence decay rates allow time-gated detection, which separates background scattering and fluorescence from the lanthanide emission. A stable and highly luminescent terbium complex based on a tetraisophthalamide (TIAM) chelate is paired with a polyaromatic-azo dark quencher (referred to as a Black Hole Quencher or BHQ) to prepare a series of 5'TIAM(Tb)/3'BHQ dual-labeled oligonucleotide probes with no secondary structure. Luminescence quenching efficiency within terbium/BHQ probes is very dependent on the terbium-BHQ distance. In an intact probe, the average terbium-BHQ distance is short, and Tb --> BHO energy transfer is efficient, decreasing both the terbium emission intensity and lifetime. Upon hybridization or nuclease digestion, which spatially separate the Tb and BHO moieties, the Tb luminescence intensity and lifetime increase. As a result, time-gated detection increases the emission intensity ratio of the unquenched probe/quenched probe due to the shorter lifetime of the quenched species. A 40-mer probe that has a 3-fold increase in steady-state luminescence upon digestion has a 50-fold increase when gated detection is used. This study demonstrates that time gating with lanthanide/clark quencher probes in energy transfer assays is an effective means of improving sensitivity.