| Abstract
| - 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. Astable and highly luminescent terbium complex based on a tetraisophthalamide (TIAM) chelate is pairedwith a polyaromatic-azo dark quencher (referred to as a Black Hole Quencher or BHQ) to prepare a seriesof 5‘TIAM(Tb)/3‘BHQ dual-labeled oligonucleotide probes with no secondary structure. Luminescencequenching efficiency within terbium/BHQ probes is very dependent on the terbium−BHQ distance. In anintact probe, the average terbium−BHQ distance is short, and Tb → BHQ energy transfer is efficient,decreasing both the terbium emission intensity and lifetime. Upon hybridization or nuclease digestion, whichspatially separate the Tb and BHQ moieties, the Tb luminescence intensity and lifetime increase. As aresult, time-gated detection increases the emission intensity ratio of the unquenched probe/quenched probedue 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 studydemonstrates that time gating with lanthanide/dark quencher probes in energy transfer assays is an effectivemeans of improving sensitivity.
|
