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 --> BHQ energy transfer is efficient, decreasing both the terbium emission intensity and lifetime. Upon hybridization or nuclease digestion, which spatially separate the Tb and BHQ 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/dark quencher probes in energy transfer assays is an effective means of improving sensitivity.
Johansson
Mary Katherine
MK
Biosearch Technologies, 81 Digital Drive, Novato, California 94949, USA. marykat@biosearchtech.com
Cook
Ronald M
RM
Xu
Jide
J
Raymond
Kenneth N
KN
D000577:Amides / Q000138:chemical synthesis / Q000737:chemistry; D002614:Chelating Agents / Q000138:chemical synthesis / Q000737:chemistry*; D004735:Energy Transfer; D008163:Luminescent Measurements; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000737:chemistry*; D009942:Organometallic Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D010795:Phthalic Acids / Q000138:chemical synthesis / Q000737:chemistry; D011071:Poly T / Q000737:chemistry; D013725:Terbium / Q000737:chemistry*
D000577:Amides; D002614:Chelating Agents; D015345:Oligonucleotide Probes; D009942:Organometallic Compounds; D010795:Phthalic Acids; D013725:Terbium; C059768:isophthalate; D011071:Poly T