Time gating improves sensitivity in energy transfer assays with terbium chelate/dark quencher oligonucleotide probes
Time gating improves sensitivity in energy transfer assays with terbium chelate/dark quencher oligonucleotide probes
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DOI：
10.1021/ja0452368
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发表时间：
2004-12-22
影响因子：
15
通讯作者：
Raymond, KN
中科院分区：
文献类型：
--
作者：
Johansson, MK;Cook, RM;Raymond, KN
镧系元素作为生物标记是有吸引力的，因为它们的长发光衰减率允许时间门控检测，这将背景散射和荧光从镧系元素发射中分离出来。以四异眼酰胺（TIAM）螯合物为基础，制备了稳定、高发光的铽配合物，并与多芳偶氮暗猝灭剂（称为黑洞猝灭剂或BHQ）配对，制备了一系列无二级结构的5'TIAM(Tb)/3'BHQ双标记寡核苷酸探针。铽/BHQ探针内的发光猝灭效率很大程度上取决于铽-BHQ的距离。在一个完整的探针中，铽- bhq的平均距离很短，而Tb -> BHO的能量传递效率很高，从而降低了铽的发射强度和寿命。杂交或酶切使Tb和BHO在空间上分离后，Tb的发光强度和寿命增加。因此，时间门控检测增加了未淬火探针/淬火探针的发射强度比，因为淬火物质的寿命较短。当使用门控检测时，40-mer探针在消化时具有3倍的稳态发光增加，具有50倍的增加。该研究表明，在能量传递分析中使用镧系/克拉克淬灭探针进行时间门控是提高灵敏度的有效手段。
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.