Institute of Biomedicine, Department of Cell Biology and Anatomy, Laboratory of Biophysics, University of Turku, Tykistökatu 6A, FI-20520, Turku, Finland.
Materials Chemistry and Chemical Analysis, Department of Chemistry, University of Turku, Vatselankatu 2, FI-20500, Turku, Finland.
Anal Chim Acta. 2019 Dec 27;1092:93-101. doi: 10.1016/j.aca.2019.09.045. Epub 2019 Sep 19.
A novel homogeneous assay system QTR-FRET (Quencher modulated Time-Resolved Förster Resonance Energy Transfer) combining quenching resonance energy transfer (QRET) and time-resolved Förster resonance energy transfer (TR-FRET) was developed to reduce background signal in the conventional energy transfer applications. The TR-FRET functionality is often limited by the lanthanide donor background signal leading to the use of low donor concentration. QTR-FRET reduces this background by introducing soluble quencher molecule, and in this work the concept functionality was proven and compared to previously introduced QRET and TR-FRET technologies. Comparison was performed with three different Eu-chelates exhibiting different luminescent lifetime and stability. The side-by-side comparison of the three signaling systems and Eu-chelates was demonstrated in a model assay with Eu-chelate conjugated biotin and streptavidin (SA) or Cy5-SA conjugate. Comparison of the methodologies showed increased signal-to-background ratios when comparing QTR-FRET to TR-FRET, especially at high Eu-biotin concentrations. Quenching the non-bound Eu-biotin improved the assay performance, which suggests that an improved assay performance can be attained with the QTR-FRET method. QTR-FRET is expected to be especially useful for Eu-labeled ligands with low affinity or assays requiring high Eu-ligand concentration. The QTR-FRET indicated potential for multi-analyte approaches separately utilizing the direct QRET-type Eu-chelate signal and energy transfer signal readout in a single-well. This potential was hypothesized with Avi-KRAS nucleotide exchange assay as a second biologically relevant model system.
一种新的均相分析系统 QTR-FRET（淬灭调制时间分辨荧光共振能量转移）结合了淬灭共振能量转移（QRET）和时间分辨荧光共振能量转移（TR-FRET），旨在减少传统能量转移应用中的背景信号。TR-FRET 的功能通常受到镧系元素供体背景信号的限制，导致使用低供体浓度。QTR-FRET 通过引入可溶性淬灭分子来减少这种背景，在这项工作中证明了该概念的功能，并与之前介绍的 QRET 和 TR-FRET 技术进行了比较。使用三种不同的 Eu-螯合物进行了比较，这些螯合物具有不同的发光寿命和稳定性。在 Eu-螯合物缀合生物素和链霉亲和素（SA）或 Cy5-SA 缀合物的模型测定中，并排比较了三种信号系统和 Eu-螯合物。与 TR-FRET 相比，QTR-FRET 时比较了方法学，显示出信号与背景的比值增加，尤其是在高 Eu-生物素浓度时。猝灭未结合的 Eu-生物素可改善测定性能，这表明 QTR-FRET 方法可获得更好的测定性能。QTR-FRET 有望特别适用于具有低亲和力的 Eu 标记配体或需要高 Eu-配体浓度的测定。QTR-FRET 具有利用单个孔中单个直接 QRET 型 Eu-螯合物信号和能量转移信号读出分别进行多分析物方法的潜力。该潜力假设 Avi-KRAS 核苷酸交换测定作为第二个生物学相关模型系统。
