State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Changsha, China.
Anal Chem. 2011 Feb 15;83(4):1356-62. doi: 10.1021/ac102710w. Epub 2011 Jan 19.
Ultrasensitive fluorescent analysis or monitoring of significant molecules in complex samples is important for many biological studies, clinical diagnosis, and forensic investigations, the major obstacle for which is the background signals from ubiquitous endogenous fluorescent components of the environments. Herein, a room-temperature phosphorescence (RTP)-based molecular beacon (MB), employing a Eu(3+) complex of chlorosulfonylated tetradentate β-diketone (L) and the quencher BHQ-2, was engineered for highly sensitive detection of DNA sequences in biological fluids. Complexation of Eu(3+) with the ligand L formed a strongly luminescent complex EuL(2). But when EuL(2) and BHQ-2 were labeled to two ends of a DNA molecule with hairpin structure, the luminescence of EuL(2) was quenched by BHQ-2 due to the stem-closed conformation of the beacon. Due to very low background luminescence from the probe molecule, >200-fold signal enhancement was achieved when nanomolar target sequence was introduced. This sensitivity is about 20-fold higher than the level achieved with conventional fluorescence-based molecular beacons. Furthermore, because the Eu(3+) complex has a much longer luminescence lifetime (≈0.8 ms) than that of the background (<10 ns), RTP measurements were used to directly detect as low as 500 pM DNA in cell media quantitatively without any sample pretreatment.
在复杂样本中对重要分子进行超灵敏荧光分析或监测对于许多生物学研究、临床诊断和法医学调查非常重要，但主要的障碍是环境中无处不在的内源性荧光成分的背景信号。在此，设计了一种基于室温磷光（RTP）的分子信标（MB），该信标采用氯磺酰化四齿β-二酮（L）和淬灭剂 BHQ-2 的 Eu(3+) 配合物，用于在生物流体中高度灵敏地检测 DNA 序列。Eu(3+)与配体 L 的络合形成强发光的 EuL(2)配合物。但是，当 EuL(2)和 BHQ-2 标记到具有发夹结构的 DNA 分子的两个末端时，由于信标的茎闭合构象，EuL(2)的发光被 BHQ-2 猝灭。由于探针分子的背景发光非常低，当引入纳摩尔级别的靶序列时，实现了>200 倍的信号增强。这种灵敏度比传统荧光分子信标高约 20 倍。此外，由于 Eu(3+) 配合物的荧光寿命（≈0.8 ms）比背景（<10 ns）长得多，因此可以直接检测细胞培养基中低至 500 pM 的 DNA，而无需任何样品预处理，定量检测。
