Song Bo, Vandevyver Caroline D B, Deiters Emmanuel, Chauvin Anne-Sophie, Hemmilä Ilkka, Bünzli Jean-Claude G
Laboratory of Lanthanide Supramolecular Chemistry, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
Analyst. 2008 Dec;133(12):1749-56. doi: 10.1039/b807959e. Epub 2008 Sep 5.
A versatile and robust method for the determination of DNA and PCR products (<500 bp) is presented, based on a mix of an Eu(III) chelate and acridine orange (AO). The nucleic acid selective stains acridine orange (AO) and ethidium bromide (EB) quench the luminescence of the bimetallic [Eu(2)(L(C2))(3)] and of other monometallic chelates such as the macrocyclic complex [Eu(L(kel))], even at very low molar ratios. Stern-Volmer plots of the metal-centered emission intensities (F(0)/F) and Eu((5)D(0)) lifetimes (tau(0)/tau) show the AO quenching being purely dynamic with K(D) = 6.7 x 10(5) M(-1) for [Eu(2)(L(C2))(3)] and 1.6 x 10(6) M(-1) for [Eu(L(kel))], and bimolecular rate constants k(q) = 2.7 x 10(8) M(-1) s(-1) and 3.4 x 10(9) M(-1) s(-1), respectively. On the other hand, EB quenching is due to both dynamic and static mechanisms. In the presence of various types of DNA > 0.1 ng microL(-1) (dsDNA, ssDNA or circular DNA), the quenched luminescence is reinstated, AO and EB intercalating into DNA, which removes the interaction with the Eu(III) complexes. The best results are obtained with [Eu(2)(L(C2))(3)]/AO with detections limits in the range 0.18-0.66 ng microL(-1); detection limits for the [Eu(L(kel))]/AO system are slightly larger; simpler monometallic Eu(III) complexes with dipicolinate derivatives do not follow suit in that they decompose in the presence of DNA. The Eu(III)/AO method is shown to be pH insensitive in the range 3-10; furthermore it is essentially insensitive to 1000-fold excesses of potential interfering substances, e.g. BSA, glucose, chelating agents and anions, alkaline earth and transition metal cations, variations in luminescence intensity being < 5%, (10 analytes) or 5-10% (4 analytes); only Co(II) and Cu(II) interfere substantially.
本文介绍了一种基于铕（III）螯合物和吖啶橙（AO）混合物的通用且强大的测定DNA和PCR产物（<500 bp）的方法。核酸选择性染料吖啶橙（AO）和溴化乙锭（EB）即使在非常低的摩尔比下也能淬灭双金属[Eu(2)(L(C2))(3)]和其他单金属螯合物（如大环配合物[Eu(L(kel))]）的发光。以金属为中心的发射强度（F(0)/F）和Eu((5)D(0))寿命（tau(0)/tau）的Stern-Volmer图表明，AO淬灭是纯粹的动态淬灭，对于[Eu(2)(L(C2))(3)]，K(D)=6.7×10(5) M(-1)，对于[Eu(L(kel))]，K(D)=1.6×10(6) M(-1)，双分子速率常数k(q)分别为2.7×10(8) M(-1) s(-1)和3.4×10(9) M(-1) s(-1)。另一方面，EB淬灭是由于动态和静态机制共同作用。在存在各种类型的DNA>0.1 ng microL(-1)（双链DNA、单链DNA或环状DNA）的情况下，淬灭的发光会恢复，AO和EB插入DNA中，从而消除与Eu(III)配合物的相互作用。使用[Eu(2)(L(C2))(3)]/AO可获得最佳结果，检测限在0.18 - 0.66 ng microL(-1)范围内；[Eu(L(kel))]/AO系统的检测限略高；含有吡啶二甲酸酯衍生物的更简单的单金属Eu(III)配合物则不然，它们在DNA存在下会分解。Eu(III)/AO方法在3 - 10的pH范围内对pH不敏感；此外，它对1000倍过量的潜在干扰物质基本不敏感，例如牛血清白蛋白、葡萄糖、螯合剂和阴离子、碱土金属和过渡金属阳离子，发光强度变化<5%（10种分析物）或5 - 10%（4种分析物）；只有Co(II)和Cu(II)有显著干扰。
