A versatile method for quantification of DNA and PCR products based on time-resolved Euiii luminescence

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.