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 EuIII chelate and acridine orange (AO). The nucleic acid selective stains acridine orange (AO) and ethidium bromide (EB) quench the luminescence of the bimetallic [Eu2(LC2)3] and of other monometallic chelates such as the macrocyclic complex [Eu(Lkel)], even at very low molar ratios. Stern–Volmer plots of the metal-centered emission intensities (F0/F) and Eu(5D0) lifetimes (τ0/τ) show the AO quenching being purely dynamic with KD = 6.7 × 105 M−1 for [Eu2(LC2)3] and 1.6 × 106 M−1 for [Eu(Lkel)], and bimolecular rate constants kq = 2.7 × 108 M−1 s−1 and 3.4 × 109 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 μL−1 (dsDNA, ssDNA or circular DNA), the quenched luminescence is reinstated, AO and EB intercalating into DNA, which removes the interaction with the EuIII complexes. The best results are obtained with [Eu2(LC2)3]/AO with detections limits in the range 0.18–0.66 ng μL−1; detection limits for the [Eu(Lkel)]/AO system are slightly larger; simpler monometallic EuIII complexes with dipicolinate derivatives do not follow suit in that they decompose in the presence of DNA. The EuIII/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 CoII and CuII interfere substantially.