Infoscience

Journal article

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 $Eu(^5D_0)$ lifetimes ($\tau_0/\tau$) show the AO quenching being purely dynamic with $K_D = 6.7 \times 10^5 M^{-1}$ for $[Eu_2(L^{C2})_3]$ and $1.6 \times 10^6 M^{-1}$ for $[Eu(L^{kel})]$, and bimolecular rate constants $k_q = 2.7 \times 10^8 M^{-1}s^{-1}$ and $3.4 \times 10^9 M^{-1} s^{-1}$, respectively. On the other hand, EB quenching is due to both dynamic and static mechanisms. In the presence of various types of DNA > $0.1 ng \, \mu L^{-1}$ (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 $0.18\mbox{\--}0.66 ng \, \mu L^{-1}$; 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.

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