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research article

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

Song, Bo  
•
Vandevyver, Caroline D. B.  
•
Deiters, Emmanuel
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2008
Analyst

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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Type
research article
DOI
10.1039/b807959e
Web of Science ID

WOS:000261005100015

Author(s)
Song, Bo  
Vandevyver, Caroline D. B.  
Deiters, Emmanuel
Chauvin, Anne-Sophie  
Hemmilä, lkka
Bünzli, Jean-Claude G.  
Date Issued

2008

Published in
Analyst
Volume

133

Start page

1749

End page

1756

Editorial or Peer reviewed

REVIEWED

Written at

EPFL

EPFL units
LCSL  
Use this identifier to reference this record
https://infoscience.epfl.ch/handle/20.500.14299/31367
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