Determination of DNA and RNA by their quenching effect on the fluorescence of the Tb 3+-Tiron complex

Abstract

The fluorescence quenching of the Tb 3+-Tiron complex by nucleic acids is reported. Studies involving calf thymus(CT) DNA, salmon(SM) DNA, herring sperm(HS) DNA and yeast RNA revealed that they compete with Tiron for the Tb 3+ ion, which resulted in the fluorescence quenching of the Tb 3+-Tiron complex because the Tb 3+-nucleic acid complex is non-fluorescent under the same conditions. The ratio of molar concentrations of Tb 3+ and Tiron appeared to be a key factor, a Tb 3+: Tiron ratio of 3: 10 being the most suitable. The maximum fluorescence was produced at pH 6.9, with maximum excitation and emission wavelengths at 317 and 546 nm, respectively. Under optimal conditions, the differential value of fluorescence intensity in the absence and presence of nucleic acids was proportional to the concentration of nucleic acids over the range of 0.005–1.0 μg ml −1 for CT DNA, SM DNA, HS DNA, and 0.005–0.7 μg ml −1 yeast RNA. The detection limits were 1 ng ml −1 for CT DNA, 1 ng ml −1 for SM DNA, 0.9 ng ml −1 for HS DNA, and 0.6 ng ml −1 for yeast RNA, respectively. The relative standard deviations (6 replicates) were within 2.5% in the middle of the linear range. The mechanism for the fluorescence quenching was also studied.