Determination of nucleic acids based on the fluorescence quenching of Hoechst 33258 at pH 4.5

It was found the strong fluorescence emitted by the bis-benzimidazole derivative Hoechst 33258 at 490 nm could be efficiently quenched in pH 4.5 buffer when nucleic acids were added. Analysis of fluorescence intensity showed that the procedure was a static quenching dominated one, which was also demonstrated by the electron absorption spectra and lifetime of the excited state. The binding constant and numbers of binding sites were obtained from the Scatchard plot. The decreased fluorescence intensity was in proportion to the concentration of nucleic acids in the range 40-1800 ng ml⁻¹ for dsDNA and 26-1700 ng ml⁻¹ for ssDNA. The limits of detection were 12 and 8 ng ml⁻¹, respectively. The sensitivity of the method was about 3.4 times higher for dsDNA detection and 5.4 times higher for ssDNA detection compared with the widely used fluorescence enhancement method using the same dye. Application results to synthetic samples showed simplicity, rapidity and satisfactory reproducibility of the presented method. Measurement of real samples extracted from leaves of Crassula argentea and E. coli genome also gave satisfactory results, which were in good agreement with those obtained using spectrophotometric method.