Determination of Micro Amounts of Nucleic Acids Based on Shifting the Aggregate‐Monomer Equilibrium of Fluorescent Dye

Abstract

Abstract A sensitive spectrofluorimetric method for the determination of nucleic acids based on shifting the aggregate‐monomer equilibrium of the fluorescent dye phenosafranine (PF) was proposed. Formation of aggregate induced by the pre‐micellar aggregation of an anionic surfactant, sodium dodecyl sulfate (SDS), was observed. The possibility of using the in situ–formed aggregate as a fluorescent probe for nucleic acids was also studied. The results showed that the fluorescence intensity of the system increased dramatically when nucleic acids were added to the solution. The fluorescence enhancement effect was thought to be based on the nucleic acid‐modulated shift of the aggregate‐monomer equilibrium of PF in the anionic surfactant solution. Intercalation of the monomer in nucleic acids caused the dissociation of the aggregate and led to a very high fluorescence enhancement. It seemed that the aggregate molecules acted as a source of monomer molecules ready for interaction with nucleic acids. A linear dependence of fluorescence intensity enhancement on the concentration of fish sperm DNA (fsDNA) over the range 0.04–20 µg/mL fsDNA allowed sensitive quantification of fsDNA by a simple fluorescence method. The detection limit was 17 ng/mL and the RSD was 1.2% for 2 µg/mL fsDNA (n=7). Furthermore, calibration graphs for calf thymus DNA (ctDNA) and yeast RNA were also obtained.