DNA and RNA play important roles in living organisms as components of substances such as coenzymes, high-energy phosphate compounds, and genes. The determination of nucleic acids is very important in many biological studies. The conventional determination of nucleic acids involves absorption at 260 nm, but the sensitivity is poor and the method lacks specificity. Although a fluorescent-dye technique using ethidium bromide has been developed, an accurate determination is difficult because the fluorescence intensity varies along with differences in the conformation of nucleic acids. Now the reaction between adenine and glyoxal has already been studied 1 , but the reaction time was long and the background noise was high. From the chemical point of view, a major challenge is the development of a direct chemiluminescence (CL) assay that requires minimum physical or chemical pretreatment of the sample.In this research, it was discovered that chemiluminescence was generated during oxidation of Ru(bipy) 3 2+ by cerium(IV) in a sulfuric acid medium. The emission intensity and the sensitivity were greatly enhanced when nucleic acids were also present in reaction system. Corresponding to the interferences of coexisting subtances, six synthetic samples are made; the results of determination are satisfactory. This method is simple, rapid and specific.
ExperimentalApparatus and reagents LKB-1251 luminometer, dispenser SVD and dispenser controller DC (Pharmacia LKB Biotechnology AB, Sweden), with an Epson LX-800 printer (Seiko Epson, Japan) were used.Stock solutions of nucleic acids were prepared by directly dissolving a certain amount of calf thymus DNA (Sigma), fish sperm DNA (Sigma) and yeast RNA (Sigma) in water at 0 -4˚C. Their concentrations were determined by the absorbance at 260 nm. The working solutions were prepared by diluting the stock solution to a proper concentration. Thermally denatured DNA was prepared by submerging the volumetric flask containing DNA solution in boiling water for 15 min, and then chilling in ice water for 10 min to prevent the renaturation of DNA. A 1.2 mg/ml stock solution of Ru(bipy) 3 Br 2 (prepared in our laboratory 2 ) was prepared by dissolving a required amount of the salt in water and diluting with water to 1000 ml.Cerium(IV) sulfate, Ce(SO 4 ) 2 ·4H 2 O stock solution, (0.01 mol/l), was prepared from commercial products (Shanghai Chemical Co. P. R. China) by dissolving a required amount of the salt in a 0.1 mol/l solution of sulfuric acid. The working solution 1.0 mmol/l was prepared by dissolving a stock solution in 0.1 mol/l solution of sulfuric acid.All other reagents were of analytical grade or better, and water used was doubly distilled in a fused-silica apparatus. A direct chemiluminescent method for the determination of nucleic acids has been developed. The method is based on the enhancement of nucleic acids on the chemiluminescence light emission of the reaction between Ru(bipy)3 2+ and Ce(IV). Under the optimum conditions, the calibration graphs are linear over the ranges 7...