Synchronous fluorescence determination of DNA based on the interaction between methylene blue and DNA.
The fluorescence intensity of methylene blue (MB) quenched by DNA in the pH range of 6.5-8.0 was studied with synchronous fluorescence technology. A novel method for detecting single-stranded and double-stranded DNA was developed. The decreased fluorescence intensity at 664 nm is in proportion to the concentration of DNA in the range of 0.28-11.0 micromol L(-1) for ctDNA, 0.14-8.25 micromol L(-1) for thermally denatured ctDNA and 0.28-8.25 micromol L(-1) for hsDNA. The detection limits (S/N=3) are 0.11, 0.04 and 0.04 micromol L(-1), respectively. The method is rapid, selective, and the reagents are lower toxic. It has been used for the determination of DNA in synthetic samples with good satisfaction. In addition, the interaction modes between MB and ctDNA and the mechanism of the fluorescence quenching were also discussed in detail. The experimental results from absorption spectra and fluorescence polarization indicate that the possible interaction modes between MB and DNA are the electrostatic binding and the intercalation binding.
Hu
Zhou
Z
Institute of Environmental Science, Zhejiang University, Hangzhou 310029, China.
Tong
Changlun
C
D001703:Biophysics / Q000379:methods; D002138:Calibration; D002627:Chemistry, Physical / Q000379:methods; D004247:DNA / Q000032:analysis*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000494:pharmacology; D008751:Methylene Blue / Q000494:pharmacology*; D008956:Models, Chemical; D012492:Salts / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D013053:Spectrophotometry; D055672:Static Electricity; D013997:Time Factors
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D012492:Salts; D004247:DNA; D008751:Methylene Blue