Stopped-flow kinetic method for the fluorimetric determination of DNA traces in biological samples based on the interaction long-wavelength fluorophor, surfactant and nucleic acid.
A simple and rapid method for the determination of DNA, involving the interaction between a surfactant, a long-wavelength fluorophor (LWF) and the nucleic acid, is presented. Different chemical systems based on the local effective charge of the surfactant/LWF system with DNA were tested, choosing cetyltrimethyl ammonium bromide (CTAB) and indocyanine green (ICG) for the development of the method. The fluorescence of ICG increases in the presence of CTAB, but it rapidly decreases in the presence of deoxyribonucleic acid. The initial reaction-rate (v(0)) and signal at a prefixed-time (DeltaIF(20)) are monitored at 780 and 802 nm as excitation and emission wavelengths, respectively, using stopped-flow mixing technique, which makes the method applicable to automate routine analysis. Each measurement was obtained in about 30 s, being the integration time 0.1 s. The dynamic range of the calibration graph was 10-1500 ng mL(-1), with a detection limit of 5 ng mL(-1). The precision of the method, expressed as relative standard deviation, ranged between 2.1% and 4.5%. After a sample treatment consisting on a conventional extraction, the method was applied to the determination of DNA in several samples from different biological materials.
Manzano-Ayala
H R
HR
Department of Analytical Chemistry, Faculty of Sciences, University of Córdoba, Marie Curie Annex Building, Campus of Rabanales, E-14071 Córdoba, Spain.
Fernández Romero
J M
JM
Gómez-Hens
A
A
D004247:DNA / Q000032:analysis*; D005456:Fluorescent Dyes / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010957:Plasmids / Q000032:analysis; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D013501:Surface-Active Agents / Q000032:analysis*
D005456:Fluorescent Dyes; D013501:Surface-Active Agents; D004247:DNA