Laser induced-thermal lens spectrometry for determination of DNA based on the interaction with methylene blue

Determination of DNA is of great importance in biochemistry, biotechnology, pharmacodynamics, and many other areas. However, the fluorometric methods have attracted more attention. It is well known that many of these fluorescent DNA probes are highly toxic and mutagenic [1]. In this study, the application of thermal lens spectrometry (TLS) [2] to determination of DNA based on the interaction between methylene blue and DNA was examined. The thermal lens technique is based on measurement of the temperature rise that is produced in a sample as a result of nonradiative relaxation of the energy absorbed from a laser. Methylene blue (MB) is a phenothiazinyl dye, which can interact easily with DNA, has a low toxicity and has been used as a stain agent for DNA [3-4]. The fluorescence intensity of methylene blue (MB) quenched by DNA in the pH range of 6.5–8.0. When fluorescence of a fluorophore decreased by a quencher, the energy of fluorescence radiation turns to internal energy and eventually increases the thermal energy. In this work, the fluorescence intensity of MB was quenched by DNA so TLS signal was inhanced and can be used for determination of DNA. The maximum excitation wavelength of MB is 664 nm therefore to obtain TLS signal, the diode laser (665 nm, 50 mW) was used as pump/probe source. The increased TLS intensity at 664 nm is in proportion to the concentration of DNA. The factors affecting the determination of DNA based on its interaction with methylene blue were investigated and optimized. These factors include pH, methylene blue concentration, salt concentration and stability test. This method is a promising tool for studying the biological properties of DNA and determining DNA.