Improved detection of reactive oxygen species by DCFH-DA: New insight into self-amplification of fluorescence signal by light irradiation

Dichlorodihydrofluorescein diacetate (DCFH-DA) has been widely used in detection of reactive oxygen species (ROS). Unfortunately, the detection process is easily affected by light irradiation during measurement, which cannot be overlooked especially for samples with a small amount of ROS. In the study, we revealed several factors that influence the ROS detection process and developed a new ROS detection method by DCFH-DA with higher stability and sensitivity. After light irradiation, the fluorescence signal of DCFH-DA solution increased significantly with pH value higher than 7.4. The increase of fluorescence signal was attributed to a selfoxidization process of DCFH-DA, which transferred dichlorodihydrofluorescein (DCFH) to 2?,7?-dichlorofluorescein (DCF) with the presence of DCF. At the beginning stage of light irradiation, i.e., when the amount of DCF was negligible compared to DCFH (DCFH-DA), the ratio of fluorescence density between samples keeps unchanged after light irradiation. On this basis, we developed a stable and sensitive ROS detection method via light irradiation. The method was successfully applied to detect ROS in HeLa cells which was induced by hydrogen peroxide (H2O2) and plastic nanoparticles. A new insight into the proportional increase of fluorescence signal by light irradiation was revealed.

Automated summary

The study identified factors influencing the detection process of ROS by DCFH-DA and developed a new method with higher stability and sensitivity. A significant increase in fluorescence signal of DCFH-DA solution under light irradiation was observed, leading to the development of a reliable and sensitive ROS detection method. This method successfully detected ROS in HeLa cells induced by H2O2 and plastic nanoparticles, providing new insights into the proportional increase of fluorescence signal under light irradiation.