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 self-oxidization 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 (H 2 O 2 ) and plastic nanoparticles. A new insight into the proportional increase of fluorescence signal by light irradiation was revealed.