Optimization of the Measurement of Particle-Bound Reactive Oxygen Species with 2′,7′-dichlorofluorescin (DCFH)

Abstract

The 2′,7′-dichlorofluorescin (DCFH) assay is widely used to measure particle-bound reactive oxygen species (ROS), which are considered as a major contributor leading to the adverse health effects upon exposure to atmospheric particulate matter. DCFH, a non-fluorescent substance that can be oxidized to highly fluorescent dichlorofluorescein (DCF) in the presence of horseradish peroxidase (HRP), is usually used as a probe for ROS determination due to its response to diverse and relevant oxidant species. However, there is limited literature that reports the effects of different experimental conditions in the performance of this assay. In our work, various experimental conditions, such as pH value, incubation temperature, reagent concentration and stability, reaction time, linearity range, and extraction method, were examined and optimized as a pilot study for developing an online system for atmospheric ROS measurement. The results showed that pH value, reagent concentration, and extraction method significantly affect the performance of DCFH assay, while incubation at a specified temperature (37 °C) did not increase the oxidization extent of DCFH. After optimization, some practical samples were measured using different experimental parameters to check the performance of the optimized assay. The comparisons of these measurements showed that optimization can greatly improve the detection limit and reproducibility of the DCFH assay, which can then be employed to better the accuracy of offline and online ROS measurement.