Optimization and application of the DPPH assay for evaluating reducing properties of particulate matter

Different acellular assays were recently developed to measure particulate matter (PM) oxidative potential (OP) [1]. Common OP methods include dithiothreitol assay (OPDTT), ascorbic acid assay (OPAA) and 2′,7′-dichlorofluorescin (OPDCFH). OP assays can provide observations on the relationship between PM characteristics and its ability to generate oxidative stress. However, along with oxidizing species, experimental OP values suggest the possible presence of reducing species in PM. Therefore, the obtained data drives the need to deepen knowledge on redox properties of PM. The DPPH (2,2-Diphenyl-1-picrylhydrazyl; Figure 1) assay is a commonly used spectrophotometric method to estimate antioxidant activity of several matrices (e.g. food [2], plants [3]) by measuring the decrease of absorbance at 517 nm over time. This assay estimates the overall antioxidant capacity of the samples and offers the advantages of being simple and rapid [2] and easily applicable to intensive PM monitoring campaigns. For these reasons, in this work the DPPH procedure was tailored and optimized to be applied to both PM field samples and to dusts produced by specific sources (e.g. brake dust, incinerator dust, urban dust). To the best of our knowledge, this is the first study describing the application of an antioxidant capacity assay on PM. The optimized procedure shows good analytical performance and allows the analysis of conventional 24-h airborne PM samples with a repeatability included in the range 5-10%. The assay was applied to samples (Figure 2) and compared with OP results in order to gain more knowledge about the redox properties of PM and to deepen the relationship between the composition of PM and its toxicity. First results suggest a competition between oxidizing and reducing processes during the extraction step, which needs to be carefully evaluated in interpreting the results.