High-throughput, semi-automated dithiothreitol (DTT) assays for oxidative potential of fine particulate matter

Fine particulate matter (PM2.5) air pollution exposure is a leading risk factor for adverse health outcomes, including cardiovascular and respiratory morbidity, and premature mortality. Quantification of PM2.5 oxidative potential (i.e., the ability of PM to promote oxidative reactions in solution) is a relatively new paradigm for exploring health risks associated with the various chemical compositions of ambient PM2.5. PM2.5 oxidative potential is commonly measured with the dithiothreitol (DTT) assay, where the DTT loss rate is measured when mixed with a PM2.5 sample extract. However, the DTT assay is time consuming and laborious, with only a few reported automation attempts. We introduce and evaluate a semi-automated DTT assay using a traditional HPLC combined with either UV/vis absorbance or electrochemical detection that has comparable accuracy and sensitivity to manual approaches. Commercial and custom-made electrochemical detectors are also compared before measuring ambient PM2.5 filter samples. The optimized, semi-automated assay can process six samples per hour (an 83% time savings compared to manual analysis). Cost becomes significant for large-scale studies and was also considered; electrochemical detection saved 40% on consumables cost compared to UV/vis detection. The presented liquid-handling automation can be applied to a variety of autosamplers in other laboratories for DTT assay semi-automation.