Development of a Simple DTT Assay for Determining the Oxidative Potential of PM2.5 Samples
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Abstract
The research aimed to determine the optimal conditions for a simplified dithiothreitol (DTT) assay using a microplate reader to assess the oxidative potential of PM2.5 samples. The method is based on measuring the rate of DTT consumption as it reacts with reactive oxygen species (ROS) present in the particulate matter over different time intervals. The remaining DTT is quantified by its absorbance after reacting with 5,5'-dithio-bis-[2-nitrobenzoic acid] (DTNB). The optimal conditions were as follows: PM2.5 samples, collected on quartz fiber filters (amount less than 0.5 mg), were extracted with 4 mL of phosphate buffer solution using a shaker at room temperature for 30 minutes. Subsequently, 100 µL of the extracted solution was aliquoted to react with 50 µL of 0.10 mM DTT solution at time intervals of 0, 5, 10, 15, 20, 25, and 30 minutes, respectively. Then, 100 µL of 0.40 mM DTNB was added, and the absorbance was measured at lmax 410 nm. The slope and intercept of the absorbance decline with reaction time were used to calculate the oxidative potential (OP). Twelve PM2.5 samples collected during the daytime in March–April 2022 in Chiang Mai Province (57.8 ± 14.4 µg/m³) were tested. The average oxidative potential of the particulate matter, calculated relative to air volume (OPv) and particulate mass (OPm), was 0.304 ± 0.133 nmol/min·m³ and 5.05 ± 1.65 pmol/min·µg, respectively, indicating low oxidative toxicity of the substances in the dust samples.
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