Detection and Analysis of Reactive Oxygen Species (ROS): Buffer Components Are Not Bystanders

Reactive oxygen species (ROS) play critical roles in pathophysiological processes. Therefore, there is widespread interest in learning ROS concentrations under various conditions. However, literature numbers in ROS concentration vary significantly, and most cannot be readily compared against each other, largely because of the lack of understanding of the effects of various factors that significantly impact the experimental outcome. In this study, we examine an overlooked factor: the chemical reactivity of commonly used organic buffer molecules toward ROS and how such reactivity affects the results interpretation. Specifically, we examined HEPES, Tris, MES, citrate, ammonium acetate, and phosphate-buffered saline (PBS) and found that most organic buffer components can rapidly consume NaOCl (the second most abundant ROS) and/or directly interact with certain ROS probes such as a boronate for H₂O₂ determination, leading to significant errors in experimental findings and interpretations of results. For example, 20 mM HEPES, MES, ammonium acetate, and Tris are found to consume 1 mM hypochlorite within 1 s, leading to false negative results. Additionally, these organic buffer components have been found to cause false negative results in the detection of ONOO^- when using a boronate-based probe. As such, these organic buffers should be avoided in the determination of ROS concentrations. We use these examples to draw attention to the profound effects of buffer components on ROS detection and examine chemistry issues in detail. We hope the findings described will lead to improved rigor in designing ROS experiments by considering factors that were previously considered as nothing but bystanders or benign.