Interference from carbonaceous particles in reactive oxygen species measurements: A case study with Printex 90 and BPEAnit

Abstract. Reactive oxygen species (ROS) and oxidative potential (OP) are strongly associated with adverse health outcomes and have emerged as a more health-relevant metric for assessing the toxicity of air pollution than particle number or mass concentration. Acellular ROS and OP measurements rely on specific chemical probes that enable quantification through fluorescence or absorbance. During measurements using a real-time ROS monitor with 9,10-bis (phenylethynyl) anthracene-nitroxide (BPEAnit) as the probe, we observed a reduction in the fluorescent response when sampling particles containing high levels of black carbon, ostensibly indicating negative ROS values, although there is scientific evidence that black carbon can generate ROS. In this study, we investigated this interference from black carbon particles in BPEAnit solution in order to understand the underlying mechanisms and propose a method of correcting for the artefacts during ROS measurements.

We suspended Printex 90 particles, known to induce OP, at three different concentrations (1 µg mL^-1, 5 µg mL^-1 and 25 µg mL^-1) in solutions containing either BPEAnit, its fluorescent and non-reactive methylated derivative BPEAnit-Me, or the solvent used in the probe solutions, dimethyl sulfoxide (DMSO), alone. The fluorescence of the suspensions was measured in the flow-through measurement cell from the real-time ROS monitor at different times after the addition of the particles. For suspension in BPEAnit and BPEAnit-Me, we found that fluorescence decreased with increasing particle concentrations, whereas no decrease was observed in solvent-only suspensions. The fluorescence response of BPEAnit with particles was consistently higher than for the same particle concentrations in BPEAnit-Me. Based on the results, we suggest that the main mechanism for the decrease in fluorescence is interactions between the particle surfaces and BPEAnit/BPEAnit-Me, and not light scattering or absorption. We attribute the difference in fluorescence between BPEAnit-Me and BPEAnit to ROS formation.

The observed interaction mechanisms are likely not specific to BPEAnit, and similar artefacts should be considered when assessing ROS or OP of carbonaceous, and possibly other insoluble particles, in both online and offline assays. For online ROS measurements with BPEAnit, we suggest regular use of BPEAnit-Me to detect and potentially correct for artefacts caused by insoluble particles.