High levels of reactive oxygen species (ROS) can impede cellular homeostasis by increasing oxidative stress which results in lipid peroxidation, protein oxidation, and DNA damage. Recent investigations have associated the redox potential of engineered nanomaterials (ENMs) with ROS induction. These studies used plate readers and DCF, an indicating fluorescent dye, to measure ENM production of ROS. While several findings have confirmed the production of ROS by some ENMs, some data is questionable and there are contradicting results between different groups. These studies used ENMs that were either sonicated in a working solution of DCF leading to early auto-oxidation of the dye or they were prepared in a DCF-horseradish peroxidase (HRP) solution. HRP can catalyze the formation of ROS in the presence of H2O2 without ENMs. We have developed a system in which we measure the induction of ROS in real time devoid of HRP in the system or the use of a DCF solution as an ENM dispersant. Using a Promega plate reader with a dual injector system we are able to measure the fluorescence of DCF to indicate ROS induction by several ENMs with environmentally relevant doses. The injector system introduces the light-sensitive dye into wells containing ENMs without exposing it to ambient light and a second injector shoots H2O2 into the system. Through these injections, absorption of the dye ono the ENM is minimized and the oxidative reactivity of the ENM can be measured in real time. The materials were synthesized by the Engineered Nanomaterials Resource and Coordination Core (ERCC) and the physiochemical properties (i.e.: diameter, hydrodynamic diameter, zeta potential) were characterized following a standardized and meticulous protocol developed for collaborative research. Preliminary results show that metal, graphene and metal-oxide nanoparticles, ranging in diameter size and core material, result in statistically significant induction of ROS through the oxidation of DCF. Using this data, the protein corona effect on ROS induction by the nanomaterials will be compared and correlations between protein composition and cytotoxicity will be established.

Rapid microplate assay for acellular reactive oxygen species generation induced by engineered nanomaterials in real-time

256th National Meeting (2018)

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