An Assessment of Fluorescence- and Absorbance-Based Assays to Study Metal-Oxide Nanoparticle ROS Production and Effects on Bacterial Membranes

The production and inevitable release of engineered nanoparticles requires rapid approaches to screen for their potential effects in environmental organisms, including bacteria. In bacteria, engineered nanoparticle effects can initiate at the cell membrane, for example by structurally damaging membranes or inhibiting energy transduction. Commercially available fluorescence- and absorbance-based assays could allow for rapidly assaying engineered nanoparticle effects on bacterial membranes, but there are limitations, including that: 1) assays are not currently configured to operate as part of a comprehensive high-throughput screening system, since assay conditions vary widely and formats are mostly high-volume and thus low-throughput, and; 2) engineered nanoparticles can interfere with assay reagents or function, yielding false-negative or -positive outcomes. Here, key assays to study reactive oxygen species (total ROS, and superoxide) production, and impacts on bacterial membrane integrity, membrane potential, and electron transport chain activity, are assessed for their potential use as a comprehensive system to test for nanoparticle effects in bacteria. To address (1), assays are adapted for simultaneous use in 96-well microplates under harmonized conditions. To address (2), a general scheme to test for engineered nanoparticle interferences with assay reagents and function is conceived, and used to study assay interferences by three nanoscale metal-oxides: nano-TiO2, nano-CeO2, and nano-ZnO. The results show that the selected assays can be used as a suite, and that nanoparticle interferences, when they occur, can be systematically investigated and often accounted for.