Evaluating ROS Production in E. coli after ZnO Exposure

The production of ROS by E. coli ATCC 25922 after treatment with ZnO nanorods was evaluated based on the previous reports.^{33 (link)} Briefly, the adjusted bacterial culture (0.5 McFarland turbidity) in PBS buffer by Sensititre™ Nephelometer (Thermo Scientific) was treated with different concentrations of nanomaterials corresponding to 0, 125, 200, and 250 μg mL⁻¹ in the presence of 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA) (Sigma-Aldrich) at a final concentration of 30 μM in PBS buffer. The mixtures in 96-well plate were incubated at 37 °C with vigorous shaking (500 rpm) using Incu-Mixer™ MP (Benchmark Scientific) for 45 min. Then, the fluorescence intensity was measured by a FLUOstar Omega (BMG Labtech) with an excitation and emission wavelengths of 485 and 520 nm, respectively. Untreated bacterial culture was used as a negative control and the background fluorescence of PBS and autofluorescence of the bacterial cells incubated without the probe was measured to calculate the net fluorescence emitted from the assay itself. The data was further analyzed using MARS Data Analysis software (ver. 3.02 R2, BMG Labtech) and the relative ROS production of samples treated with increasing concentrations of NPs was compared to non-NP treated samples from triplicate experiments with p < 0.05.