3.6. Escherichia coli Growth Inhibition Assay

The antibacterial activity of QPEI-functionalized oxCNTs was obtained by a bacteria growth inhibition assay. Escherichia coli XL1-blue bacteria expressing red fluorescent protein (RFP), from a plasmid-encoded gene, were grown in Luria–Bertani (LB) broth at 37 °C overnight, in a Stuart SI500 orbital shaker at approximately 200 rpm shaking speed in aerobic conditions. The culture was subsequently diluted to an optical density (O.D.) of 0.4 at 600 nm. The QPEI-functionalized oxCNTs were homogeneously dispersed in distilled water by sonication and added to the bacterial culture at concentrations ranging from 5 to 400 μg/mL. The assay was performed in a 96-well plate format in a 200 μL final volume. Fluorescence intensity over growth of untreated bacteria revealed that the optimum incubation time was 6 h, as the intensity reached a plateau (Figure S4). Thus, plates were incubated at 37 °C, shaking at 100 rpm in aerobic conditions for 6 h, and bacterial growth was monitored using the fluorescence intensity of red fluorescent protein, which was recorded at an emission wavelength of 590 nm by an Infinite M200 plate reader (Tecan group Ltd., Männedorf, Switzerland) at an excitation wavelength of 545 nm. In order to eliminate the effect of CNTs in the measured intensities, the initial values (at 0 h), although minor compared to those obtained after 6 h, were subtracted from the final measurements. For each treatment eight replicates were used and three independent experiments were performed. Untreated bacteria were used as control, representing 100% fluorescence intensity in Figure 8.