Dosage- and time-dependent antibacterial effect of zinc oxide nanoparticles determined by a highly uniform SERS...

Massive production of nanomaterials poses a high risk to environmental ecology and human health. However, comprehensive understanding of nanotoxicity is still a major challenge due to the limitations of assessment methods, especially at the molecular level. We developed a new, sensitive, and robust fingerprinting surface-enhanced Raman spectroscopy (SERS) approach to interrogate both dose- and time-dependent phenotypic bacterial responses to zinc oxide nanoparticles (ZnO NPs). SERS enhancement was provided by biocompatible Au NPs. Additionally, a novel vacuum filtration-based strategy was adopted to fabricate bacterial samples with highly uniform SERS signals, ensuring the acquisition of robust and independent spectral changes from ZnO NPs-impacted bacteria without undesirable spectral variations. Combined with multivariate analysis, clear and informative spectral alteration profiles were obtained. Much greater alterations were found in low-dose ranges than high-dose ranges, indicating a reduction in the bioavailability of ZnO NPs with doses. Time-resolved bacterial responses provided important information on toxic dynamics, i.e., rapid action of ZnO NPs within 0.5 h was identified, and ZnO NPs at low doses and long exposure time exerted similar effects to high doses, indicating the concerns associated with low-dose exposure. Further analysis of biochemical changes revealed metabolic activity decrease over both incubation time and doses. Meanwhile, a short-term protection strategy of bacteria by producing lipid-containing outer membrane vesicles to mitigate the cell of toxic NPs was suggested. Finally, Zn(2+) ions released from NPs were demonstrated to be irrelevant to bacterial responses on both dose and time scales. The new SERS methodology can potentially profile a large variety of toxic NPs and advance our understanding of nanotoxicity. Graphical Abstract A highly uniform SERS signal of bacteria negating undesired spectral variation via a novel vacuum filtration-based strategy, combined with multivariate PCA-LDA analysis, was utilized to interrogate both dose- and time-dependent antibacterial effect of zinc oxide nanoparticles, and can be extended to a variety of other toxic nanoparticles.