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

Abstract

Massive production of nanomaterials posesa 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 0.5h 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.