Cobalt Release from a Nanoscale Multiphase Lithiated Cobalt Phosphate Dominates Interaction with Shewanella oneidensis MR-1 and Bacillus subtilis SB491.

Cobalt phosphate engineered nanomaterials (ENMs) are an important class of materials that are used as lithium ion battery cathodes, catalysts, and potentially as super capacitors. As production of these nanomaterials increases, so does the likelihood of their environmental release; however, to date, there are relatively few investigations of the impact of nanoscale metal phosphates on biological systems. Furthermore, nanomaterials used in commercial applications are often multiphase materials, and analysis of the toxic potential of mixtures of nanomaterials has been rare. In this work, we studied the interactions of two model environmental bacteria, Shewanella oneidensis MR-1 and Bacillus subtilis, with a multiphase lithiated cobalt phosphate (mLCP) nanomaterial. Using a growth-based viability assay, we found that mLCP was toxic to both bacteria used in this study. To understand the observed toxicity, we screened for production of reactive oxygen species (ROS) and release of Co2+ from mLCP using three abiotic fluorophores. We also used Newport Green DCF dye to show that cobalt was taken up by the bacteria after mLCP exposure. Using transmission electron microscopy, we noted that the mLCP was not associated with the bacterial cell surface. In order for us to further probe the mechanism of interaction of mLCP, the bacteria were exposed to an equivalent dose of cobalt ions that dissolved from mLCP, which recapitulated the changes in viability when the bacteria were exposed to mLCP, and it also recapitulated the observed bacterial uptake of cobalt. Taken together, this implicates the release of cobalt ions and their subsequent uptake by the bacteria as the major toxicity mechanism of mLCP. The properties of the ENM govern the release rate of cobalt, but the toxicity does not arise from nanospecific effects-and importantly, the chemical composition of the ENM may dictate the oxidation state of the metal centers and thus limit ROS production.

Linking nanomaterial properties to biological outcomes: analytical chemistry challenges in nanotoxicology for the next decade.

The field of nanotoxicology has evolved rapidly in the past two decades. Starting from simple nanomaterials and established toxicity assays, researchers' foci have shifted towards understanding the mechanisms underlying nanotoxicity. Furthermore, an important goal has been linking nanomaterial properties to biological responses to build predictive models for safer nanomaterial design. Here, we provide our perspectives, as analytical chemists, on the analytical challenges in nanotoxicology as the field is entering its third decade. We have identified these challenges to include understanding causal relationships in mechanistic studies of nanotoxicity, overcoming nanomaterial interferences for accurate nanotoxicity assays, connecting nanoparticle interactions to cellular responses at the single-cell level, and making chemical measurements at the nano-bio interface in real-time and in situ.

Growth-Based Bacterial Viability Assay for Interference-Free and High-Throughput Toxicity Screening of Nanomaterials.

Current high-throughput approaches evaluating toxicity of chemical agents toward bacteria typically rely on optical assays, such as luminescence and absorbance, to probe the viability of the bacteria. However, when applied to toxicity induced by nanomaterials, scattering and absorbance from the nanomaterials act as interferences that complicate quantitative analysis. Herein, we describe a bacterial viability assay that is free of optical interference from nanomaterials and can be performed in a high-throughput format on 96-well plates. In this assay, bacteria were exposed to various materials and then diluted by a large factor into fresh growth medium. The large dilution ensured minimal optical interference from the nanomaterial when reading optical density, and the residue left from the exposure mixture after dilution was confirmed not to impact the bacterial growth profile. The fractions of viable cells after exposure were allowed to grow in fresh medium to generate measurable growth curves. Bacterial viability was then quantitatively correlated to the delay of bacterial growth compared to a reference regarded as 100% viable cells; data analysis was inspired by that in quantitative polymerase chain reactions, where the delay in the amplification curve is correlated to the starting amount of the template nucleic acid. Fast and robust data analysis was achieved by developing computer algorithms carried out using R. This method was tested on four bacterial strains, including both Gram-negative and Gram-positive bacteria, showing great potential for application to all culturable bacterial strains. With the increasing diversity of engineered nanomaterials being considered for large-scale use, this high-throughput screening method will facilitate rapid screening of nanomaterial toxicity and thus inform the risk assessment of nanoparticles in a timely fashion.