Abstract
Increasing consumer use of engineered nanomaterials has led to significantly increased efforts to understand their potential impact on the environment and living organisms. Currently, no individual technique can provide all the necessary information such as their size, distribution, and chemistry in complex biological systems. Consequently, there is a need to develop complementary instrumental imaging approaches that provide enhanced understanding of these "bio-nano" interactions to overcome the limitations of individual techniques. Here we used a multimodal imaging approach incorporating dark-field light microscopy, high-resolution electron microscopy, and nanoscale secondary ion mass spectrometry (NanoSIMS). The aim was to gain insight into the bio-nano interactions of surface-functionalized silver nanoparticles (Ag-NPs) with the green algae Raphidocelis subcapitata, by combining the fidelity, spatial resolution, and elemental identification offered by the three techniques, respectively. Each technique revealed that Ag-NPs interact with the green algae with a dependence on the size (10 nm vs 60 nm) and surface functionality (tannic acid vs branched polyethylenimine, bPEI) of the NPs. Dark-field light microscopy revealed the presence of strong light scatterers on the algal cell surface, and SEM imaging confirmed their nanoparticulate nature and localization at nanoscale resolution. NanoSIMS imaging confirmed their chemical identity as Ag, with the majority of signal concentrated at the cell surface. Furthermore, SEM and NanoSIMS provided evidence of 10 nm bPEI Ag-NP internalization at higher concentrations (40 μg/L), correlating with the highest toxicity observed from these NPs. This multimodal approach thus demonstrated an effective approach to complement dose-response studies in nano-(eco)-toxicological investigations.
Highlights
NanoSIMS imaging confirmed their chemical identity as Ag, with the majority of signal concentrated at the cell surface
For a given surface functionality, the smaller 10 nm Ag-NPs were more toxic than their 60 nm counterparts, while the positively charged branched polyethylenimine coating imparted greater toxicity than the negatively charged tannic acid (TA) coatings (Figure S1). These observations are in agreement with previously published findings, where smaller Ag-NPs and positively charged bPEI-Ag-NPs were consistently reported to be more toxic based on equivalent Ag mass concentrations.[14,33]
We have demonstrated a complementary multimodal methodology for analyzing Ag-NPs interactions with the green algae
Summary
NanoSIMS imaging confirmed their chemical identity as Ag, with the majority of signal concentrated at the cell surface. Resolution is limited by diffraction (for visible light, d ≈ λ/2 ≈ 250 nm) so that objects that are less than d apart cannot be distinguished from one another This is inadequate to resolve ENMs, whose sizes, by definition, are less than 100 nm in at least one dimension.[7] This limitation can be overcome by using electron microscopy (EM) techniques: both scanning and transmission electron microscopies (SEM and TEM) are capable of providing visual identification of ENMs at the nanoscale, and they are the most widely used methods for imaging bio-nano interactions. While EM has traditionally required fixed, dry samples, the development of environmental (low-vacuum) SEM8,9 or cryo-EM10 has enabled high-resolution imaging of samples under “hydrated” conditions Despite these technological advances, elemental identification at the nanoscale is still challenging. Complementary approaches are needed that combine techniques with strengths in imaging and elemental characterization to overcome the individual limitations and provide a comprehensive understanding of ENM biointeractions
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