Separation, Sizing, and Quantitation of Engineered Nanoparticles in an Organism Model Using Inductively Coupled Plasma Mass Spectrometry and Image Analysis

Abstract

The increased use of engineered nanoparticles (ENPs) in consumer products and the relative lack of understanding of their associated hazards necessitate further investigation of the interactions between ENPs and environmental and biological systems. For environmental studies assessing uptake of orally ingested ENPs, a key step in ensuring accurate quantification of ingested ENPs is efficient separation of the organism from ENPs that are either nonspecifically adsorbed to the organism and/or suspended in the dispersion following exposure. Here, we measure the uptake of 30 nm and 60 nm gold nanoparticles (AuNPs) by the free-living nematode, Caenorhabditis elegans, using a sucrose density gradient centrifugation protocol to remove non-ingested AuNPs. Both conventional inductively coupled plasma-mass spectrometry (ICP-MS) and single particle (sp) ICP-MS are utilized to measure the total mass and size distribution, respectively, of ingested AuNPs. Scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS) imaging confirmed that traditional nematode washing procedures were ineffective at removing excess suspended and/or adsorbed AuNPs after exposure. Quantification of total Au uptake was performed following acidic digestion of non-exposed and Au-exposed nematodes, while an alkaline digestion procedure was optimized for the liberation of ingested AuNPs for spICP-MS characterization. Size distributions and particle number concentrations were determined for AuNPs ingested by nematodes with corresponding confirmation of nematode uptake via high-pressure freezing/freeze substitution resin preparation and large-area SEM imaging. Methods for the separation and in vivo quantification of ENPs in multicellular organisms will facilitate robust studies of ENP uptake, biotransformation, and hazard assessment in the environment.

Sucrose density gradient centrifugation for efficient separation of engineered nanoparticles from a model organism, Caenorhabditis elegans

“…[6-8, 10, 11, 15, 16] When our research group tested and employed a similar rinsing strategy, we found that water rinsing was unsuitable for the efficient removal of non-ingested NPs, even at low, environmentally relevant exposure concentrations (ng mL -1 or part per billion in gold mass fraction). [17] Therefore, it was necessary to devise a postexposure rinsing strategy that, unlike conventional water rinsing protocols, ensured greater efficiency in regard to the removal of excess, non-ingested ENPs.…”

“…[3,24,25] However, sucrose density gradient centrifugation (SDGC) separation had not been employed until recently for the separation of C. elegans from NPs after exposure. [17] This protocol details an SDGC procedure for the efficient separation of AuNPs from C. elegans.…”

“…Details regarding C. elegans culturing and maintenance practices can be found in the Experimental section of Johnson et al [17] See Figure 1A.…”

“…With an n of 4, the repeatability of the separation in our lab, data displayed in Table 2 and Table 3, was evaluated by calculating the average concentration of Au found not only in each individual layer, but also as an average sum of Au found in each individual gradient sample. This approach was successfully employed in two published studies for the separation of mixtures of AuNPs at varying sizes, [17], [27] as well as at low and high concentration levels.…”

“…This relates to an average AuNP-removal efficiency of 97.3 %. [17] Table 2. Total Au, in ng, found in gradient sample fractions following centrifugation of a mixture of 30 nm and 60 nm AuNPs (NIST) at 100 ng mL -1 , respectively.…”