Separation, Sizing, and Quantitation of Engineered Nanoparticles in an Organism Model Using Inductively Coupled Plasma Mass Spectrometry and Image Analysis Monique E. Johnson Shannon K. Hanna Antonio R. Montoro Bustos Christopher M. Sims Lindsay C. C. Elliott Akshay Lingayat Adrian C. Johnston Babak Nikoobakht John T. Elliott R. David Holbrook Keana C. K. Scott Karen E. Murphy Elijah J. Petersen Lee L. Yu Bryant C. Nelson 10.1021/acsnano.6b06582.s001 https://acs.figshare.com/articles/journal_contribution/Separation_Sizing_and_Quantitation_of_Engineered_Nanoparticles_in_an_Organism_Model_Using_Inductively_Coupled_Plasma_Mass_Spectrometry_and_Image_Analysis/4499111 For environmental studies assessing uptake of orally ingested engineered nanoparticles (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 and 60 nm gold nanoparticles (AuNPs) by the nematode, Caenorhabditis elegans, using a sucrose density gradient centrifugation protocol to remove noningested 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. Water rinsing procedures had AuNP removal efficiencies ranging from 57 to 97% and 22 to 83%, while the sucrose density gradient procedure had removal efficiencies of 100 and 93 to 98%, respectively, for the 30 and 60 nm AuNP exposure conditions. Quantification of total Au uptake was performed following acidic digestion of nonexposed and Au-exposed nematodes, whereas 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 <i>via</i> high-pressure freezing/freeze substitution resin preparation and large-area SEM imaging. Methods for the separation and <i>in vivo</i> quantification of ENPs in multicellular organisms will facilitate robust studies of ENP uptake, biotransformation, and hazard assessment in the environment. 2016-12-16 00:00:00 Engineered Nanoparticles acidic digestion hazard assessment Image Analysis sucrose density gradient centrifugation protocol removal efficiencies Caenorhabditis elegans AuNP removal efficiencies particle number concentrations SEM imaging size distribution 60 nm gold nanoparticles multicellular organisms vivo quantification 60 nm AuNP exposure conditions nematode uptake plasma mass spectrometry digestion procedure Organism Model Size distributions water rinsing procedures spICP-MS characterization Plasma Mass Spectrometry sucrose density gradient procedure ENP uptake Au-exposed nematodes noningested AuNPs