An electron microscopy based method for the detection and quantification of nanomaterial number concentration in environmentally relevant media

Abstract

Improved detection and characterization of nanomaterials (NMs) in complex environmental media requires the development of novel sampling approaches to improve the detection limit to be close to environmentally realistic concentrations. Transmission electron microscopy (TEM) is an indispensable metrological tool in nanotechnology and environmental nanoscience due to its high spatial resolution and analytical capabilities when coupled to spectroscopic techniques. However, these capabilities are hampered by the conventional sample preparation methods, which suffer from low NM recovery. The current work presents a validated, fully quantitative sampling technique for TEM that overcomes conventional sample preparation shortcomings, and thus enables the use of TEM for measurement of particle number concentration and their detection in complex media at environmentally realistic concentrations. This sampling method is based on ultracentrifugation of NMs from suspension onto a poly-l-lysine (PLL) functionalized TEM grid, using active deposition (by ultracentrifugation) and retention (by PLL interactions with NM surface) of NMs on the substrate, enabling fully quantitative analysis. Similar analysis with AFM was satisfactory in simple media but the lack of chemical-selectivity of AFM limits its applicability for the detection of NMs in complex environmental samples. The sampling approach was validated using both citrate- and PVP-coated AuNMs in pure water, which demonstrated an even distribution of NM on the TEM grid and high NM recovery (80-100%) at environmentally relevant NM concentrations (ca. 0.20-100 μg L(-1)). The applicability of the sampling method to complex environmental samples was demonstrated by the quantification of particle number concentration of AuNMs in EPA soft water (with and without Suwannee River fulvic acid) and lake water. This sample preparation approach is also applicable to other types of NMs with some modifications (e.g. centrifugation force and time) to insure full sample recovery. This TEM sampling method is key to the accurate quantification of NM number concentration, and therefore to improving our understanding of environmental fate, behavior, effects and dose of NMs.

Sewage spills are a major source of titanium dioxide engineered (nano)-particle release into the environment

“…Overall, samples from 7 sites (Crane C1, C2, Gills G1, Stoop S1, S2, S3, and S4) were observed during two sampling campaigns (December 15 th and January 18 th ). TEM samples were prepared by ultracentrifugation of natural waters (4 mL) at 150,000 g for 60 minutes using a Sorvall TM MTX 150 Micro-Ultracentrifuge (ThermoFisher Scientific, USA) with a S52-ST swinging-Bucker Rotor on a TEM grid 36 . A Teflon insert was placed at the bottom of the centrifuge tubes to create a flat surface that supports the 300 mesh Cu TEM grid (Ted Pella, Pelco ® , USA).…”

“…However practical limitations including the demanding operator time, the presence of high concentrations of natural particles, the poor statistical power due to limited number of particles that can be imaged and analyzed within a reasonable time and cost frame, hamper the quantification of ENMs in complex matrices 7 . These limitations can potentially be overcome through better sample preparation e.g., particle disaggregation, followed by density-based separation to concentrate the particle of interest, followed by total particle deposition on the TEM grids 36 . Therefore, pragmatically, TEM should be used to provide complementary (qualitative data) to support more statistically powerful techniques such as ME-SP-ICP-MS.…”

Improvements in Nanoparticle Tracking Analysis To Measure Particle Aggregation and Mass Distribution: A Case Study on Engineered Nanomaterial Stability in Incineration Landfill Leachates

“…5,6 Transmission electron microscopy (TEM) is another popular option to visualize particle size distribution and agglomeration with particles, even in complex media, but without being quantitative, the technique offers little in terms of being able to robustly characterize the ENM in a system of interest, though validation methods for quantifying particle number have been investigated. 7 Field flow fractionation, while requiring extensive method development, is another analytical option for researchers studying ENM in natural systems. [8][9][10] While not measuring particles on an individual basis like the previously mentioned techniques, the ability to provide an initial particle size distribution, ENM agglomeration and/or complexation to natural materials is possible.…”

The concentration-dependent behaviour of nanoparticles

“…[10] A third study suggested an increased dissolution of Tween coated-Ag NPs after dilution to near environmentally realistic Ag NP concentrations (,400 mg L À1 ) in ultra-high purity water. [11] Here, following advances in sample preparation for microscopy analysis in order to lower concentration detection limits, [12,13] and the use of long path UV-Vis cell cuvettes to monitor poly(vinylpyrrolidinone) (PVP)-coated Ag NPs at nearenvironmentally relevant concentrations (100-1000 mg L À1 ), we highlight that these concentration-dependent behaviours of nanomaterials become even more pronounced at the microgram per litre levels. This kind of behaviour should be thoroughly considered in any assessment of NP fate and effects for materials with potential for dissolution.…”