International Interlaboratory Comparison of Thermogravimetric Analysis of Graphene-Related Two-Dimensional Materials.

Research on graphene-related two-dimensional (2D) materials (GR2Ms) in recent years is strongly moving from academia to industrial sectors with many new developed products and devices on the market. Characterization and quality control of the GR2Ms and their properties are critical for growing industrial translation, which requires the development of appropriate and reliable analytical methods. These challenges are recognized by International Organization for Standardization (ISO 229) and International Electrotechnical Commission (IEC 113) committees to facilitate the development of these methods and standards which are currently in progress. Toward these efforts, the aim of this study was to perform an international interlaboratory comparison (ILC), conducted under Versailles Project on Advanced Materials and Standards (VAMAS) Technical Working Area (TWA) 41 "Graphene and Related 2D Materials" to evaluate the performance (reproducibility and confidence) of the thermogravimetric analysis (TGA) method as a potential new method for chemical characterization of GR2Ms. Three different types of representative and industrially manufactured GR2Ms samples, namely, pristine few-layer graphene (FLG), graphene oxide (GO), and reduced graphene oxide (rGO), were used and supplied to ILC participants to complete the study. The TGA method performance was evaluated by a series of measurements of selected parameters of the chemical and physical properties of these GR2Ms including the number of mass loss steps, thermal stability, temperature of maximum mass change rate (Tp) for each decomposition step, and the mass contents (%) of moisture, oxygen groups, carbon, and impurities (organic and non-combustible residue). TGA measurements determining these parameters were performed using the provided optimized TGA protocol on the same GR2Ms by 12 participants across academia, industry stakeholders, and national metrology institutes. This paper presents these results with corresponding statistical analysis showing low standard deviation and statistical conformity across all participants that confirm that the TGA method can be satisfactorily used for characterization of these parameters and the chemical characterization and quality control of GR2Ms. The common measurement uncertainty for each parameter, key contribution factors were identified with explanations and recommendations for their elimination and improvements toward their implementation for the development of the ISO/IEC standard for chemical characterization of GR2Ms.

Thickness measurements of graphene oxide flakes using atomic force microscopy: results of an international interlaboratory comparison.

Flake thickness is one of the defining properties of graphene-related 2D materials (GR2Ms), and therefore requires reliable, accurate, and reproducible measurements with well-understood uncertainties. This is needed regardless of the production method or manufacturer because it is important for all GR2M products to be globally comparable. An international interlaboratory comparison on thickness measurements of graphene oxide flakes using atomic force microscopy has been completed in technical working area 41 of versailles project on advanced materials and standards. Twelve laboratories participated in the comparison project, led by NIM, China, to improve the equivalence of thickness measurement for two-dimensional flakes. The measurement methods, uncertainty evaluation and a comparison of the results and analysis are reported in this manuscript. The data and results of this project will be directly used to support the development of an ISO standard.

Improved Metrological Traceability of Particle Size Values Measured with Line-Start Incremental Centrifugal Liquid Sedimentation.

Line-start incremental centrifugal liquid sedimentation (disc-CLS) is a powerful method to determine particle size based on the principles of Stokes' law. Because several of the input quantities of the Stokes equation cannot be easily determined for this case of a rotating disc, the disc-CLS approach relies on calibrating the sedimentation time scale with reference particles. To use these calibrant particles for establishing metrological traceability, they must fulfill the same requirements as those imposed on a certified reference material, i.e., their certified Stokes diameter and density value must come with a realistic measurement uncertainty and with a traceability statement. As is the case for several other techniques, the calibrants do not always come with uncertainties for the assigned modal diameter and effective particle density. The lack of such information and the absence of a traceability statement make it difficult for the end-user to estimate the uncertainty of the measurement results and to compare them with results obtained by others. We present the results of a collaborative study that aimed at demonstrating the traceability of particle size results obtained with disc-CLS. For this purpose, the particle size and effective particle density of polyvinyl chloride calibrants were measured using different validated methods, and measurement uncertainties were estimated according to the Guide to the Expression of Uncertainty in Measurement. The results indicate that the modal Stokes diameter and effective particle density that are assigned to the calibrants are accurate within 5% and 3.5%, respectively, and that they can be used to establish traceability of particle size results obtained with disc-CLS. This conclusion has a great impact on the traceability statement of certified particle size reference materials, for which the traceability is limited to the size and density values of the calibrant particles.

Quantification of nanoparticle concentration in colloidal suspensions by a non-destructive optical method.

The estimation of nanoparticle number concentration in colloidal suspensions is a prerequisite in many procedures, and in particular in multi-stage, low-yield reactions. Here, we describe a rapid, non-destructive method based on optical extinction and dynamic light scattering (DLS), which combines measurements using common bench-top instrumentation with a numerical algorithm to calculate the particle size distribution (PSD) and concentration. These quantities were derived from Mie theory applied to measurements of the optical extinction spectrum of homogeneous, non-absorbing nanoparticles, and the relative PSD of a colloidal suspension. The work presents an approach to account for PSDs achieved by DLS which, due to the underlying model, may not be representative of the true sample PSD. The presented approach estimates the absolute particle number concentration of samples with mono-, bi-modal and broad size distributions with <50% precision. This provides a convenient and practical solution for number concentration estimation required during many applications of colloidal nanomaterials.

Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification.

Droplet digital polymerase chain reaction (ddPCR) is a new technology that was recently commercialized to enable the precise quantification of target nucleic acids in a sample. ddPCR measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions. This novel ddPCR format offers a simple workflow capable of generating highly stable partitioning of DNA molecules. In this study, we assessed key performance parameters of the ddPCR system. A linear ddPCR response to DNA concentration was obtained from 0.16% through to 99.6% saturation in a 20,000 droplet assay corresponding to more than 4 orders of magnitude of target DNA copy number per ddPCR. Analysis of simplex and duplex assays targeting two distinct loci in the Lambda DNA genome using the ddPCR platform agreed, within their expanded uncertainties, with values obtained using a lower density microfluidic chamber based digital PCR (cdPCR). A relative expanded uncertainty under 5% was achieved for copy number concentration using ddPCR. This level of uncertainty is much lower than values typically observed for quantification of specific DNA target sequences using currently commercially available real-time and digital cdPCR technologies.

Single particle inductively coupled plasma-mass spectrometry: a performance evaluation and method comparison in the determination of nanoparticle size.

Sizing engineered nanoparticles in simple, laboratory systems is now a robust field of science; however, application of available techniques to more complex, natural systems is hindered by numerous challenges including low nanoparticle number concentrations, polydispersity from aggregation and/or dissolution, and interference from other incidental particulates. A new emerging technique, single particle inductively coupled plasma-mass spectrometry (spICPMS), has the potential to address many of these analytical challenges when sizing inorganic nanoparticles in environmental matrices. However, to date, there is little beyond the initial feasibility studies that investigates the performance characteristics and validation of spICPMS as a nanoparticle sizing technique. This study compares sizing of four silver nanoparticle dispersions (nominal diameters of 40, 60, 80, and 100 nm) by spICPMS to four established sizing techniques: dynamic light scattering, differential centrifugal sedimentation, nanoparticle tracking analysis, and TEM. Results show that spICPMS is able to size silver nanoparticles, across different sizes and particle number concentrations, with accuracy similar to the other commercially available techniques. Furthermore, a novel approach to evaluating particle coincidence is presented. In addition, spICPMS size measurements were successfully performed on nanoparticles suspended in algal growth media at low concentrations. Overall, while further development of the technique is needed, spICPMS yields important advantages over other techniques when sizing nanoparticles in environmentally relevant media.

Determining transport efficiency for the purpose of counting and sizing nanoparticles via single particle inductively coupled plasma mass spectrometry.

Currently there are few ideal methods for the characterization of nanoparticles in complex, environmental samples, leading to significant gaps in toxicity and exposure assessments of nanomaterials. Single particle-inductively coupled plasma-mass spectrometry (spICPMS) is an emerging technique that can both size and count metal-containing nanoparticles. A major benefit of the spICPMS method is its ability to characterize nanoparticles at concentrations relevant to the environment. This paper presents a practical guide on how to count and size nanoparticles using spICPMS. Different methods are investigated for measuring transport efficiency (i.e., nebulization efficiency), an important term in the spICPMS calculations. In addition, an alternative protocol is provided for determining particle size that broadens the applicability of the technique to all types of inorganic nanoparticles. Initial comparison, using well-characterized, monodisperse silver nanoparticles, showed the importance of having an accurate transport efficiency value when determining particle number concentration and, if using the newly presented protocol, particle size. Ultimately, the goal of this paper is to provide improvements to nanometrology by further developing this technique for the characterization of metal-containing nanoparticles.

New Multiscale Characterization Methodology for Effective Determination of Isolation-Structure-Function Relationship of Extracellular Vesicles.

Extracellular vesicles (EVs) have been lauded as next-generation medicines, but very few EV-based therapeutics have progressed to clinical use. Limited clinical translation is largely due to technical barriers that hamper our ability to mass produce EVs, i.e., to isolate, purify, and characterize them effectively. Technical limitations in comprehensive characterization of EVs lead to unpredicted biological effects of EVs. Here, using a range of optical and non-optical techniques, we showed that the differences in molecular composition of EVs isolated using two isolation methods correlated with the differences in their biological function. Our results demonstrated that the isolation method determines the composition of isolated EVs at single and sub-population levels. Besides the composition, we measured for the first time the dry mass and predicted sedimentation of EVs. These parameters were likely to contribute to the biological and functional effects of EVs on single cell and cell cultures. We anticipate that our new multiscale characterization approach, which goes beyond traditional experimental methodology, will support fundamental understanding of EVs as well as elucidate the functional effects of EVs in in vitro and in vivo studies. Our findings and methodology will be pivotal for developing optimal isolation methods and establishing EVs as mainstream therapeutics and diagnostics. This innovative approach is applicable to a wide range of sectors including biopharma and biotechnology as well as to regulatory agencies.

Does gas supersaturation by a chemical reaction produce bulk nanobubbles?

HYPOTHESIS: Supersaturation of dissolved gas is the most commonly reported method for generating long-lived bulk nanobubbles. However, these reports are treated with skepticism because of the lack of techniques that directly show that these particles are gas filled bubbles. Therefore, this work has tested the hypothesis that supersaturation obtained by a chemical reaction produces long-lived nanosized bubbles in bulk using an established protocol that relies on evaluating the density of nanoparticles and measuring their response to external pressure. EXPERIMENTS: Nanoparticles were generated using a chemical reaction between aqueous solutions of ammonium chloride and sodium nitrite. Standard nanoparticle sizing techniques, such as nanoparticle tracking analysis and dynamic light scattering, were utilized to determine the size and stability of the nanoparticles. Resonant mass measurement was used to measure the buoyant mass of the nanoparticles, and their compressibility was investigated by measuring their size under the application of external pressure. FINDINGS: The formation of nanoparticles was consistent with the kinetics of nitrogen gas evolution produced in the reaction, where the nanoparticle size was shown to be dependent on the pH and concentration of the reactants. However, the chemical reaction was found to generate incompressible nanoparticles with a density larger than that of the solvent, confirming that these particles were not gas-filled bubbles.

Size and shape distributions of primary crystallites in titania aggregates.

The primary crystallite size of titania powder relates to its properties in a number of applications. Transmission electron microscopy was used in this interlaboratory comparison (ILC) to measure primary crystallite size and shape distributions for a commercial aggregated titania powder. Data of four size descriptors and two shape descriptors were evaluated across nine laboratories. Data repeatability and reproducibility was evaluated by analysis of variance. One-third of the laboratory pairs had similar size descriptor data, but 83% of the pairs had similar aspect ratio data. Scale descriptor distributions were generally unimodal and were well-described by lognormal reference models. Shape descriptor distributions were multi-modal but data visualization plots demonstrated that the Weibull distribution was preferred to the normal distribution. For the equivalent circular diameter size descriptor, measurement uncertainties of the lognormal distribution scale and width parameters were 9.5% and 22%, respectively. For the aspect ratio shape descriptor, the measurement uncertainties of the Weibull distribution scale and width parameters were 7.0% and 26%, respectively. Both measurement uncertainty estimates and data visualizations should be used to analyze size and shape distributions of particles on the nanoscale.

A comparative study of submicron particle sizing platforms: accuracy, precision and resolution analysis of polydisperse particle size distributions.

The particle size distribution (PSD) of a polydisperse or multimodal system can often be difficult to obtain due to the inherent limitations in established measurement techniques. For this reason, the resolution, accuracy and precision of three new and one established, commercially available and fundamentally different particle size analysis platforms were compared by measuring both individual and a mixed sample of monodisperse, sub-micron (220, 330, and 410 nm - nominal modal size) polystyrene particles. The platforms compared were the qNano Tunable Resistive Pulse Sensor, Nanosight LM10 Particle Tracking Analysis System, the CPS Instruments's UHR24000 Disc Centrifuge, and the routinely used Malvern Zetasizer Nano ZS Dynamic Light Scattering system. All measurements were subjected to a peak detection algorithm so that the detected particle populations could be compared to 'reference' Transmission Electron Microscope measurements of the individual particle samples. Only the Tunable Resistive Pulse Sensor and Disc Centrifuge platforms provided the resolution required to resolve all three particle populations present in the mixed 'multimodal' particle sample. In contrast, the light scattering based Particle Tracking Analysis and Dynamic Light Scattering platforms were only able to detect a single population of particles corresponding to either the largest (410 nm) or smallest (220 nm) particles in the multimodal sample, respectively. When the particle sets were measured separately (monomodal) each platform was able to resolve and accurately obtain a mean particle size within 10% of the Transmission Electron Microscope reference values. However, the broadness of the PSD measured in the monomodal samples deviated greatly, with coefficients of variation being ~2-6-fold larger than the TEM measurements across all four platforms. The large variation in the PSDs obtained from these four, fundamentally different platforms, indicates that great care must still be taken in the analysis of samples known to have complex PSDs. All of the platforms were found to have high precision, i.e. they gave rise to less than 5% variance in PSD shape descriptors over the replicate measurements.

Tissue distribution of inhaled micro- and nano-sized cerium oxide particles in rats: results from a 28-day exposure study.

In order to obtain more insight into the tissue distribution, accumulation, and elimination of cerium oxide nanoparticles after inhalation exposure, blood and tissue kinetics were investigated during and after a 28-day inhalation study in rats with micro- and nanocerium oxide particles (nominal primary particle size: < 5000, 40, and 5-10 nm). Powder aerosolization resulted in comparable mass median aerodynamic diameter (1.40, 1.17, and 1.02 µm). After single exposure, approximately 10% of the inhaled dose was measured in lung tissue, as was also estimated by a multiple path particle dosimetry model (MPPD). Though small differences in pulmonary deposition efficiencies of cerium oxide were observed, no consistent differences in pulmonary deposition between the micro- and nanoparticles were observed. Each cerium oxide sample was also distributed to tissues other than lung after a single 6-h exposure, such as liver, kidney, and spleen and also brain, testis, and epididymis. No clear particle size-dependent effect on extrapulmonary tissue distribution was observed. Repeated exposure to cerium oxide resulted in significant accumulation of the particles in the (extra)pulmonary tissues. In addition, tissue clearance was shown to be slow, and, overall, insignificant amounts of cerium oxide were eliminated from the body at 48- to 72-h post-exposure. In conclusion, no clear effect of the primary particle size or surface area on pulmonary deposition and extrapulmonary tissue distribution could be demonstrated. This is most likely explained by similar aerodynamic diameter of the cerium oxide particles in air because of the formation of aggregates and irrespective possible differences in surface characteristics. The implications of the accumulation of cerium oxide particles for systemic toxicological effects after repeated chronic exposure via ambient air are significant and require further exploration.