Victoria A. Coleman

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.

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 Instrumentss 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.

Challenges in the size analysis of a silica nanoparticle mixture as candidate certified reference material

A new certified reference material for quality control of nanoparticle size analysis methods has been developed and produced by the Institute for Reference Materials and Measurements of the European Commission’s Joint Research Centre. The material, ERM-FD102, consists of an aqueous suspension of a mixture of silica nanoparticle populations of distinct particle size and origin. The characterisation relied on an interlaboratory comparison study in which 30 laboratories of demonstrated competence participated with a variety of techniques for particle size analysis. After scrutinising the received datasets, certified and indicative values for different method-defined equivalent diameters that are specific for dynamic light scatteringxa0(DLS), centrifugal liquid sedimentationxa0(CLS), scanning and transmission electron microscopy (SEM and TEM), atomic force microscopyxa0(AFM), particle tracking analysis (PTA) and asymmetrical-flow field-flow fractionation (AF4) were assigned. The value assignment was a particular challenge because metrological concepts were not always interpreted uniformly across all participating laboratories. This paper presents the main elements and results of the ERM-FD102 characterisation study and discusses in particular the key issues of measurand definition and the estimation of measurement uncertainty.

A comparative study of the physical and chemical properties of nano-sized ZnO particles from multiple batches of three commercial products

Given the broad commercial applications for ZnO nanomaterials, accurate attribution of physicochemical characteristics that induce toxic effects is particularly important. We report on the physicochemical properties of three commercial nano-ZnO products: Z-COTE and Z-COTExa0HP1 from BASF, andxa0Nanosun from Micronisers, and, for reference, “bulk” ZnOxa0from Sigma-Aldrich. Z-COTE, Nanosun and “bulk” consist of uncoated particles with different sizes, while Z-COTExa0HP1 consists of nanoparticles with a hydrophobic coating. Specific batches of these ZnO products were included in the OECD Sponsorship Programme to test manufactured nanomaterials. In order to identify properties potentially susceptible to variations between production runs, three additional batches of Z-COTE and Nanosun and two additional batches of Z-COTExa0HP1 were also investigated here. In general, all products showed little variation between batches for properties measured from powdered samples, but batch variations in the amount of surface coating were evident for the coated Z-COTExa0HP1. Properties measured with samples dispersed in liquids (agglomeration, photocatalytic activity, dissolution) were highly dependent on dispersion protocols, and this made it difficult to differentiate between differences due to dispersion and due to batches. However, batch-sensitive properties did appear to be present in Z-COTE and Z-COTExa0HP1 (photocatalytic activity), and Nanosun (dissolution). Intra-batch time and/or storage-dependent changes in the applied surface coating, noted specifically for the OECD batch of Z-COTExa0HP1, highlight the need for best practice when storing and accessing stocks of nano products. Awareness of inter-batch and intra-batch variability is essential for commercial applications and for nanotoxicological studies aimed at identifying links between physicochemical properties and any adverse effects in biological systems.

A tiered approach

information is provided without hampering innovation and driving new development of chemicals out of the European Union. We cannot see reasons to treat nanomaterials differently in this respect, particularly in light of the conclusion of the Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR) that nanomaterials as a category are not more or less hazardous than other chemicals. ❐ Antonio Tajani Vice-President of the European Commission, Enterprise and Industry, B-1049 Brussels, Belgium. e-mail: Antonio.Tajani@ec.europa.eu

Nanoparticles and metrology: a comparison of methods for the determination of particle size distributions

Nanoparticles and products incorporating nanoparticles are a growing branch of nanotechnology industry. They have found a broad market, including the cosmetic, health care and energy sectors. Accurate and representative determination of particle size distributions in such products is critical at all stages of the product lifecycle, extending from quality control at point of manufacture to environmental fate at the point of disposal. Determination of particle size distributions is non-trivial, and is complicated by the fact that different techniques measure different quantities, leading to differences in the measured size distributions. In this study we use both mono- and multi-modal dispersions of nanoparticle reference materials to compare and contrast traditional and novel methods for particle size distribution determination. The methods investigated include ensemble techniques such as dynamic light scattering (DLS) and differential centrifugal sedimentation (DCS), as well as single particle techniques such as transmission electron microscopy (TEM) and microchannel resonator (ultra high-resolution mass sensor).

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.

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.

Characterisation of gold agglomerates: size distribution, shape and optical properties

The slow agglomeration of gold colloids of approximate diameter 30 nm in the presence of a small concentration of L-cysteine·HCl has been followed by multiple techniques, namely particle tracking analysis (PTA), differential centrifugal sedimentation (DCS), UV-visible spectroscopy (UV), second order spectroscopy (SOS) and transmission electron microscopy (TEM). The citrate-stabilized Au nanoparticles were characterized by PTA, DCS, UV and dynamic light scattering (DLS) prior to exposure to the cysteine. Hydrodynamic forces during centrifugation can cause the disintegration of weakly held agglomerates. TEM reveals small linear agglomerates that become open linked chains of fractal-like structures after several hours of agglomeration. Second order Rayleigh scattering observed at the harmonic wavelength of 680 nm was resonantly enhanced by surface plasmon excitation of the growing agglomerates. During the initial stage of the agglomeration process, when chainlike or quasi-chainlike agglomerates were the dominant species, the SOS signal goes up by a factor of about three before reaching saturation. This study of a model nanoparticle system provides insights into the information obtained from a range of measurement techniques, with recommendations for characterisation of agglomerating nanoparticles under end-use conditions.