Publications

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NM-Series of Representative Manufactured Nanomaterials Zinc Oxide NM-110, NM-111, NM-112, NM-113 Characterisation and Test Item Preparation

Abstract

The European Commission Joint Research Centre (JRC) provides scientific support to European Union policy regarding nanotechnology. Over the last three years, the JRC, in collaboration with international public and private partners, focused part of its work on establishing and applying a priority list (NM-Series) of Representative Manufactured Nanomaterials (RMNs) in support of one of the most comprehensive nanomaterial research programmes that is currently being carried out: the Organisation for Economic Co-operation and Development’s (OECD) Working Party on Manufactured Nanomaterials (WPMN) Sponsorship Programme; this collaborative programme ultimately enables the development and collection of data on characterisation, measurement, toxicological and eco-toxicological testing, as well as risk assessment and safety evaluation of nanomaterials (NMs). It is of utmost timely importance to make representative nanomaterials available to the international scientific community, in order to enable innovation and development of safe materials and products. The present report describes the characterisation of NM-110, NM-111, NM-112, and NM-113, RMN Zinc Oxide substances, originating from defined batches of commercially manufactured material, respectively. The NM-Series materials were subsampled in collaboration with Fraunhofer Institute for Molecular and Applied Ecology (Fh IME), in order to be made available for measurement and testing for hazard identification, risk and exposure assessment studies. The results for more than 15 endpoints are addressed in the present report, including physical-chemical properties, such as size and size distribution, crystallite size and electron microscopy images. Sample and test item preparation procedures are addressed. The RMNs are studied by a number of international laboratories. The properties of the Zinc Oxide RMNs NM-110, NM-111, NM-112, and NM-113 studied and described in this report demonstrate their relevance for use in measurement and testing studies of nanomaterials. The studies were performed in close collaboration between the PROSPECT consortium partners, the JRC, the Fraunhofer Institute for Molecular and Applied Ecology (Fh-IME), BASF AG Ludwigshafen, LGC standards, the National Physical Laboratory (NPL) as national metrology institute of the United Kingdom, the National Research Centre for the Working Environment, Denmark, CSIRO and the National Measurement Institute of Australia.

Determination of the volume-specific surface area by using transmission electron tomography for characterization and definition of nanomaterials

Abstract

Background

Transmission electron microscopy (TEM) remains an important technique to investigate the size, shape and surface characteristics of particles at the nanometer scale. Resulting micrographs are two dimensional projections of objects and their interpretation can be difficult. Recently, electron tomography (ET) is increasingly used to reveal the morphology of nanomaterials (NM) in 3D. In this study, we examined the feasibility to visualize and measure silica and gold NM in suspension using conventional bright field electron tomography.

Results

The general morphology of gold and silica NM was visualized in 3D by conventional TEM in bright field mode. In orthoslices of the examined NM the surface features of a NM could be seen and measured without interference of higher or lower lying structures inherent to conventional TEM. Segmentation by isosurface rendering allowed visualizing the 3D information of an electron tomographic reconstruction in greater detail than digital slicing. From the 3D reconstructions, the surface area and the volume of the examined NM could be estimated directly and the volume-specific surface area (VSSA) was calculated. The mean VSSA of all examined NM was significantly larger than the threshold of 60 m2/cm3.

The high correlation between the measured values of area and volume gold nanoparticles with a known spherical morphology and the areas and volumes calculated from the equivalent circle diameter (ECD) of projected nanoparticles (NP) indicates that the values measured from electron tomographic reconstructions are valid for these gold particles.

Conclusion

The characterization and definition of the examined gold and silica NM can benefit from application of conventional bright field electron tomography: the NM can be visualized in 3D, while surface features and the VSSA can be measured.

Quantitative characterization of agglomerates and aggregates of pyrogenic and precipitated amorphous silica nanomaterials by transmission electron microscopy

Abstract

Background

The interaction of a nanomaterial (NM) with a biological system depends not only on the size of its primary particles but also on the size, shape and surface topology of its aggregates and agglomerates. A method based on transmission electron microscopy (TEM), to visualize the NM and on image analysis, to measure detected features quantitatively, was assessed for its capacity to characterize the aggregates and agglomerates of precipitated and pyrogenic synthetic amorphous silicon dioxide (SAS), or silica, NM.

Results

Bright field (BF) TEM combined with systematic random imaging and semi-automatic image analysis allows measuring the properties of SAS NM quantitatively. Automation allows measuring multiple and arithmetically complex parameters simultaneously on high numbers of detected particles. This reduces operator-induced bias and assures a statistically relevant number of measurements, avoiding the tedious repetitive task of manual measurements. Access to multiple parameters further allows selecting the optimal parameter in function of a specific purpose.

Using principle component analysis (PCA), twenty-three measured parameters were classified into three classes containing measures for size, shape and surface topology of the NM.

Conclusion

The presented method allows a detailed quantitative characterization of NM, like dispersions of precipitated and pyrogenic SAS based on the number-based distributions of their mean diameter, sphericity and shape factor.

Quantitative characterization of aggregated and agglomerated titanium dioxide nanomaterials by transmission electron microscopy

Abstract

The physical properties of TiO2 nanomaterials are determined quantitatively using a method that combines imaging by transmission electron microscopy (TEM) with semi-automatic particle detection and analysis. The method is applied on four powdered TiO2 nanomaterials, NM-102, NM-103, NM-104 and NM-105, dispersed in distilled water.

Qualitative analysis shows that the stability and polydispersity of the dispersed nanomaterials are influenced by the material from which the vial used for dispersion, is made. In glass vials, the uncoated nanomaterials, NM-102 and NM-105, precipitate immediately after sonication, while the coated nanomaterials, NM-103 and NM-104, remain stable in dispersion. In polypropylene vials, stable dispersions are obtained for all nanomaterials. It is shown that the vial material alters the pH of the dispersions, which in turn influences the agglomeration state of the nanomaterials.

Quantitative analysis of stable dispersions, based on TEM imaging combined with semi-automatic image analysis, results in number-based distributions of characteristic parameters, measuring the size, shape and surface topology of the unbound, aggregated and agglomerated TiO2 particles. Iterative curve fitting is applied to the number-based distributions of selected parameters and allows objective comparison of the distributions based on the properties of the fitted curves. Using this method, it is shown that the size, the shape and the surface properties of NM-102 and NM-105 and of the coated nanomaterials, NM-103 and NM-104, are significantly different. The physical characteristics of NM-103 and NM-104 are similar. This supports the validity of the method as these are in fact the same material with a different coating.

TEM and SP-ICP-MS analysis of the release of silver nanoparticles from decoration of pastry

Abstract

Metallic silver is an EU approved food additive referred to as E174. It is generally assumed that silver is only present in bulk form in the food chain. This work demonstrates that a simple treatment with water of “silver pearls”, meant for decoration of pastry, results in the release of a subfraction of silver nanoparticles. The number-based size and shape distributions of the single, aggregated, and/or agglomerated particles released from the silver pearls were determined by combining conventional bright-field TEM imaging with semiautomatic particle detection and analysis. In addition, the crystal structure of the particles was studied by electron diffraction and chemical information was obtained by combining HAADF-STEM imaging with EDX spectroscopy and mapping. The TEM results were confirmed by SP-ICP-MS. The representative Ag test nanomaterial NM-300K was used as a positive control to determine the uncertainty on the measurement of the size and shape of the particles.

Quantitative characterization of synthetic amorphous silica nanomaterials, dispersed in different media, by transmission electron microscopy.

Abstract

A quantitative method based on TEM is developed by studying SAS NM dispersed in water in their most disperse form. The SAS NM are prepared using the generic NANOGENOTOX dispersion protocol which was developed for preparation of general batch dispersions for in vitro and in vivo toxicity testing. Application of the method allowed characterization of SAS NM in different media, used for in vitro and in vivo toxicity testing.

Quantitative analysis of the physical characteristics of manufactured silica NP used in food by advanced transmission electron microscopy.

International Symposiom on nanotechnology in the food chain, Brussels, Belgium

Abstract

Manufactured silicon dioxide (silica) nanoparticles (NP) are chemically inert, pure white and free-flowing with a neutral pH. They do not affect the color, taste, odor or nature of food. Silica NP are applied in concentrations of up to 2 % of the end product weight to make granular and powdered food materials free-flowing, as anti-caking agents for food products high in oils or fats and to convert liquids into free flowing powders. Silica NP are effective across a wide variety of food applications, including cheese, non-dairy creamers, food flavors, powdered drink mixes, seasonings and as tabletting aid for vitamin supplements.

Physical characteristics of silica NP are important factors to evaluate their effectivity and the possible health risk of their application. By its high resolution, transmission electron microscopy (TEM) is one of the few techniques that allow direct visualization of nanomaterials. Conventional sample preparation techniques coupled to TEM imaging and (semi)automatic, threshold-based detection of NP in electron micrographs are evaluated to measure the physical properties of silica NP used in food on a per-particle-basis, and standard operating procedures were developed.

Conventional TEM imaging using a Tecnai Spirit TEM (FEI, Eindhoven, The Netherlands) operating at 120 kV allows directly visualizing the agglomeration state of the silica NP and the structure, size and shape of their composing primary subunits (Figure A). Digital micrographs were made using a 4*4 k Eagle CCD-camera (FEI) and stored in an iTEM database (Olympus, Münster, Germany) together with imaging and sample preparation data and with (intermediate) results.

Threshold-based detection of projected particles in micrographs using the ‘Detection module’ of iTEM allows measuring hundreds of individual particles simultaneously.These quantitative measurements include the electron density, size, shape, perimeter and surface area. Distributions of these parameters can be expressed on a number basis (Figure B).

Electron tomographic reconstruction allowed visualizing the morphology of the silica NP in three dimensions. Tilt series of micrographs were recorded semi-automatically assisted by Explore 3D (FEI). These were aligned and reconstructed using Inspect 3D (FEI). Reconstructions were visualized using AMIRA (Mercury). Artifacts inherent to the analysis of projections of NP were detected and interpreted. An estimation of the surface area, volume (Figure C) and volume specific surface area of NP in suspension was obtained.

Characterization of colloidal gold and silver nanomaterials by advanced transmission electron microscopy.

European Microscopy Congress 2012.

Abstract

Silver and gold nanomaterials (NM) are commercially distributed worldwide and applied in a variety of end-products. A detailed physical characterization of these metallic NM allows controlling the quality of different products and batches. This information is also essential in a legislatory framework and allows analyzing possible health risks. By its high resolution, transmission electron microscopy (TEM) is one of the few techniques that allow direct visualization of such NM. The conventional grid-on-drop sample preparation method coupled to TEM imaging and (semi-)automatic, threshold-based detection of NM in electron micrographs are applied to measure the physical properties of Au and Ag NM on a per-particle-basis. Such an approach allows measuring hundreds of individual particles simultaneously.

Different types and batches of colloidal Ag and Au NM are characterized and compared. These include two samples of spherical Ag particles of the same batch, and two samples of spherical, branched and rod-like Au particles of different batches.

We demonstrates the results of the characterization of the NM, and shows number based distributions of selected properties. The mean diameter is chosen as a measure for the NM size, the sphericity is an estimate of the NM shape, and the shape factor estimates the topology of the NM surface. It can be derived that spherical NM are smaller, more symmetrical and smoother than branched and rod-like NM. Furthermore, Au rod-like NM can clearly be distinguished from spherical and branched particles based on their sphericity. For all parameters, spherical NM have a relatively small polydispersity compared with branched and rod-like NM, i.e. the variation based on mean diameter, sphericity and shape factor is small between the spherical Au NM (between batches) and Ag NM (between samples of the same batch). The larger polydispersities between batches of branched and rod-like Au NM reflect the difficulties to control their production processes. We also demonstrates that the two batches of branched NM can be distinguished based on their shape factor. In addition, the quantitative TEM analyses support the observation that in the rod-like NM batches, two subpopulations are present. In both batches approximately 8 % of the particles are spherical instead of rod-like.

In conclusion, TEM imaging combined with image analysis contributes to the detailed characterization and batch control of dispersed Au and Ag NM.

Characterization of aggregated and agglomerated titanium oxide nanomaterials by transmission electron microscopy.

European Microscopy Congress 2012.

Abstract

Nano-sized titanium oxide (TiO2) is widely used in cosmetics and skin care products, e.g. as a pigment, sunscreen or a thickener. In addition, TiO2 is applied in coatings, and as a photo catalyst in air and water cleaning. A different behavior of nano-scaled TiO2 compared to the bulk material has been observed, and can often be attributed to the change in surface-to-volume ratio with nanoparticle size. The interaction between a nanomaterial (NM) and a biological system depends on the characteristics of the unbound, aggregated and agglomerated particles of the material, such as size, morphology, surface topology, coating and charge. To monitor TiO2 applications and for risk analysis and regulatory purposes, methods allowing a detailed physical characterization of TiO2 aggregates and agglomerates are required. In this study, such a method is developed by examining TiO2 nanomaterials NM-102, NM-103, NM-104 and NM-105 obtained from the JRC NM repository (Ispra, Italy). These nanomaterials are dispersed in distilled water, and analyzed using transmission electron microscopy (TEM).

First, sample preparation conditions (type of vial, sonication time, charge of the grid, dilution) are examined. It is demonstrated that the type of vial can influence the aggregation status of the materials: dispersed and sonicated NM-102 and NM-105 precipitate in glass vials, but remain stable in polyethylene vials. On the contrary, NM-103 and NM-104 remain stable in both glass and polyethylene vials.

Subsequently, a method which consists of recording bright-field TEM images, processing the images by semi-automatic detection of nanoparticles, and automated measurement of nanoparticle properties resulting in number based distributions of characterization parameters, is explored. This method is in line with the work of De Temmerman et al. for the characterization of silica nanomaterials. The method is extended with iterative curve fitting of the number based distributions, which allows comparing nanomaterials and excludes the effects of the chosen bin size.

The materials are characterized by number based distributions of 23 physical parameters. Principle component analysis (PCA) indicates that these parameters can be classified into three classes containing measures for size, shape and surface topology. The curves fitted to the number based distributions of representatives of the components, namely mean diameter, sphericity and convexity, are compared for each TiO2 nanomaterial.

Effects of sample preparation in glass and polyethylene vials are assessed using the proposed method. In agreement with the observations, large differences are found for NM-102 and NM-105, while only minor differences are found for NM-103 and NM-104.

Furthermore, comparison of the TiO2 nanomaterials, prepared in polyethylene vials, support the validity of the method. As expected, the physical characteristics of the coated nanomaterials NM-103 and NM-104 are similar: they are in fact the same material with a different monomolecular coating. The mean diameter, the sphericity and the convexity of NM-102, NM-105 and of both coated NM are different.

Quantitative analysis of the physical characteristics of gold and silver nanoparticles by advanced transmission electron microscopy.

Abstract

Silver and gold nanoparticles (NP) are commercially distributed on the internet at an international level as food supplements.

The question of toxicological risks arises directly, because from a nutritional physiology point of view, noble metals are not required and because gold NP have catalytic effects, while silver NP have a biocidal effect.

The latter is the basis of the use of Ag NP as antimicrobial agents in food packaging, refrigerators and kitchen appliances.

To evaluate the possible health risks of these applications, knowledge of the physical characteristics of these metallic NP is an important factor.

By its high resolution, transmission electron microscopy (TEM) is one of the few techniques that allow direct visualization of such nanomaterials.

Conventional sample preparation techniques coupled to TEM imaging and (semi)automatic, threshold-based detection of NP in electron micrographs are evaluated to categorize the particles as NP and to measure their physical properties on a per-particle-basis.

Standard operating procedures were developed for this purpose.

Conventional TEM imaging using a Tecnai Spirit TEM (FEI, Eindhoven, The Netherlands) operating at 120 kV allows directly visualizing the structure, size and shape and agglomeration state of preparations of gold and silver NP (Figure A). Digital micrographs were made using a 4*4 k Eagle CCD-camera (FEI) and stored in an iTEM database (Olympus, Münster, Germany) together with imaging and sample preparation data and of (intermediate) results.

Threshold-based detection of projected particles in micrographs using the ‘Detection module’ of iTEM allows to measure hundreds of individual particles simultaneously. These quantitative measurements include the electron density, size, shape, perimeter and surface area. Distributions of these parameters can be expressed on a number basis (Figure B).

Electron tomographic reconstruction allowed visualizing the morphology of the gold and silver NP in three dimensions. Tilt series of micrographs were recorded semi-automatically assisted by Explore 3D (FEI). These were aligned and reconstructed using Inspect 3D (FEI). Reconstructions were visualized using AMIRA (Mercury). Artifacts inherent to the analysis of projections of NP were detected and interpreted. An estimation of the surface area, volume (Figure C) and volume specific surface area of particles in suspension was obtained and a correlation with the area and volume calculated from the equivalent circle diameter of projected NP was demonstrated.

Defining of a nanomaterial using advanced transmission electron microscopy by quantitative analysis of its physical characteristics.

Abstract

Manufactured silicon dioxide (silica) nanoparticles (NP) are chemically inert, pure white and free-flowing with a neutral pH. They do not affect the color, taste, odor or nature of food. Silica NP are applied in concentrations of up to 2 % of the end product weight to make granular and powdered food materials free-flowing, as anti-caking agents for food products high in oils or fats and to convert liquids into free flowing powders. Silica NP are effective across a wide variety of food applications, including cheese, non-dairy creamers, food flavors, powdered drink mixes, seasonings and as tabletting aid for vitamin supplements.

Physical characteristics of silica NP are important factors to evaluate their effectivity and the possible health risk of their application. By its high resolution, transmission electron microscopy (TEM) is one of the few techniques that allow direct visualization of nanomaterials. Conventional sample preparation techniques coupled to TEM imaging and (semi)automatic, threshold-based detection of NP in electron micrographs are evaluated to measure the physical properties of silica NP used in food on a per-particle-basis, and standard operating procedures were developed.

Conventional TEM imaging using a Tecnai Spirit TEM (FEI, Eindhoven, The Netherlands) operating at 120 kV allows directly visualizing the agglomeration state of the silica NP and the structure, size and shape of their composing primary subunits (Figure A). Digital micrographs were made using a 4*4 k Eagle CCD-camera (FEI) and stored in an iTEM database (Olympus, Münster, Germany) together with imaging and sample preparation data and with (intermediate) results.

Threshold-based detection of projected particles in micrographs using the ‘Detection module’ of iTEM allows measuring hundreds of individual particles simultaneously. These quantitative measurements include the electron density, size, shape, perimeter and surface area. Distributions of these parameters can be expressed on a number basis (Figure B).

Electron tomographic reconstruction allowed visualizing the morphology of the silica NP in three dimensions. Tilt series of micrographs were recorded semi-automatically assisted by Explore 3D (FEI). These were aligned and reconstructed using Inspect 3D (FEI). Reconstructions were visualized using AMIRA (Mercury). Artifacts inherent to the analysis of projections of NP were detected and interpreted. An estimation of the surface area, volume (Figure C) and volume specific surface area of NP in suspension was obtained.