physicochemical characterisation of nanoparticles
3. Calibration standards and test-cases
Calibration standards and test-cases for performance assessment are a further point.
In order to calibrate the npSCOPE instrument and to provide the end users with the means to assess, at any time, the performances of the instrument in different application fields, the npSCOPE consortium will select a certain number of industry relevant nanomaterials with different physico-chemico properties and will develop test-case biological samples, based on SOPs, covering different application fields.
1. Calibration standards: Nanomaterials representative of industrial materials will be selected and used to calibrate the instruments in its different components and to verify its performances over time.
2. Test-cases representative of different application fields will be developed together with the respective SOPs, in order to allow future users of the npSCOPE instrument to produce reference biological materials to be used to verify the performances of the instrument.
4 Objectives
The npSCOPE project will develop a new instrument that couples the extraordinarily high resolution of the recently commercialised helium-ion microscope with sensors for composition (a mass spectrometer) and 3D visualisation (transmitted ion detector) in order to more fully characterise individual nanoparticles and their interaction with their environment (tissue, cells, etc.) and to better understand the risks they might pose to human health or the environment. It is well known that the risks posed by particles depend not only on their composition and surface chemistry, but also upon their shape. The prototype instrument (nanoparticle 'scope npSCOPE) will be thoroughly benchmarked against existing techniques, and its performance validated using representative materials and calibration standards. Software and sample-handling techniques and hardware will be developed to allow high-throughput analysis (for useful statistical descriptions of real-world samples of many nanoparticles), and to facilitate the use of correlative microscopy techniques for a complete understanding of the context of nanoparticles in biological materials. These techniques and protocols will be tested on a suite of well-characterised representative materials and matrices.
In the last twenty years, engineered nanomaterials have become the focus of intensive research and financial investment in a
multitude of areas. However, the same properties that make nanomaterials desirable in these various applications have the potential to alter biological properties, and thus to have an impact on the environment, health, and safety. Nano-specific health risks could result from altered environmental fate, availability, bio-persistence, etc. when compared with the larger or bulk forms of the same material, thus the risks are not simple to extrapolate from similar bulk materials. As nanomaterials and products incorporating these materials are more and more widely adopted, it becomes more and more important to evaluate their potential environmental and human health risks - preferably as early on as possible in the value chain and certainly throughout their life cycle.
Therefore (nano)toxicologists need to have a precise and complete idea of the characteristics of the particles, both ex situ and in situ, that could interact with the biological system of concern.
Both for raw materials and in biological environments, nanoparticle characterisation needs can be classified according to 3 domains:
1. What does the material look like?
• Particle size / size distribution (3 dimensions).
• Grain, particle, film morphology (shape, roughness, topography, crystal structure).
• Agglomeration state/aggregation.
2. What is the material made of?
• Overall composition (chemical composition).
• Surface composition.
• Purity (including levels of impurities).
3. What factors affect how a material interacts with its surroundings?
• Surface area, surface per volume (curvature).
• Surface energy, reactivity, hydrophobicity, porosity, crystallinity.
• Surface charge.
In addition, for biological systems, the context in which the particle is found is critical (e.g. in which organs does it accumulate, or in which parts of a cell).
The overarching objective of the npSCOPE project is to develop a single instrument in combination with dedicated methodologies that will answer these three questions in a rapid, accurate and reproducible way, both for nanoparticles in their pristine form and embedded in complex matrices (Error! Reference source not found.). While a particular focus will be on inorganic nanoparticles, as these account for the most abundant group as outlined above, we will also investigate the potential of npSCOPE on organic nanoparticles using two test cases.
The npSCOPE instrument will provide a full dataset about an investigated sample containing nanoparticles with the following specifications:
Size distribution of the nanoparticles: range from 0.5 nm to several hundreds of nm.
Morphology of the nanoparticles: 3D resolution of 0.5 nm.
Semi-quantitative chemical composition of the nanoparticles with excellent detection limits: from 10-3 (i.e. 0.1 at%) for nanoparticles having a volume of 100 nm3, to ppm (i.e. 10-4 at%) for nanoparticles having a volume of 105 nm3, parallel detection of all elements.
The number of nanoparticles in the investigated matrix:
automatic and accurate determination of the concentration of nanoparticles (i.e. number of nanoparticles per unit volume).
Precise localisation of the nanoparticles (e.g. in biological samples): 3D resolution of 0.5 nm.
Interaction of the nanoparticles with their environment:
detection of changes in surface composition, detection of changes in morphology, etc.
This overarching objective of the npSCOPE project can be differentiated into the following sub-objectives:
1. Development of hardware based on the Gas Field Ion Source (GFIS) as a unique key enabling technology to enable an original in situ real-time combination of Scanning Transmission Helium Ion Microscopy (STIM), Secondary Electron (SE) imaging and Secondary Ion Mass Spectrometry (SIMS) in one single platform. A cryo-stage compatible with the above described characterisation techniques will be part of the final solution. The GFIS can be operated with He+ and Ne+ ion beams and has a high brightness of 4.109 A.cm-2.sr-1 with an energy spread of less than 1 eV, enabling very small spot sizes (He+ spot sizes of 3 Å have been demonstrated) while maintaining an ion current that is appropriate for imaging and analytics.
2. Development of protocols for sample preparation and instrument operation, and correlative methodologies and software tools that will allow the automatic and accurate correlation of high sensitivity and high resolution chemical data with morphological information obtained at 0.5 nm resolution.
3. Development of analytical standards, and standardized exposure scenarios as representative test-cases for validation and cross-checking and benchmarking purposes of the npSCOPE instrument and, in general, for analytical nanomaterial characterization technologies.
4. Development of go-to-market strategies for the npSCOPE instrument taking into account performance criteria, cost, easiness to operate, level of automation, intellectual property and freedom to operate aspects.
5 Organisation
npSCOPE consists of four technical work packages WP1 through WP4, and two non-technical work packages - WP5 for
dissemination/exploitation and WP6 for
management/coordination. The relationship between these work packages, the EC and other stakeholders is sketched in Figure 4.
Figure 4 : Schematic of the projects components (work packages) and how they are linked to each other and to the funding agencies, the project's advisory board and to other stakeholders.
It is planned that the npSCOPE project will be completed within 4 years of its start. The specification and detailed design phase of WP1 is expected to last 12 calendar months, finishing with the design blueprints for the instrumentation. Building the npSCOPE instrument and upgrading some existing instruments (for their later use within the round-robin studies performed within WP4) will be performed in WP2, which will run until month 28 and finish with the functional testing of npSCOPE instrument. WP3, which will focus on workflows, software and validation of the performance specifications of the npSCOPE instrument will start at an early stage so that workflow and software development can be done in parallel to the instrument designing and building effort.
WP3 will run until month 45 to allow that the software and algorithms can be continuously optimised and upgraded based on the findings of WP4. Two major steps in WP3 are the milestones
"Alpha version of analysis software" to facilitate the round-robin and benchmarking work, and “All performance specifications of the npSCOPE instrument met” fixed at month 32. WP4 is the work package with the largest effort in terms of manpower, consisting of testing, validating and benchmarking the npSCOPE instrument Figure 3 : In situ correlation of STIM, SIMS and SE datasets to provide rapid, accurate and reproducible data of the physico-chemical properties of industrial nanomaterials, both in their pristine form and in complex matrices.
and methodologies using test cases in nanotoxicology, as well as - to a lesser extent – demonstrating applications beyond nanotoxicology as suggested by the expected impacts in the call.
The dissemination / exploitation (WP5) and management / coordination (WP6) work packages last for as long as the project.
6 Expected Impact
The development of the npSCOPE will reinforce the leading position of Europe in the field of nanotechnology and in the field of advanced instrumentation.
Identification of key descriptors that can be used to reveal correlations associated with health and environmental impacts and meaningful basis for grouping, read-across and QSARs purposes Several attempts have been made to try to group nanomaterials and to model their toxicity using QSAR approaches. The COST action MODENA, of which LIST was a member, analysed over 170 datasets in order to develop coherent approaches for the modelling of toxicity of nanomaterials (report under preparation).
Recently one OECD working group on the categorisation of manufactured nanomaterials highlighted that nanomaterials should be considered separately from chemicals and that different parameters should be considered for categorization and (Q)SAR modelling. Parameters such as size, shape, aggregation state, fate exposure, etc. should all be considered in order to develop a successful approach. The technology that will be developed within the npSCOPE project, in combination with standardized test-cases and analytical representative materials that will be produced, will allow addressing most of the critical points highlighted by the OECD working group. One barrier to developing Quantitative Structure-Activity Relationships (QSARs) that would allow the prediction of risks from nanoparticles has been the difficulty of properly characterising the materials used, both in their original form and in tissue samples or organisms after exposure. The npSCOPE instrument will go some way towards reducing this barrier by making a fairly comprehensive characterisation possible on a single instrument that can handle several sample formats, including cryo-prepared tissue sections, and perform both imaging and chemical mapping at nanometre scales. The ability to measure many particles in tissue sections (using automated sample analysis with load-lock chamber, customised ample stage and new software) will allow a better estimate of the true nanoparticle dose in particular organs or cell-types to be made; the estimation of effective doses has been one of the difficulties facing QSAR development. The npSCOPE approach will build upon the recommendations for read-across made by the European Chemicals Agency and a recent OECD survey.
Given the low sample quantities needed and the strong potential of the platform to generate high-quality physico-chemical data on nanomaterials, both ex situ and in situ, a major step forward in defining key descriptors for read-across, grouping, in silico modelling and creating meaningful relationships with biological activity data is expected. In particular, parameters describing the nanomaterials’ interface with the environment, as well as the subcellular localisation and quantification of intracellular dose are currently difficult to describe in combination with basic physico-chemical parameters, so that npSCOPE will establish an important currently missing link. The resulting QSAR models for the production of new safe-by-design nanomaterials will represent a
major improvement for R&D strategies from an industrial perspective, with a considerable reduction of the cost and time needed to ensure the safety of the new products. Another major advantage of the npSCOPE instrument as compared to the state-of-art approach, which relies on ex situ multi-technique analysis on separate standalone instruments, is that the newly developed instrument will allow a correlative in situ approach using one single ion beam, hence significantly increasing the reproducibility and the accuracy of the data acquisition and processing. The use of this innovative approach will allow an increased confidence in the determination of physico-chemical features, thus providing more reliable and consistent data for safety evaluation of nanomaterials.
The performance specifications of the npSCOPE instrument, in particular 0.5 nm resolution imaging combined with simultaneous elemental/chemical information of major elements and trace elements, together with representative nanomaterials for its calibration and verification, will make it a very suitable tool for quality control of nanomaterials during their development, production, processing, transport and storage, and detection of counterfeiting applications. Information on nanomaterials’
stability over their life time is important to avoid loss of desired product properties or development of undesired functionalities.
Another application area of the platform concerns reliable traceability of nanomaterials in consumer products, which allows for correct product labelling (cf. cosmetics directive, novel foods regulation). The obtained technical expertise as well as the developed detection systems and data analysis strategies are not restricted to nanoparticle characterisation. Many biological and technological processes rely on the unambiguous, efficient and reliable analysis of microscopic samples, sample-structure and sample-composition. Research areas such as 2D materials and traditional materials science (materials for nuclear applications, inorganic and polymer nano-composite materials, etc.) will benefit from the developed analysis equipment.
Increased confidence in nanosafety studies and findings through sound physico-chemical characterisation methods and standard operating procedures
Nanosafety studies must not only assess the effects (toxicity) of nanomaterials upon the environment and human and animal health, but for these results to be useful and transferable the nature of the nanomaterials and the dose must be understood.
Because a variety of instrumental techniques and sample preparation techniques have been used to carry out physico-chemical characterisation (but not always enough or the appropriate techniques), there has been some scatter and uncertainty in interpretation of nanosafety studies. The npSCOPE project addresses this problem in two ways. Firstly, by using a common instrument platform to carry out a number of high resolution imaging and characterisation tasks on the same sample(s) and particles, with no need for sample transfer between instruments or institutions (especially important for cryo-sections).
Secondly, by developing a set of protocols and workflows for dosing, sampling, sample preparation, imaging and image analysis and by benchmarking and validating these protocols and workflows using well-characterised materials, repeated experiments and independently repeated measurements. The npSCOPE project has selected several application area and exposure scenarios. e.g. for ingested food additives (or nanoparticles from packaging or appliances), or for dermal exposure to cosmetics. This project will help safety authorities by
improving the analytical information available for materials that are already on the market, while providing relevant test-cases based on standard methodologies (in vitro and in vivo) for further reliable and reproducible toxicity testing.
The expected outcome is to propose standard conditions for the physico-chemical characterisation of representative nanoparticles (using multi-technique analysis in one platform) in realistic conditions for tissue translocation of nanoparticles. If these approaches are widely adopted, we are certain that increased confidence in study results will follow. The increased confidence (through reproducible and validated results) in studies and findings will apply not only to the authors and readers of such studies, but will eventually be apparent to the wider public as consensus in the field emerges on the risks of some categories of nanomaterials.
Examples of synergies with other applications of the method Nanomedicine will rely on the fact that both translocation and subsequent potential toxicity in tissues and cells are highly
dependent on the surface properties and coatings of nanoparticles. e.g. nanomaterials may be 'protected' in part of the body whereas intake by particular cells elsewhere may be favoured, thus increasing oral bioavailability of nanomedicines.
Future requirements for the clinical quality control of nanosized delivery systems (including metal nanoparticles) will depend on the accuracy of physico-chemical characterization of nanoparticles, which will be improved by npSCOPE's outputs.
The npSCOPE technology can provide a valuable tool for quality control and process analysis in technical nanosystems such as fuel cells, batteries, solar cells or semiconductor nanostructures;
including the analysis of undesired contaminations. The high lateral resolution in 3D and the possibility to analyze light elements (H, Li), make the npSCOPE instrument a powerful tool to solve issues such as lithium diffusion in battery applications, catalyst dispersion or interfacial diffusion which is crucial for the effective proliferation of an electric economy and for the implementation of many renewable energy technologies, or reliability of the materials.
7 Directory
Table 1 Directory of people involved in this project.
First Name Last Name Affiliation Address e-mail
Akif Emre TÜRELI MJR Überherrn, Germany e.tuereli@mjr-pharmjet.com
Arkady KRASHENINNIKOV HZDR Dresden, Germany a.krasheninnikov@hzdr.de
David DOWSETT LIST Belvaux, Luxembourg david.dowsett@list.lu
Elisa BOUTET TOXALIM Toulouse, France elisa.boutet-robinet@inra.fr
Eric HOUDEAU TOXALIM Toulouse, France eric.houdeau@inra.fr
Fabrice PIERRE TOXALIM Toulouse, France fabrice.pierre@inra.fr
Falk LUCAS ETHZ Zürich, Switzerland falk.lucas@scopem.ethz.ch
Gregor HLAWACEK HZDR Dresden, Germany g.hlawacek@hzdr.de
Inge NELISSEN VITO Mol, Belgium inge.nelissen@vito.be
Jean-Nicolas AUDINOT LIST Belvaux, Luxembourg jean-nicolas.audinot@list.lu
Julien VIGNARD TOXALIM Toulouse, France julien.vignard@inra.fr
Lasse KLING FAU Nürnberg, Germany lasse.kling@mpl.mpg.de
Michael STEIGERWALD ZEISS Jena, Germany michael.steigerwald@zeiss.com
Miriam LUCAS ETHZ Zürich, Switzerland miriam.lucas@scopem.ethz.ch
Nazende GÜNDAY TÜRELI MJR Überherrn, Germany n.guenday@mjr-pharmjet.com
Nicolas BLANC ETHZ Zürich, Switzerland nicolas.blanc@scopem.ethz.ch
Olivier BOUTON LIST Belvaux, Luxembourg olivier.bouton@list.lu
Olivier DE CASTRO LIST Belvaux, Luxembourg olivier.decastro@list.lu
Patrick PHILIPP LIST Belvaux, Luxembourg patrick.philipp@list.lu
Peter GNAUCK ZEISS Jena, Germany peter.gnauck@zeiss.com
Peter ROZEMA PHOTONIS Roden, Netherlands p.rozema@photonis.com
Rachid BARRAHMA LIST Belvaux, Luxembourg rachid.barrahma@list.lu
Russ MELLO ZEISS Jena, Germany russ.mello@zeiss.com
Sebastian TACKE ETHZ Zürich, Switzerland sebastian.tacke@scopem.ethz.ch
Serge DUARTE PINTO PHOTONIS Roden, Netherlands s.duartepinto@photonis.com
Sévérine IFFLAND LIST Belvaux, Luxembourg severine.iffland@list.lu
Silke CHRISTIANSEN FAU Nürnberg, Germany silke.christiansen@mpl.mpg.de
Tom VENKEN VITO Mol, Belgium tom.venken@vito.be
Tom WIRTZ LIST Belvaux, Luxembourg tom.wirtz@list.lu
Tommaso SERCHI LIST Belvaux, Luxembourg tommaso.serchi@list.lu
Uwe MICK FAU Nürnberg, Germany uwe.mick@mpl.mpg.de
Véronique BECKER LIST Belvaux, Luxembourg veronique.becker@list.lu
8 Copyright
© 2017, Luxembourg Institute of Science and Technology on behalf of the npSCOPE consortium.
npSCOPE is a Research and Innovation Action under the European Commission's Horizon 2020 Programme.
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