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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - ACEnano (Analytical and Characterisation Excellence in nanomaterial risk assessment: A tiered approach)

Teaser

Summary

Horizon2020-funded ACEnano project is working towards introducing confidence, adaptability and clarity into NMs risk assessment by developing a widely implementable and robust tiered approach to NM physico-chemical characterisation that will simplify and facilitate contextual (hazard or exposure) description and its transcription into a reliable NMs grouping framework. This is implemented by the creation oftiered approach to cost efficient NMs analysis that will facilitate decision-making in choice of techniques and protocols. The ACEnano toolbox includes methods for sample preparation and measurement of all physicochemical variables and a combination of methods to ensure robust characterisation.
The Objectives of ACEnano are:
1. To innovate a carefully selected set of analytical techniques, instrumentation and equipment for the testing of NM properties/descriptors that are: (i) most diagnostic to nanosafety, (ii) linked to key descriptors (ensembles of particle sizes, complex shapes, surface area and surface chemistry, coating stability or multiple composition (multicomposites), (iii) facilitate assessment of the longer term fate of particles following their interactions within complex matrices, i.e. in living systems, or the environment; (iv) have the potential to resolve analytical uncertainty and/or reduce testing costs. Among the innovations are minimisation of inconsistencies by streamlining and automating sample delivery (to analytical detection methods), providing an integrated modular screening regime (ACEnano toolbox), available as virtual tools for SMEs deciding which characterisation is needed.
2. To optimise the existing techniques/ instrumentation, miniaturise and simplify where possible (to reduce costs and enhance accessibility for SMEs) and support their use by SMEs through training and documentation.
3. To benchmark key components of the ACEnano toolbox and set criteria for future benchmarking of further components, thereby building the necessary confidence of all stakeholders. ACEnano will create a cohort of trained experts in a core of partner facilities, who will participate in all round robins and will deliver a series of training events to the nanosafety community.
4. To link the methodological advancements of ACEnano to a mechanistic ontology framework, importantly considering fate and exposure as well as hazard, and create an interface between the ACEnano Data Warehouse and the ACEnano toolbox, thereby creating the ACEnano â€œvirtual toolboxâ€ and underpinning decision tree to guide users through selection of the most appropriate (combination of) methods to address their specific research or regulatory question.
5. To embed all above into a quality assurance and risk assessment framework to increase confidence in NMs characterisation, grouping, prediction of impacts and risk assessment that will facilitate uptake by industry and legislators.
6. To ensure dissemination and exploitation of the ACEnano project, its innovations and outcomes to the spectrum of stakeholders in industry (SME and enterprise, both NMs producers and users), regulatory and policy communities including those responsible for implementation, and the scientific community.

Work performed

The second period of effort was very much about delivering the technological innovations, optimisations and benchmarking, as appropriate, and addressing all other peripheral challenges, such as moving forward with their implementation, when appropriate via interlaboratory testing and standardization, dissemination and training. A very exciting aspect of the ACEnano project is the strong collaborative element throughout technologies, interlaboratory comparisons, and data or training solutions, including multi-industry partner solutions. In terms of a technical focus, ACEnano specifically addresses 9 physico-chemical properties or measurement challenges, namely: polydisperse samples, ultrasmall nanomaterial sizes, shape, surface chemistry, coating stability, multiple compositions, interactions with living systems, and dissolution/environmental fate. Excellent progress is being made towards addressing all of these parameters individually, and towards optimization of methods that can address several of them in parallel or in sequence (i.e. via hyphenation).

Final results

A summary of some of the key advances to date include:

New hardware and/or software developed:
- Implementation of different sample introduction systems (syringe pump, autosampler) into PNV software to gain more stability during measurements and higher throughput in ICP-MS studies.
- TofWerk Tofpilot module for single particle and single cell analysis developed and now integrated into their instruments â€“ already shipped to first customers.
- The newly designed Vitrocell 12/1 analytical modules (miniaturised ALI system) are integrated into the prototype of miniaturised automated exposure station.
- Horiba developed a new liquid cell for TERS measurements in liquid.
- An ultrasound cross-coupling device was developed and successfully integrated in a universal sample introduction system (CSEM).
- A mixing and readout system was developed that encompasses the microfluidic well plate, initiates the mixing and incubation process and automatically measures the absorbance of the 24 reactivity assays.
- Use of hydrogen in helium as the collision cell technology gas for ICP-MS led to a decrease of the limit of detection (LOD) for 56Fe a of at least an order of magnitude.
- Reduction of false positives/negatives in spICP-MS, i.e. distinguish accurately dissolved ions from particle events, was achieved by developing the nanoCount algorithm further to utilize the shape of particle peaks for distinction of dissolved ion/particle events.

Hyphenation of methods:
- FFF-SP-TOF-MS evaluated using Postnova FFF system.
- Offline coupling of FFF and NTA results show that sample concentration, nature of the solvent and NTA flow rate need to be optimized in order to obtain analytically meaningful results. A preliminary application note is already drafted.

New assays developed:
- A microfluidic well plate was developed that allows for 24 assays to be performed fully automatically and in parallel assessing NM dissolution and for NM reactivity.
- Preliminary TERS measurements of lipid bilayers in liquid using side illumination. This work shows that the NMs are readily absorbed by cell membranes.
- Hydrophobic interaction chromatography using CSEM-owned technology (waveguide interrogated optical sensor) measures the adhesion of NMs directly on the surface of a chip. Adhesion is promoted by specific hydrophilic and hydrophobic coatings on the chip.
- LA-sp-ICP-MS method was successfully applied to soil samples containing AuNPs with various concentrations and sizes. Particle size distributions of AuNP in the soil samples were similar to the particle size distribution of the reference Au NP suspension.

The method development and optimisation work has resulted in 9 published articles during the second period.

Expected results until the end of the project and potential impacts
The ACEnano tiered characterization concept and ACEnano methods Toolbox will bring an enhanced efficiency in method selection, reduce unnecessary costs and effort, whilst simultaneously increasing the confidence of industry and regulators in the quality, reliability and relevance of the data produced utilizing the ACEnano toolbox.