Nanoparticles in food:
Analytical methods for detection and characterisation
NanoLyse is a European collaborative research project which is partly funded by the European Commission under the 7th Framework Programme (contract no. 245162). The project addresses the increasing need for analytical tools for the measurement of engineered nanomaterials in food. The bi-annual NanoLyse newsletter keeps stakeholders, scientific communities and the interested public up to date with aims and progress of the project. Furthermore, NanoLyse dissemination activities such as Open Days and training workshops will be announced. The newsletter is completed by a compilation of external events related to the topic. A full version of this newsletter as well as further information is available via the project’s website: nanolyse.eu
The joint second/third issue describes the first outcomes of the project focused on development of validated methods and reference materials for the analysis of engineered nanoparticles in food and beverages.
I wish you a pleasant reading and especially would like to draw your attention to the first NanoLyse Open Day. This event will take place in conjunction with the 5th International Symposium on Recent Advances in Food Analysis (RAFA 2011) in November in
Prague and will offer the possibility to get familiar with goals and progress of the project and to discuss with NanoLyse scientists.
Coordinator of the NanoLyse project
WP1: Reference materials for engineered nanoparticles
In the first phase of the project Work package 1 focused on the procurement and characterisation of suitable nanoparticles for use in method and reference material development.
Silver and silica nanoparticles were obtained from several commercial suppliers and the most suitable ones in terms of size, dispersibility and stability were chosen. The selected materials were silver particles (43 ± 15 nm, spherical, PVP stabilised) from NANOGAP (Spain) and silica particles (150 ± 63 nm; Aerodisp® W 7520 N) from Evonik (Germany). Silica particles are an approved and widely used food additive (E551) and the chosen material is largely equivalent to OECD testing material NM203. Dispersions of defined concentration levels were prepared. They were distributed to the project partners and will be used in the development of reference materials.
The initial intention to include hydrophobic surface modified nanoparticles which are applied i.a. in food packaging was hampered by stability and dispersibility issues. A number of commercially available and custom synthesised particles have been investigated, but none was suitable for addition to the target matrix (edible oil). Finally, it was opted to switch to C60 fullerenes. These hydrophobic particles are present as contaminants in the environment and thus may enter and enrich in the food chain.
Stability issues prevented so far the selection of suitable organic nanoparticles. Organic particles on the market tend to dissolve in the medium term, thus making them unsuitable for reference materials. However, initial experiments with research stage organic nanoparticles show promising results.
Furthermore, production of stable labelled inorganic materials was successful. Labelled analogues for silica and silver nanomaterials are available. These materials are intended as internal standard for the methods to be developed.
Finally, a common validation approach for the determination of nanoparticles in food was developed. The proposed approach will be published as a basis for discussion among scientific and regulatory communities and the iterative development and advancement of robust and widely accepted validation guidelines.
WP2: Development of rapid imaging and screening methods for engineered nanoparticles
Work on the development of analytical imaging techniques for the detection of engineered nanoparticles in food is initially focused on nanosilica, which is present in fumed silica used as a free-flowing agent in various powered foods, such as coffee creamer, soups etc. As nanoparticles of silica tend to agglomerate when dispersed in aqueous media, the current work is aimed at establishing whether this does happens in food, and not as a result of the procedures used during analysis. The aim is to obtain a true depiction of nanosilica form in food products. Attempts have therefore been made to stabilise nanosilica with proteins such as, bovine serum albumin (BSA), and/or by changing pH of the media. The stability of the dispersions over longer periods of storage is also being investigated. The methods being used for detection of nanosilica include light dispersion, electron microscopy (TEM, SEM) and ICP-MS for total Si content.
To streamline the imaging methods, an object-based image recognition software system is being developed. The scrutiny of electron microscopy images is currently carried out manually, which is a time consuming process that may also lead to human errors. The prototype of the software has been developed, and is being further refined. The software system will enable a semi-automated screening of a large number of samples using the imaging methods.
In parallel, screening assays for nanoparticles in food are developed based on two different platforms: Surface Plasmon Resonance (SPR) and ELISA. In both cases, the development of specific binders for nanoparticles is crucial. On the one hand, efforts have been focussed on the generation of polyclonal antibodies for commercial and research stage organic nanoencapsulates. A high quality polyclonal antibody against one of these NPs was obtained and served to develop a sensitive and selective sandwich ELISA. The application of the developed procedure to food matrices such as apple, orange and grape juice, milk and soft juice has been initiated. On the other hand, specific metal binding proteins or synthetic peptides are explored as recognition elements for inorganic nanoparticles. A silver-NP binding protein was successfully coated onto an SPR sensorchip surface. The chip can be used for detection and quantification of silver nanoparticles in food and water samples at parts per billion level. The applicability of this SPR assay to measurements in complex matrices, its robustness and selectivity are currently being studied and optimised.
WP3: Development of coupled separation / characterisation methods for inorganic nanoparticles
The work of WP3 was focused on a strategy and methodology for representative sampling of solid or liquid foods for inorganic engineered nanoparticles (ENP) analysis and adaption and optimisation of analytical methods for ENP in food matrices.
Principles for nanoparticle sampling procedures from food matrices based on statistical considerations have been established. Separation methods based on asymmetric flow field flow separation (AF4) were developed for different Ag and SiO2 nanopaticles.
Various detection devices coupled to Field Flow Fractionation (FFF) have been evaluated. These included UV vis, multi-angle laser light scattering (MALLS), dynamic light scattering (DLS) and inductively coupled plasma mass spectrometry (ICP-MS). Optimizations were carried out towards analysis of silver and silica nanoparticles in food matrices, resulting i.a. in recommendations concerning the type of detector and the setting to be used. ICP-MS and light scattering detection present the most promising outcome. Besides, improvements of the quality of the results are expected with the optimization of the separation technique.
WP4: Development of coupled separation / characterisation methods for organic and functionalised nanoparticles
Work aimed at development of sampling, sample preparation and chromatographic separation and detection methods for organic ENP in food has been initiated.
A differential mobility analyser (DMA) to separate nanoparticles on size is operational and being tested. Nanoparticles in suspension are being nebulised and ionized with a system that is comparable with electron spray ionisation in LC/MS analysis. The resolution of the system appears to be good, also for small (>20 nm) particles.
At the same time a nanoparticle database for MALDI-TOF is being prepared and additional particles are tested to add them to the database. Suspensions of these particles, and later on sample extracts, are separated using hydrodynamic chromatography to isolate the fraction containing the particles of interest. These are then off-line analysed with MALDI-TOF.
In addition to that, if the MALDI-TOF is not specific enough, or if compounds are not ionised in MALDI-TOF, mass fingerprinting using TOFMS is being studied. First results with casein as a model particle show that it can be characterised by MALDI-TOF, but much more in detail (type of casein) by fingerprinting/TOFMS.
WP5: Dissemination and training activities
The intra consortium training programme has been launched. It aims at the exchange of expertise among the project participants and supporting young scientists in the development of their careers. Two trainings for two trainees were held in DTU, Denmark during March 2011, while further are already scheduled in the coming months.
The NanoLyse consortium members represented the project at several events with the aim to disseminate information both about the project goals, its activities and knowledge generated within the project. More details on WP2 activities and WP5 activities are available from the project website. Furthermore, the NanoLyse profile was also contributed to the NanoSafety Cluster Compendium, which gives a good overview of current European projects in this area.
NanoLyse authors contributed in total five review papers to a special issue of Trends in Analytical Chemistry reflecting research topics of individual NanoLyse WPs in:
· WP1: Thomas P.J. Linsinger, Gert Roebben, Conxita Solans, Roland Ramsch (2011). Reference materials for measuring the size of nanoparticles, Trends in Analytical Chemistry 30:18-27.
· WP2: Agnieszka Dudkiewicz, Karen Tiede, Alistair B.A. Boxall, Katrin Loeschner, Louise Helene Soegaard Jensen, Eric Jensen, Rafal Wierzbicki, Kristian Molhave (2011). Characterisation of nanomaterials in food by electron microscopy, Trends in Analytical Chemistry 30:28-43.
· WP3: Frank Von der Kammer, Samuel Legros, Thilo Hofmann, Erik H. Larsen, Katrin Loeschner (2011). Separation and characterization of nanoparticles in complex food and environmental samples by field-flow fractionation, Trends in Analytical Chemistry 30: 425-436.
· WP4: Ruud Peters, Guillaime ten Dam, Hans Bouwmeester, Hans Helsper, Stefan Weigel, Günter Allmaier, Frank vd Kammer, Roland Ramsch, Conxita Solans, Monika Tomaniova, Jana Hajslova (2011). Identification and characterization of organic nanoparticles in food, Trends in Analytical Chemistry 30:100-112.
Günter Allmaier, Anne Maißer, Christian Laschober, Paul Messner, Wladyslaw W. Szymanski (2011). Parallel differential mobility analysis for electrostatic characterization and manipulation of nanoparticles and viruses, Trends in Analytical Chemistry 30:123-132.
The 1st NanoLyse Open day will be organised as satellite event of the 5th International Symposium on Recent Advance in Food Analysis (RAFA 2011), 1-4 November, 2011, in Prague, Czech Republic. The objectives and first results of the project will be presented through posters, (video) demonstration activities, handouts and oral explanations. Opportunities for discussions on the presented project activities with the involved scientists will be available. Please register as stakeholder at the NanoLyse website in order to receive update information on the Open Day.
5th International Conference on Nanotechnology – Occupational and Environmental Health
9-12 August, 2011, Boston, MA, USA More >>>
6th International Conference on the Environmental Effects of Nanoparticles and Nanomaterials
19-21 September, 2011, London, United Kingdom More >>>
21-23 September 2011, Brno, Czech Republic More >>>
1st Annual World Congress of Nano-S&T
23-26 October, 2011, Dalian, China More >>>
5th International Symposium on Recent Advances in Food Analysis
Session on “Analysis of nanoparticles in food”
1-4 November 2011, Prague, Czech Republic More >>>
SETAC North America 32nd Annual Meeting
Session on “Metrology tools for studying the behavior and effects of nanomaterials in the environment and biological systems”
13-17 November 2011, Boston, Massachusetts, USA More >>>
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The work leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 245162.
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