1st NanoLyse OPEN DAY – A BIG SUCCESS !
The first NanoLyse Open Day had been organised on the 2nd November 2011, during the 5th International Symposium on Recent Advances in Food Analysis (RAFA 2011), one of the biggest food analysis events in the world, held from the 1st to the 4th November 2011 in Prague, Czech Republic.
The Open day was attended by more than 50 participants from various countries and different sectors as well as from national and European authorities. They actively engaged in discussions with the present NanoLyse scientists. The level of interaction was remarkably high and there was an intense exchange not only on the presented goals and results of the project, but also about future needs and possible collaborations.
WP1: Reference materials for engineered nanoparticles
Tomato soup has been prepared from basic ingredients (bones, fresh onions, tomatoes etc.) to ensure absence of engineered nanoparticles. This soup was spiked by silica nanoparticles to achieve final mass fractions of 1 and 4%. This soup corresponds to for example soups in powder form containing silica as anti-caking agent (approved food additive E551), which may also be small enough to fulfil the definition of nanomaterial.
Rapeseed oil was spiked with fullerenes at mass fractions of 10 and 100 mg/L. These materials have been distributed to the other project partners for further experiments.
Experiments to stabilise meat, the target matrix for the silver particles, have been executed: freezing is not possible because of potential change and agglomeration of particles. Steam sterilisation was also not successful, but gamma-irradiation was found to prevent degradation. Ag nanoparticles in aqueous suspension remained unchanged by gamma-irradiation, hinting that it should not cause any problems also for the matrix material. Currently, meat spiked with Ag nanoparticles, mimicking e.g. diffusion from Ag-impregnated cutting boards, is being prepared.
Labelled particles have been synthesised and characterised: Silica nanoparticles containing a small amount of GeO2 and Ag nanoparticles containing a small amount of Au were produced. The materials were characterised with respect to their particle size, dispersion behaviour and Au/Ge mass fraction. The particles show sufficient stability and are intended as internal standards and/or as recovery control for the methods being developed.
WP2: Development of rapid imaging and screening methods for engineered nanoparticles
One of widely used approaches for identification of engineered nanoparticles (ENPs) is electron microscopy (EM). EM allows not only to acquire images for subsequent sizing of ENPs but also to characterize ENPs morphology, elemental composition (when coupled to spectrometric techniques) and interactions with the food components. Therefore it has a potential of numerous applications for visualization of ENPs in food samples. In this project EM is explored as a screening technique for ENPs in food. Major challenge was development of suitable sample preparation protocols, which would allow to image ENPs in unaltered state. In comparison to some standard specimen preparation approaches, methods developed within the project activities allowed to:
1. avoid artefact of particle clustering,
2. increase particle recovery from the sample
3. improve contrast of ENPs in EM
4. shorten sample preparation time from 4-5 days to 1-2 hours
WP2 is subsequently dealing with optimization of imaging conditions for liquid and solid food samples containing ENPs of different chemical composition. Suitable imaging and sample preparation protocols will then be combined into validated approach for screening on ENPs in food.
A major bottleneck in the analysis of nanomaterials in complex matrices by electron microscopy is the time and effort required to assess each of the images manually. This issue is being addressed in WP2 through the development of an object-based image analysis system to streamline the processing of EM images in a (semi)automated way. The development of the image analysis system, based on eCognition platform (Trimble, Germany), involved a large number of EM images in different nanoparticle-matrix scenarios. A crucial aspect was to understand and ‘teach’ the programme those basic criteria that an expert would use in manual processing of the image to distinguish a nanoparticle from background or from other matrix-related objects. Developed software when tested and fine-tuned will be a very useful tool in rapid processing of a lot of EM images to provide information on the presence/absence of nanoparticles, as well as various parameters related to their size/number/agglomeration state, etc.
In parallel, two screening assays based on two different approaches for determination of nanoparticles in food are developed.
For ELISA, one specific polyclonal antibody has been raised against crosslinked gelatin NP. Gelatin NP can be detected in fruit juice (apple and orange), milk and soft drink matrices with limit of detection below 0.5 ppm. Sample preparation has been optimized to allow detection of the NP in all matrices. Validation of the ELISA prototype will start after complete characterisation of the NP by the partners.
A second ELISA has been developed for nano-carrier systems made from beta-lactoglobulin (loaded with the antioxidant epigallocatechin gallate). An antibody for the native beta-LG can be used and allows detection in fruit juice (apple and orange), and soft drink matrices with the limit around 0.25 ppm.
A Surface Plasmon Resonance biosensor for rapid detection of nanosilver in food has been developed. The biosensor has been characterized in terms of selectivity and sensitivity towards different types of silver nanoparticles and was applied for measurements in fresh vegetables and river water. Immobilisation of a metal-binding protein on the sensor surface enables screening for potentially biologically active silver nanoparticles with parts per billion sensitivity, displaying the highest sensitivity towards larger and uncoated AgNPs. The findings suggest that this SPR sensor has the potential to be utilized as a routine screening method for silver nanoparticles, providing rapid and automated analysis dedicated to environmental and food safety monitoring.
WP3: Development of coupled separation / characterisation methods for inorganic nanoparticles
WP3 aims to develop methods based on Field Flow Fractionation (FFF) for the separation and characterization of inorganic nanoparticles in food matrices, namely silver nanoparticles in lean meat and silica nanoparticles in tomato soup.
At first, UV-Vis absorbance, dynamic (DLS) and multi-angle laser light scattering (MALLS) and inductively coupled plasma mass spectrometry (ICP-MS) were tested for the detection of the nanoparticles following FFF separation. Experiments were performed with standard silver and silica nanoparticles suspensions. UV-Vis absorbance permits to detect Ag NPs and SiO2 NPs dispersed in water, but the application for food extracts will be difficult since the signal also includes size and material dependent effects. Quantification with high sensitivity could be achieved by FFF-ICP-MS; Ag NPs had a low limit of detection (LOD) of a few 100 pg/ml. Difficulties for SiO2 NPs determination due to high Si background have been suppressed using collision/reaction cell decreasing the LOD especially in high carbon background.
Direct size information could be obtained for Ag NPs in the size range of 20 to 70 nm based on FFF-DLS measurements. The optimum silver mass concentration for DLS of Ag NPs was around 100 ng/ml. Direct size determination of 100 nm SiO2 NP for Si concentrations down to a few µg/ml was possible with FFF-MALLS. However, the light scattering signal is not specific for SiO2 NPs and has to be used with care in complex samples.
Secondly, separation methods were developed for the aqueous suspensions of silver and silica nanoparticles provided by WP1. Various FFF separation parameters were optimized including carrier liquid composition, flow conditions, membrane material and channel height. The separation was evaluated based on retention times and relative recoveries. The optimization of the separation methods is about to be finished.
Finally, the development of sample preparation methods for the two different food matrices, meat and tomato soup, has been initiated. For FFF separation the matrix has to be removed without changing the nanoparticle characteristics. Acid digestion and colloidal extraction procedure of tomato soup containing silica nanoparticles was successful with relatively high recoveries. Further work will focus on optimization of the sample preparation to achieve high recoveries and to reduce preparation time. Enzymatic digestion was tested for the extraction of silver nanoparticles from meat. Further work will investigate the stability of the nanoparticles during enzymatic digestion; alternative preparation methods will be also tested.
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 continued. Protein-, carbohydrate- and lipid-based particles are used as model nanoparticles. In addition, C60 fullerenes have been added as a carbon-based nanoparticle.
Filtration and size exclusion chromatography show potential as a sample preparation technique for organic nanoparticles. However, further research is required when the analytical methods are in place. For sampling of nanoparticles, a theoretical study was performed to study the relation between sample size and sampling error.
A parallel differential mobility analysis (pDMA) method is developed for the separation of organic nanoparticles in liquid suspensions. Detection is done using a Faraday cup or a condensation particle counter (CPC) and fractions are collected in an electro nanoparticle sampler for further analysis with techniques like electron microscopy (EM), atomic force microscopy (AFM) or matrix assisted laser desorption ionization mass spectrometry (MALDI-TOFMS). The resolution of the system appears to be good, also for small (>20 nm) particles.
Hydrodynamic chromatography (HDC) shows good results for size separation of protein, lipid and carbohydrate based nanoparticles. Reversed phase chromatography (HPLC) is suitable for the separation of carbon-based nanoparticles. Characterization and identification of organic nanoparticles is done using MALDI-TOFMS. A combination of HDC and off-line MALDI-TOFMS is used for size and chemical characterization and identification of organic nanoparticles employing a nanoparticle database.
Since the possibilities of sample preparation methods may be limited, two TOFMS based screening methods for detection of organic nanoparticle components in the whole sample are tested; ultrahigh-performance liquid chromatography with time-of-flight mass spectrometry (UHPLC-TOFMS) and direct analysis in real time (DART) in combination with TOFMS. With both techniques it is possible to detect the presence of organic nanoparticle components in fruit juices at levels below 1%.
WP5: Dissemination and training activities
The intra consortium training programme, aimed at the exchange of expertise among the project participants and supporting young scientists in the development of their careers, continues; training for one trainee was held in UVIE, Vienna, Austria, during September 2011.
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 WP1 activities, WP2 activities, WP3 activities, WP4 activities and WP5 activities are available from the project website.
Session on the analysis of nanoparticles in food at RAFA 2011 received a lot of attention
NanoLyse organised and chaired an oral session on “Analysis of Nanoparticles in Food” within the highly recognised symposium on Recent Advances in Food Analysis (RAFA), held in Prague, in November 1-4, 2011. The presentations from international experts (both from within and outside the NanoLyse project) gave an overview of the state of the art in analysis of engineered nanoparticles in food. NanoLyse scientists contributed by 5 lectures and also by 5 posters in the poster session on Nanoparticles. In addition, NanoLyse research was awarded by one of RAFA 2011 poster awards.
1st NanoLyse OPEN DAY – A BIG SUCCESS !
At the 1st NanoLyse Open day organised on the 2nd November, 2011 as satellite event of the 5th International Symposium on Recent Advance in Food Analysis (RAFA 2011), 1-4 November, 2011, in Prague, Czech Republic, project plans, progress and major outcomes have been presented by NanoLyse scientists.
Workshop on image analysis, which was participated by partners who have expertise and/or are involved in the use of imaging techniques, was organised at Fera, UK, in September 2011.
In the last phase of the project the second NanoLyse Open Day and two training workshops will be organised. Please register as stakeholder at the NanoLyse website in order to receive more information on planned NanoLyse events.
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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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