Prioritization before risk assessment: The viability of uncertain data on food contact materials.

The shortage of data on non-intentionally added substances (NIAS) present in food contact material (FCM) limits the ability to ensure food safety. Recent strategies in analytical method development permit NIAS investigation by using chemical exploration, but this has not been sufficiently investigated in risk assessment context. Here, exploration is utilized and followed by risk prioritization on chemical compounds that can potentially migrate to food from two paperboard FCM samples. Concentration estimates from exploration are converted to tentative exposure assessment, while predicted chemical structures are assessed using quantitative structure-activity relationships (QSAR) models for carcinogenicity, mutagenicity, and reproductive toxicity. A selection of 60 chemical compounds from two FCMs is assessed by four risk assessors to classify compounds based on probable risk. For almost 60% of cases, the assessors classified compounds as either high priority or low priority. Unclassified compounds are due to disagreements between experts (18%) or due to a perceived lack of data (23%). Among the high priority substances are high-concentration compounds, benzophenone derivatives, and dyes. The low priority compounds contained e.g. oligomers from plasticizers and linear alkane amides. The classification scheme provides valuable information based on tentative data and is able to prioritize discovered chemical compounds for pending risk assessment.

Immobilization of magnetic nanoparticles onto conductive surfaces modified by diazonium chemistry.

Core-shell γ-Fe(2)O(3)@SiO(2) nanoparticles (NPs) substituted by PEG and NH(2) groups may be immobilized on metal surfaces (glassy carbon or gold) substituted by 4-carboxyphenyl groups through electrostatic interactions. Such immobilization is evidenced by (i) IRRAS owing to the Si-O band, (ii) SEM images, which show that the surface coverage by the NPs is nearly 100%, and (iii) the NPs film thickness measured by ellipsometry or AFM, which corresponds to about one NPs monolayer. Such NPs film is permeable to redox probes, which allows us to propose electrochemical methods based on direct or local measurements as a way to inspect the NPs assembly steps through their ability to alter mass and charge transfer. This process also applies to patterned polystyrene surfaces, and selective immobilization of NPs substituted by amino groups was carried out onto submillimeter patterns obtained by local oxidation. Biological applications are then expected for hyperthermia activation of the NPs to trigger cellular death. Finally, some tests were performed to further derivatize the immobilized NPs onto surfaces through either a covalent bond or electrostatic interactions. Future work will be dedicated to the recovery of such Janus NPs from the substrate surface.

Kinetic analyses and performance of a colloidal magnetic nanoparticle based immunoassay dedicated to allergy diagnosis.

In this paper, we demonstrate the possibility to use magnetic nanoparticles as immunosupports for allergy diagnosis. Most immunoassays used for immunosupports and clinical diagnosis are based on a heterogeneous solid-phase system and suffer from mass-transfer limitation. The nanoparticles' colloidal behavior and magnetic properties bring the advantages of homogeneous immunoassay, i.e., species diffusion, and of heterogeneous immunoassay, i.e., easy separation of the immunocomplex and free forms, as well as analyte preconcentration. We thus developed a colloidal, non-competitive, indirect immunoassay using magnetic core-shell nanoparticles (MCSNP) as immunosupports. The feasibility of such an immunoassay was first demonstrated with a model antibody and described by comparing the immunocapture kinetics using macro (standard microtiter plate), micro (microparticles) and nanosupports (MCSNP). The influence of the nanosupport properties (surface chemistry, antigen density) and of the medium (ionic strength, counter ion nature) on the immunocapture efficiency and specificity was then investigated. The performances of this original MCSNP-based immunoassay were compared with a gold standard enzyme-linked immunosorbent assay (ELISA) using a microtiter plate. The capture rate of target IgG was accelerated 200-fold and a tenfold lower limit of detection was achieved. Finally, the MCSNP-based immunoassay was successfully applied to the detection of specific IgE from milk-allergic patient's sera with a lower LOD and a good agreement (CV < 6%) with the microtiter plate, confirming the great potential of this analytical platform in the field of immunodiagnosis.

Separation of alpha-lactalbumin grafted- and non-grafted maghemite core/silica shell nanoparticles by capillary zone electrophoresis.

The use of nanoparticles (NPs) in immunodiagnostics is a challenging task for many reasons, including the need for miniaturization. In view of the development of an assay dedicated to an original, miniaturized and fully automated immunodiagnostics which aims to mimic in vivo interactions, magnetic zwitterionic bifunctional amino/polyethyleneoxide maghemite core/silica shell NPs functionalized with allergenic alpha-lactalbumin were characterized by CE. Proper analytical performances were obtained through semi-permanent capillary coating with didodecyldimethylammonium bromide (DDAB) or permanent capillary wall modification by hydroxypropylcellulose. The influence of experimental conditions (e.g. buffer component nature, pH, ionic strength, and electric field strength) on sample stability, electrophoretic mobility, and dispersion was investigated using either DDAB- or hydroxypropylcellulose-coated capillaries. Adsorption to the capillary wall and aggregation phenomena were evaluated according to the CE conditions. The proper choice of experimental conditions, i.e. separation under -10 kV in a 25 mM ionic strength MES/NaOH (pH 6.0) with a DDAB-coated capillary, allowed the separation of the grafted and the non-grafted NPs.

Charge-based characterization of nanometric cationic bifunctional maghemite/silica core/shell particles by capillary zone electrophoresis.

In view of employing functionalized nanoparticles (NPs) in the context of an immunodiagnostic, aminated maghemite/silica core/shell particles were synthesized so as to be further coated with an antibody or an antigen via the amino groups at their surface. Different functionalization rates were obtained by coating these maghemite/silica core/shell particles with 3-(aminopropyl)triethoxysilane and 2-[methoxy(polyethyleneoxy)propyl]-trimethoxysilane at different molar ratios. Adequate analytical performances with CE coupled with UV-visible detection were obtained through semi-permanent capillary coating with didodecyldimethyl-ammonium bromide, thus preventing particle adsorption. First, the influence of experimental conditions such as electric field strength, injected particle amount as well as electrolyte ionic strength and pH, was evaluated. A charge-dependent electrophoretic mobility was evidenced and the separation selectivity was tuned according to electrolyte ionic strength and pH. The best resolutions were obtained at pH 8.0, high ionic strength (ca. 100 mM), and low total particle volume fraction (ca. 0.055%), thus eliminating interference effects between different particle populations in mixtures. A protocol derived from Kaiser's original description was performed for quantitation of the primary amino groups attached onto the NP surface. Thereafter a correlation between particle electrophoretic mobility and the density of amino groups at their surface was established. Eventually, CE proved to be an easy, fast, and reliable method for the determination of NP effective surface charge density.

Nanomaterials in Food - Prioritisation & Assessment.

Nanomaterials (NMs) are of significant economic interest and have a huge impact on many industries including the food industry. The main application in food industry includes food additives and food packaging. However, the effects of NMs on human health are highly discussed, as well as the need of harmonised analytical methods and risk assessment methodologies. In line with these discussions, the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) has started in 2017 a 2-year project focusing on NMs in food, to which the fellow was involved under the framework of the European Food Risk Assessment Fellowship Programme (EU-FORA). This technical report contains a description of the working program, the aims and the activities to which the fellow was involved during this placement. The main aims of the programme were to be involved in different steps of risk assessment process, to improve knowledge regarding food process, analytical and toxicological methods and to learn how to conduct expert assessments. All aims were linked with different kind of activities. Gaining hands-on experience on food risk assessment was achieved mainly by collecting occurrence data and performing exposure assessment calculations for the 'of concern' NMs, while scheduled visits to laboratories specialising in analytical methods of nanoparticles and toxicological studies helped to improve knowledge in these fields. Regular participation in the Working Group (GT) related to NMs in food and interaction with experts within ANSES facilitated the learning process of how to conduct collective expertise as well as to be further trained in risk assessment processes. Furthermore, apart from knowledge gained in risk assessment and NMs, the fellow was able to obtain transferable skills and knowledge that can be used to increase the scientific capacity of the fellow's home institute as well as to expand her scientific network, which could lead to collaboration opportunities in the future well beyond this fellowship.

Chromatin immunoprecipitation in microfluidic droplets: towards fast and cheap analyses.

Genetic organization is governed by the interaction of DNA with histone proteins, and differential modifications of these proteins is a fundamental mechanism of gene regulation. Histone modifications are primarily studied through chromatin immunoprecipitation (ChIP) assays, however conventional ChIP procedures are time consuming, laborious and require a large number of cells. Here we report for the first time the development of ChIP in droplets based on a microfluidic platform combining nanoliter droplets, magnetic beads (MB) and magnetic tweezers (MT). The droplet approach enabled compartmentalization and improved mixing, while reducing the consumption of samples and reagents in an integrated workflow. Anti-histone antibodies grafted to MB were used as a solid support to capture and transfer the target chromatin from droplets to droplets in order to perform chromatin immunoprecipitation, washing, elution and purification of DNA. We designed a new ChIP protocol to investigate four different types of modified histones with known roles in gene activation or repression. We evaluated the performances of this new ChIP in droplet assay in comparison with conventional methods. The proposed technology dramatically reduces analytical time from a few days to 7 hours, simplifies the ChIP protocol and decreases the number of cells required by 100 fold while maintaining a high degree of sensitivity and specificity. Therefore this droplet-based ChIP assay represents a new, highly advantageous and convenient approach to epigenetic analyses.

Droplet Microfluidic and Magnetic Particles Platform for Cancer Typing.

Analyses of nucleic acids are routinely performed in hospital laboratories to detect gene alterations for cancer diagnosis and treatment decision. Among the different possible investigations, mRNA analysis provides information on abnormal levels of genes expression. Standard laboratory methods are still not adapted to the isolation and quantitation of low mRNA amounts and new techniques needs to be developed in particular for rare subsets analysis. By reducing the volume involved, time process, and the contamination risks, droplet microfluidics provide numerous advantages to perform analysis down to the single cell level.We report on a droplet microfluidic platform based on the manipulation of magnetic particles that allows the clinical analysis of tumor tissues. In particular, it allows the extraction of mRNA from the total-RNA sample, Reverse Transcription, and cDNA amplification, all in droplets.

Microfluidic platform combining droplets and magnetic tweezers: application to HER2 expression in cancer diagnosis.

The development of precision medicine, together with the multiplication of targeted therapies and associated molecular biomarkers, call for major progress in genetic analysis methods, allowing increased multiplexing and the implementation of more complex decision trees, without cost increase or loss of robustness. We present a platform combining droplet microfluidics and magnetic tweezers, performing RNA purification, reverse transcription and amplification in a fully automated and programmable way, in droplets of 250nL directly sampled from a microtiter-plate. This platform decreases sample consumption about 100 fold as compared to current robotized platforms and it reduces human manipulations and contamination risk. The platform's performance was first evaluated on cell lines, showing robust operation on RNA quantities corresponding to less than one cell, and then clinically validated with a cohort of 21 breast cancer samples, for the determination of their HER2 expression status, in a blind comparison with an established routine clinical analysis.

Microfluidic model of the platelet-generating organ: beyond bone marrow biomimetics.

We present a new, rapid method for producing blood platelets in vitro from cultured megakaryocytes based on a microfluidic device. This device consists in a wide array of VWF-coated micropillars. Such pillars act as anchors on megakaryocytes, allowing them to remain trapped in the device and subjected to hydrodynamic shear. The combined effect of anchoring and shear induces the elongation of megakaryocytes and finally their rupture into platelets and proplatelets. This process was observed with megakaryocytes from different origins and found to be robust. This original bioreactor design allows to process megakaryocytes at high throughput (millions per hour). Since platelets are produced in such a large amount, their extensive biological characterisation is possible and shows that platelets produced in this bioreactor are functional.

A low cost and high throughput magnetic bead-based immuno-agglutination assay in confined droplets.

Although passive immuno-agglutination assays consist of one step and simple procedures, they are usually not adapted for high throughput analyses and they require expensive and bulky equipment for quantitation steps. Here we demonstrate a low cost, multimodal and high throughput immuno-agglutination assay that relies on a combination of magnetic beads (MBs), droplets microfluidics and magnetic tweezers. Antibody coated MBs were used as a capture support in the homogeneous phase. Following the immune interaction, water in oil droplets containing MBs and analytes were generated and transported in Teflon tubing. When passing in between magnetic tweezers, the MBs contained in the droplets were magnetically confined in order to enhance the agglutination rate and kinetics. When releasing the magnetic field, the internal recirculation flows in the droplet induce shear forces that favor MBs redispersion. In the presence of the analyte, the system preserves specific interactions and MBs stay in the aggregated state while in the case of a non-specific analyte, redispersion of particles occurs. The analyte quantitation procedure relies on the MBs redispersion rate within the droplet. The influence of different parameters such as magnetic field intensity, flow rate and MBs concentration on the agglutination performances have been investigated and optimized. Although the immuno-agglutination assay described in this work may not compete with enzyme linked immunosorbent assay (ELISA) in terms of sensitivity, it offers major advantages regarding the reagents consumption (analysis is performed in sub microliter droplet) and the platform cost that yields to very cheap analyses. Moreover the fully automated analysis procedure provides reproducible analyses with throughput well above those of existing technologies. We demonstrated the detection of biotinylated phosphatase alkaline in 100 nL sample volumes with an analysis rate of 300 assays per hour and a limit of detection of 100 pM.

Colloidal nanomaterial-based immunoassay.

Nanomaterials have been widely developed for their use in nanomedicine, especially for immunoassay-based diagnosis. In this review we focus on the use of nanomaterials as a nanoplatform for colloidal immunoassays. While conventional heterogeneous immunoassays suffer from mass transfer limitations and consequently long assay time, colloidal immunosupports allow target capture in the entire volume, thus speeding up reaction kinetics and shortening assay time. Owing to their wide range of chemical and physical properties, nanomaterials are an interesting candidate for immunoassay development. The most popular colloidal nanomaterials for colloidal immunoassays will be discussed, as well as their influence on immune reactions. Recent advances in nanomaterial applications for different formats of immunoassays will be reported, such as nanomaterial-based indirect immunoassays, optical-based agglutination immunoassays, resonance energy transfer-based immunoassays and magnetic relaxation-based immunoassays. Finally, the future of using nanomaterials for homogeneous immunoassays dedicated to clinical diagnosis will be discussed.

Microchip integrating magnetic nanoparticles for allergy diagnosis.

We report on the development of a simple and easy to use microchip dedicated to allergy diagnosis. This microchip combines both the advantages of homogeneous immunoassays i.e. species diffusion and heterogeneous immunoassays i.e. easy separation and preconcentration steps. In vitro allergy diagnosis is based on specific Immunoglobulin E (IgE) quantitation, in that way we have developed and integrated magnetic core-shell nanoparticles (MCSNPs) as an IgE capture nanoplatform in a microdevice taking benefit from both their magnetic and colloidal properties. Integrating such immunosupport allows to perform the target analyte (IgE) capture in the colloidal phase thus increasing the analyte capture kinetics since both immunological partners are diffusing during the immune reaction. This colloidal approach improves 1000 times the analyte capture kinetics compared to conventional methods. Moreover, based on the MCSNPs' magnetic properties and on the magnetic chamber we have previously developed the MCSNPs and therefore the target can be confined and preconcentrated within the microdevice prior to the detection step. The MCSNPs preconcentration factor achieved was about 35,000 and allows to reach high sensitivity thus avoiding catalytic amplification during the detection step. The developed microchip offers many advantages: the analytical procedure was fully integrated on-chip, analyses were performed in short assay time (20 min), the sample and reagents consumption was reduced to few microlitres (5 µL) while a low limit of detection can be achieved (about 1 ng mL(-1)).

Magnetic core shell nanoparticles trapping in a microdevice generating high magnetic gradient.

Magnetic core shell nanoparticles (MCSNPs) 30 nm diameter with a magnetic weight of 10% are usually much too small to be trapped in microfluidic systems using classical external magnets. Here, a simple microchip for efficient MCSNPs trapping and release is presented. It comprises a bed of micrometric iron beads (6-8 µm diameter) packed in a microchannel against a physical restriction and presenting a low dead volume of 0.8 nL. These beads of high magnetic permeability are used to focus magnetic field lines from an external permanent magnet and generate local high magnetic gradients. The nanoparticles magnetic trap has been characterised both by numerical simulations and fluorescent MCSNPs imaging. Numerical simulations have been performed to map both the magnetic flux density and the magnetic force, and showed that MCSNPs are preferentially trapped at the iron bead magnetic poles where the magnetic force is increased by 3 orders of magnitude. The trapping efficiency was experimentally determined using fluorescent MCSNPs for different flow rates, different iron beads and permanent magnet positions. At a flow rate of 100 µL h(-1), the nanoparticles trapping/release can be achieved within 20 s with a preconcentration factor of 4000.

A chemometric approach for optimizing protein covalent immobilization on magnetic core-shell nanoparticles in view of an alternative immunoassay.

A chemometric approach was developed to optimize the grafting of a bovine milk allergen: alpha-Lactalbumin (alpha-Lac) on colloidal functionalized magnetic core-shell nanoparticles (MCSNP). Such nanoparticles, functionalized with polyethyleneglycol and amino groups, exhibit a 30nm physical diameter and behave as a quasi-homogeneous system. The alpha-Lac immobilization was achieved through the covalent binding between MCSNP amino groups and alpha-Lac carboxylic moieties using the well-known tandem carbodiimide (EDC) and hydroxysulfosuccinimide (NHS). In this study, a chemometric approach was employed to highlight the parameters influencing the number of grafted proteins on the MCSNP. Three factors were evaluated: the ratio in concentration between EDC and alpha-Lac, between NHS and EDC and the concentration of alpha-Lac. After a first full factorial design to delimit the region of the space where the optimum could be located, a central composite design was then carried out to predict the best grafting conditions. It was established and experimentally confirmed that the optimum parameters are [EDC]/[alpha-Lac]=25; [NHS]/[EDC]=1.55 and alpha-Lac=24.85nmolmL(-1). In these optimal conditions, MCSNP surface was successfully saturated with alpha-Lac (34 alpha-Lac/MCSNP) with a high reproducibility (RSD=2%). The colloidal stability of MCSNP grafted with alpha-Lac as well as the immunological interactions using anti alpha-Lac antibody were then investigated in different buffers. The results emphasized that a 50mM MES buffer (pH 6) allows an efficient immune capture and a satisfying colloidal stability which provide an immunological interaction in homogeneous liquid phase.