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.

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.