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Extracellular Vesicles (EVs) carry cell-derived proteins that confer functionality and selective cell uptake. However, whether proteins are packaged stochastically or co-enriched within individual EVs, and whether co-enrichment fluctuates under homeostasis and disease, has not been measured. EV abundance and protein global relative expression have been qualified by bulk analysis. Meanwhile, co-enrichment is not directly accessible via bulk measurement and has not been reported for single EV analysis. Here, we introduce the normalized index of co-enrichment (NICE) to measure protein co-enrichment. NICE was derived by (i) capturing EVs based on the expression of a membrane-bound protein, (ii) probing for the co-expression of a second protein at the population level - EV integrity underwrites the detection of single EV co-expression without the need to resolve single EVs - and (iii) normalizing measured values using two universal normalization probes. Axiomatically, NICE = 1 for stochastic inclusion or no overall co-enrichment, while for positive and negative co-enrichment NICE > 1 or < 1, respectively. We quantified the NICE of tetraspanins, growth factor receptors and integrins in EVs of eight breast cancer cell lines of varying metastatic potential and organotropism, combinatorially mapping up to 104 protein pairs. Our analysis revealed protein enrichment and co-expression patterns consistent with previous findings. For the organotropic cell lines, most protein pairs were co-enriched on EVs, with the majority of NICE values between 0.2 to 11.5, and extending from 0.037 to 80.4. Median NICE were either negative, neutral or positive depending on the cells. NICE analysis is easily multiplexed and is compatible with microarrays, bead-based and single EV assays. Additional studies are needed to deepen our understanding of the potential and significance of NICE for research and clinical uses.

International audience; Bacterial contamination and subsequent infections are a major threat to human health. An early detection in the food chain, clinics or the environment, is key to limit this threat. We present a new concept to develop low-cost hand-held devices for the ultra-sensitive and specific detection of bacteria in a one-step process of 2–8 h, directly from complex raw samples. This approach is based on a novel microfluidic magnetic fluidized bed. It reaches a 4 CFU (colony forming unit) sensitivity with high quantification accuracy in a large dynamic range of 100–107CFU/mL. The versatility of the approach was demonstrated with the detection of different bacteria strains, among which Salmonella Typhimurium and E. coli O157:H15. Additionally, the method is sensitive to infectious bacteria only, a criterion requested by main applications and currently requiring additional culture steps of one to several days.

To tackle new challenges in trace analysis, a new generation of microfluidic magnetic fluidized has been developed. This system is based on a suspension of magnetic beads inside a microfluidic channel. Particles are in suspension between two forces: the drag one created by the flow of liquid and the magnetic force due to addition of a permanent magnet to the system. In order to increase the flow rate of work, the height of the chip has been increased five times. We were able to work at 15 µL/min and the efficiency of the new system was improved by addition of a vibrator motor and a bimodal support. A proof of concept was performed on capture of biotin and DNA. A second part of my PhD work was dedicated to the application of the fluidized bed module for bioanalysis. First, extraction, pre-concentration and detection of bacteria on chip have been performed with a significant decrease of the limit of detection and of the time scale compared to usual methods. Then the system of fluidized bed was used as a platform to tests new ligands in order to perform a competitive bioassay in the context of preeclampsia. Finally, an ELISA was integrated on the fluidized bed with promising results.Those results proved the efficiency of the microfluidic magnetic fluidized bed to work as a module for extraction, pre-concentration and detection of target of low concentration., Un système de lit fluidisé magnétique microfluidique a été développé pour répondre aux besoins d’extraction et de préconcentration nécessaire comme première étape de bioanalyse. Ce système se base sur le même concept que les lits fluidisés macroscopiques en remplaçant la gravité par une force magnétique. Un nouveau module a été développé au court de ma thèse afin d’aborder de nouveaux défis en terme de détection à très basse concentration. Une augmentation de la hauteur de la puce nous a permis de travailler à hauts débits (15 µL/min). Un premier travail sur l’homogénéisation de la répartition des billes à l’intérieur de la puce microfluidique a été effectué grâce à l’implémentation d’un système de vibration et d’une matrice bimodale. Cette approche a prouvé son efficacité sur des modèles de capture de protéines et d’ADN.En parallèle, une seconde partie de mon travail a porté sur l’application du système de lit fluidisé dans à différents défis analytiques. Dans un premier temps, nous avons montré son potentiel pour l’analyse bactérienne. Le lit fluidisé peut aussi être utilisé comme plateforme de tests pour ligands pour la prise en charge de patientes souffrant de pré-éclampsie au cours de leur grossesse. Enfin un immuno-dosage de type ELISA a été réalisé entièrement dans ce système de lit fluidisé.Ces résultats montrent l’adaptabilité de ce système au-delà de ses capacités à travailler à haut débit, bas coût, en consommant peu d’échantillon, et avec une grande sensitivité.