Développement d'un immunocapteur impédimétrique pour la détection et la quantification d'une sous-population cellulaire : application au diagnostic précoce des infections

La technologie des biocapteurs et des dispositifs de type laboratoire sur puce est devenue un outil essentiel pour le diagnostic rapide et le suivi de traitement de maladies. Le travail de ce mémoire s'intègre dans un projet de recherche visant à la conception d'un laboratoire sur puce pour l'isolement, le tri et la quantification de sous populations cellulaires pour le diagnostic précoce et en temps réel des maladies infectieuses. L'objectif du travail de thèse est de développer un immunocapteur impédimétrique pour la détection des monocytes inflammatoires dont l'augmentation témoigne d'un processus infectieux. La fonctionnalisation permettant le piégeage des cellules via une liaison antigène/anticorps a dans un premier temps été optimisée sur des électrodes d'or fabriquées à partir des techniques issues des microtechnologies. Le greffage des anticorps repose sur un système multicouche comprenant des monocouches auto-assemblées et des protéines d'adhésion, chaque étape du protocole ayant été caractérisée par des méthodes électrochimiques et optiques pour différentes tailles d'électrodes. Les performances du biocapteur ont ensuite été étudiées par spectroscopie d'impédance électrochimique et ont mis en évidence une détection sélective suivant l'anticorps immobilisé et quantitative des monocytes piégés. Afin d'augmenter la sensibilité de détection, des systèmes de microélectrodes ont alors été développés. Après optimisation du procédé de fabrication et de modification des électrodes, la détection des monocytes a été validée par spectroscopie d'impédance. L'intégration des microélectrodes dans un dispositif microfluidique est en cours de validation.Biosensors and lab-on-chips are becoming important tools for diseases diagnosis and treatment monitoring. This work is a part of a research project which aims to design an integrated device for isolation, sorting and counting of cells subpopulations for early and real time diagnosis of infectious diseases. It consists in developing an impedimetric immunosensor to detect inflammatory monocytes found in larger number in the blood of patients with inflammation and infectious diseases. The functionalization for cells trapping via an immunochemical interaction has firstly been optimized on gold electrodes fabricated thanks to microtechnologies. The antibodies grafting lies on a multilayer system consisting of stacking self assembled monolayers and cells adhesion proteins and each assembly step have been characterized by optical and electrochemical techniques for different electrodes sizes. Then, the biosensor performances have been investigated by electrochemical impedance spectroscopy and we successfully demonstrated a selective and quantitative detection of trapped monocytes according to the immobilized antibody. Miniaturization enhancing the detection sensitivity, microelectrodes have been developed. After optimization of the fabrication process and functionalization of the microelectrodes, detection of monocytes has been validated by impedance. The integration of these microsystems in a microfluidic device is actually under development

Electrochemical behavior of indolone-N-oxides: Relationship to structure and antiplasmodial activity

Indolone-N-oxides exert high parasiticidal activity at the nanomolar level in vitro against Plasmodiumfalciparum, the parasite responsible for malaria. The bioreductive character of these molecules was investigated using cyclic voltammetry and EPR spectroelectrochemistry to examine the relationship between electrochemical behavior and antimalarial activity and to understand theirmechanisms of action. For all the compounds (37 compounds) studied, the voltammograms recorded in acetonitrile showed a well-defined and reversible redox couple followed by a second complicated electron transfer. The first reduction (−0.88 VbE1/2b−0.50 V vs. SCE) was attributed to the reduction of the N-oxide function to form a radical nitroxide anion. The second reduction (−1.65 VbE1/2b−1.14 V vs. SCE) was assigned to the reduction of the ketone function. By coupling electrochemistry with EPR spectroscopy, the EPR spectra confirmed the formation of the nitroxide anion radical.Moreover, the experiments demonstrated that a slowprotonation occurs at the carbon of the nitrone function and not at the NO function. A relationship between electrochemical behavior and indolone-N-oxide structure can be established for compounds with R1=―OCH3, R2=H, and electron-withdrawing substituents on the phenyl group at R3. The results help in the design of new molecules with more potent in vivo antimalarial activity

Développement d'un immunocapteur impédimétrique pour la détection et la quantification d'une sous-population cellulaire (application au diagnostic précoce des infections)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Système microfluidique et procédé pour isoler et quantifier au moins une sous-population de cellules à partir d'une population de cellules

The invention concerns a microfluidic System (1) capable of receiving cell populations and capable of simultaneously isolating and quantifying, for each of the populations, at least one sub-population of cells {ÇA), and a method of simultaneous isolation and quantification using this System. The System comprises a substrate having networks of microchannels (10) comprising a first sorting unit capable of isolating, by magnetic attraction, cells of interest (C2) in the population in at least one first sorting micro-channel. This network comprises a second unit for simultaneous sorting and counting (U2) comprising at least one second sorting microchannel (10) defined by a closed wall (S) having an inner face (9) provided with at least one functionalised électrode (El,E2) capable of trapping a sub-population, the second unit comprising means (El,E2) for counting the sub-population by means of impedance spectroscopy. According to the invention, the second sorting microchannel is provided with at least one pair of opposing functionalised électrodes and at least one pair of second micro-coils (11and12) for trapping the cells of interest arranged in the wall facing the électrodes and controlling successive attraction/release cycles that alternate between the micro-coils.L'invention concerne un système microfiuidique (1) apte à recevoir des populations de cellules et apte à isoler et à quantifier simultanément pour chacune des populations au moins une sous-population de cellules {ÇA), et un procédé d'isolement et de quantification simultanés utilisant ce système. Le système comporte un substrat à réseau de microcanaux (10) comprenant, une première unité de tri apte à isoler par attraction magnétique des cellules d'intérêt (C2) de la population dans au moins un microcanal de premier tri. Ce réseau comprend une seconde unité de tri et de comptage simultanés (U2) comportant au moins un microcanal de second tri (10) défini par une paroi fermée (S) à face interne (9) pour vue d'au moins une électrode fonctionnalisée (El,E2) apte à piéger une sous-population, la seconde unité comportant des moyens de comptage (El,E2) par spectroscopie d'impédance de la sous-population. Selon l'invention, te micfocanal de second tri est' pourvu d'au moins une paire d'électrodes fonctionnalisées opposées et d'au moins une paire de secondes micro-bobines de piégeage (11et12) des cellules d'intérêt agencées dans la paroi en regard des électrodes et commandant des cycles d'attraction/libération successifs et alternés entre lesmicro-bobines

AN INTEGRATED MAGNETIC PLANAR ACTUATOR REDEFINING MULTILEVEL (3D) MICROFLUIDIC STRATEGIES

Accepté 10th International Conference on the Scientific and Clinical Applications of Magnetic Carriers, 10-14 June 2014 Dresden, GermanyInternational audienceSuper-paramagnetic micro particles (SMPs) are broadly used in medical and biological applications (from cellular to genomic scales), but the handling tools for those particles remain mostly non-integrated. Since a decade, a few solutions for integrated actuation have been proposed and paved the way to the micro-scale control of SMPs. So far, spiral planar coils showed the best compromises between the available magnetic force and the actuator footprint. Albeit the technology to integrate this type of coils with microfluidic structures exists, it is hard to find examples of dual architectures using those two aspects for concrete applications. We therefore developed a SU-8 dry film lamination technique, which can be used with standard micro technology processes. It allows us to manufacture lab on chip integrating miniaturized electromagnets. Using that system, we achieved to separate a solution of SMPs with an 80% efficiency at a 2.5 µL/min flow rate (4.6 mm/s). It is also possible to perform trapping (all the beads stay on the coils) at lower flow rate

Impedimetric immunosensor for the detection of circulating pro-inflammatory monocytes as infection markers

International audienceCirculating blood monocytes belong to the first line of defense against pathogens and inflammation. Monocytes can be divided into three populations defined by the expression of the cell surface molecules, CD 14 and CD 16. The CD 14(++) CD 16(-) cells, called "classical" monocytes, represent 85% to 95% of the total monocytes in a healthy person whereas CD 14(-) CD 16(+), called "proinflammatory" monocytes, are found in greater numbers in the blood of patients with acute inflammation and infectious diseases. This increase in the concentration of proinflammatory monocytes can be a good indicator of an infectious state. This study presents an immunosensor based on impedance detection for specific cell trapping of classical and proinflammatory monocytes. The grafting of specific antibodies (CD 14 or CD 16) was based on the use of mixed SAM associated with protein G. Each step of the functionalization was characterized by electrochemical methods, quartz crystal microbalance and atomic force microscopy. Faradaic electrochemical impedance spectroscopy and voltametric analysis confirmed the success of the modification process with a surface coverage reaching 92% for the antibody layer. The increase in the deposited mass at each step of the modification process confirmed this results revealing that one protein G in two was bound to an antibody. The cell trapping capacity, evaluated by the variation in the film resistance using non-faradaic impedance spectroscopy revealed that the cell trapping is selective, depending on the specific antibody grafted and quantitative with the range of detection being 1000 to 30,000 infected cells. This range of detection is consistent with the application targeted

A microfluidic device for the quantization of subpopulations of cells

International audienc

Multilevel (3D) microfluidic technology for an innovative magnetic cell separation and couting platform

International audienceCurrently, the technique for the quantitative detection of cells is flow cytometry. This technique has the advantage of being sensitive and reliable but is expensive, time consuming and not suited to both routine screening and point‐of‐care diagnostics. Miniaturized cell separation devices offer many advantages such as the use of small volumes, portability and low cost.We propose a new concept of device which, by combining 3D fluid engineering and localized magnetic actuation, enables the full integration of cell tagging, magnetic separation and cell counting in a single device. The labs on chip are manufactured by laminating commercially available photosensitive dry film that fits microfluidic requirements and gives the possibility to build easily 3D microfluidic systems.We show we can tag efficiently THP1 monocytes and subsequently sort them through magnetic trapping on integrated micro-coils. The separation efficiency is studied at different flow rates. Cell counting capacity, evaluated by using non‐ faradic impedance spectroscopy revealed that the cell trapping is selective, depending on the specific antibody grafted and quantitative with the range of detection being 1000 to 30000 infected cells. This range of detection is consistent with the targeted application

Improved on-chip impedimetric immuno-detection of subpopulations of cells toward single-cell resolution

International audienceImpedance spectroscopy has gained interest for the quantitative detection of specific cells mainly due to a label-free detection and their miniaturization capability required for integration on chip and development of point-of-care diagnostics. In this paper, we report the study of impedimetric microfluidic devices with improved sensitivity targeting the immuno-detection of cells. The sensitivity of our system was evaluated in terms of the capacity of the electrodes to trap monocytes by immune-reaction with CD14 antibody immobilized on micro-electrode surface. All measurements were performed in faradic mode using a redox probe. The sensitivity was evaluated as a function of the impedance increase recorded at 100 Hz caused by the insulating character of the cell trapped on electrodes. Analyses first confirmed that the sensing performances were significantly improved by using microfluidic. This increase could originate from an increase in the probability of cell trapping and a better organization of cells on the electrode due to the laminar flow. The great sensitivity was recorded with interdigitated electrodes for which the influence of the gap value was evaluated. The maximum sensitivity was reached with the smallest inter-electrodes gap tested (50 µm). This performance was in part attributed to the redox cycling taking place between neighboring fingers that was strongly affected when cells were trapped on the electrodes edges. Furthermore we also demonstrate that the slice of cell concentration for which the sensitivity is maximized is correlated to the area of electrodes. Moreover, the smallest area of interdigitated electrode (0.1 mm length) allowed the detection of as low as 5 cells per m