Dielectrophoretic separation of blood pathogens ou Bacterial extraction from biological samples using DEP forces

International audienceWe report here a method based on DEP separation to concentrate pathogens out of a biological sample by combining positive and negative DEP to separate pathogens from the sample matrix. In this approach, we take advantage from the large tolerance of micro-organisms towards osmotic shock to perform dielectrophoretic separation in a low electric conductivity medium. This condition enables to collect micro-organisms by positive DEP, while lysed blood cells are repelled from the electrodes by negative DEP. Our microfluidic device is designed based on numerical simulation. This approach is validated on a large range of microorganisms, since it has been tested with different species of bacteria (S. epidermidis (Gram +) and E. coli (Gram -)) and yeasts (C. albicans)

Micro-organism extraction from biological samples using DEP forces enhanced by osmotic shock

1473-0197International audienceOn the road towards efficient diagnostics of infectious diseases, sample preparation is considered as the key step and remains a real technical challenge. Finding new methods for extraction of micro-organisms from a complex biological sample remains a major challenge prior to pathogen detection and analysis. This paper reports a new technique for capturing and isolating micro-organisms from a complex sample. To achieve the segregation of pathogens and blood cells, dielectrophoretic forces applied to bioparticles previously subjected to an osmotic shock are successfully implemented within a dedicated microfluidic device. Our device involves an electrode array of interdigitated electrodes, coated with an insulating layer, to minimize electrochemical reactions with the electrolyte and to enable long-time use. The electric field intensity inside the device is optimized, considering the insulating layer, for a given frequency bandwidth, enabling the separation of bioparticles by dielectrophoretic forces. Our predictions are based on analytical models, consistent with numerical simulations (using COMSOL Multiphysics) and correlated to experimental results. The method and device have been shown to extract different types of micro-organisms spiked in a blood cell sample. We strongly believe that this new separation approach may open the way towards a simple device for pathogen extraction from blood and more generally complex samples, with potential advantages of genericness and simplicity

Interaction champ électrique cellule (conception de puces microfluidiques pour l'appariement cellulaire et la fusion par champ électrique pulsé)

La fusion cellulaire est une méthode de génération de cellules hybrides combinant les propriétés spécifiques des cellules mères. Initialement développée pour la production d anticorps, elle est maintenant aussi investiguée pour l immunothérapie du cancer. L électrofusion consiste à produire ces hybrides en utilisant un champ électrique pulsé. Cette technique présente de meilleurs rendements que les fusions chimiques ou virales, sans introduire de contaminant. L électrofusion est actuellement investiguée en cuve d électroporation où le champ électrique n est pas contrôlable avec précision et le placement cellulaire impossible, produisant de faibles rendements binucléaires. Afin d augmenter le rendement et la qualité de fusion, la capture et l appariement des cellules s avèrent alors nécessaires.Notre objectif a été de développer et de réaliser des biopuces intégrant des microélectrodes et des canaux microfluidiques afin de positionner et d apparier les cellules avant leur électrofusion. Une première structure de piégeage se basant sur des plots isolants et l utilisation de la diélectrophorèse a été réalisée. Afin d effectuer des expérimentations sous flux, une méthode de scellement des canaux, biocompatible et étanche a été développée. Puis, le milieu d expérimentation a été adapté pour l électrofusion. En confrontant les résultats des expériences biologiques aux simulations numériques, nous avons pu démontrer que l application d impulsions électriques induisait la diminution de la conductivité cytoplasmique. Nous avons ensuite validé la structure par l électrofusion de cellules. Un rendement de 55% avec une durée de fusion membranaire de 6 s a été obtenu. Dans un second temps, nous avons proposé deux microstructures de piégeage pour l électrofusion haute densité. La première se base sur un piégeage fluidique, alors que la seconde, utilise ladiélectrophorèse sans adressage électrique à l aide de plots conducteurs. Jusqu à 75% des cellules fusionnent dans cette dernière structure. Plus de 97% des hybridomes produits sont binucléaires. Le piégeage étant réversible, les hybridomes peuvent ensuite être collectés pour des études ultérieures.Cell fusion is a method to generate a hybrid cell combing the specific properties of its progenitor cells. Initially developed for antibody production, it is now also investigated for cancer immunotherapy. Electrofusion consists on the production of hybridoma using electric pulses. Compared to viral or chemical methods, electrofusion shows higher yields and this system is contaminant free. Actually, electrofusion is investigated in electroporation cuvettes, where the electric field is not precisely controllable and cell placement impossible, resulting in low binuclear hibridoma yields. To improve the fusion quality and yield, cell capture and pairing are necessary.Our objective was the development and realization of biochips involving microelectrodes and microfluidic channels to place and pair cells prior to electrofusion. A first trapping structure based on insulators and the use of dielectrophoresis has been achieved. In order to perform fluidic experiments, a biocompatible irreversible packaging was developed. Then, the experimental medium was optimized for electrofusion. Confronting the biological experiments and the numerical simulations, we showed that the application of electric pulses leads to a decrease of the cytoplasmic conductivity. The microstructure was validated by cell electrofusion. A yield of 55%, with a membrane fusion duration of 6 s has been achieved. Secondly, we proposed two trapping microstructures for high density electrofusions. The first one is based on a fluidic trapping while the second one uses dielectrophoresis, free of electric wiring, thanks to conductive pads. Up to 75% of paired cells were successfully electrofused with the conductive pads. More than 97% of the hybridoma were binuclear. The trapping being reversible, the hybridoma can be collected for further analysis.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Vacuum Casting to Manufacture a Plastic Biochip for Highly Parallel Cell Transfection

International audienceA novel polymer microarray fabrication technique is presented and applied to the realization of a biochip for highly parallelized cell transfection. The proposed microfabrication technique is derived from a macroscale rapid prototyping technique called vacuum casting. It was optimized to reduce production cost, in order to produce small series (100-10 000 chip series) of chips to meet demand in today's market of cellulomics. Microfabrication technologies and rapid prototyping technologies are combined to shape the master part, which can thus involve microsized features. The corresponding female structure is moulded in a flexible silicone material. The duplicated polymer chips are obtained by casting a thermosetting plastic under vacuum. The dimensional replication accuracy between the master part and the duplicated parts is uniform over the duplicated parts and better than 1%. Advantages of the proposed technique over existing plastic microfabrication techniques are discussed in the paper. Using this microfabrication technique, we produced a plastic biochip for highly parallelized transfection of arrays of living cells. The feasibility of parallel lipofection was demonstrated: two different plasmids encoding, respectively, eGFP and DsRED2 were inserted into HEK293T cells. The transfection was monitored through fluorescence observation after 72 h showing successful expression of both genes

Electrical Detection of the Mechanical Alteration of Sickling Red Blood Cells within a Microfluidic Capillary Network

International audienceIn this paper we demonstrate the capability to detect red blood cells mechanical disorders, in particular the sickle cell disease, using the electrical signature of the cell transit within a microfluidic restriction mimicking the blood capillaries

Activity Monitoring of Functional OprM Using a Biomimetic Microfluidic Device

International audienceThis paper describes the fabrication and use of a biomimetic microfluidic device for the monitoring of a functional porin reconstituted within miniaturized suspended artificial bilayer lipid membrane (BLM). Such a microfluidic device allows for 1) fluidic and electrical access to both sides of the BLM, 2) reproducible membrane protein insertion and long-term electrical monitoring of its conductance (Gi), thanks to the miniaturization of the BLM. We demonstrate here for the first time the feasibility to insert a large trans-membrane protein through its β-barrel, and monitor its functional activity during more than 1 hour (limited by buffer evaporation). In this paper, we specifically used our device for the monitoring of OprM, a bacterial efflux channel involved in the multidrug resistance of the bacteria Pseudomonas aeruginosa. Sub-steps of the OprM channel conductance were detected during the electrical recordings within our device, which might be due to oscillations between several structural conformations (sub-states) adopted by the protein, as part of its opening mechanism. This work is a first step towards the establishing of a genuine platform dedicated to the investigation of bacterial proteins under reconstituted conditions, a very promising tool for the screening of new inhibitors against bacterial channels involved in drug resistance

Sensing of Oxygen Concentration in a Microfluidic Device mimicking Liver 3D Microarchitecture

International audienceWe designed a microfluidic structure which closely reproduces liver microarchitecture, constraining primary rat hepatocytes at a high density and in three dimensions (3D), and in which a gradient of oxygen can be generated. The device includes an oxygen sensitive membrane that could map the oxygen consumption of hepatocytes. INTRODUCTION Compared to classical two-dimensional cell culture, microfluidic devices or/and 3D culture conditions were evidenced to increase the period of time during which primary hepatocytes retain their functions [1]. Moreover, microfluidic techniques offer the opportunity to mimic the in vivo hepatocyte zonation, by subjecting hepatocytes to oxygen gradients [1-2]. Such oxygen gradients that can be estimated by numerical simulations, were recently experimentally assessed using an oxygen sensitive fluorescent membrane [3]. We proposed to include the oxygen sensitive membrane within a miniaturized fluidic device mimicking several hepatic cords in series, and inducing a gradient of oxygen on those. Moreover each of those hepatic cord units was inducing 3D organization of hepatocytes, due to the 72 µm height of culture chambers in which they can aggregate

Electric pulses: a flexible tool to manipulate cytosolic calcium concentrations and generate spontaneous-like calcium oscillations in mesenchymal stem cells

Human adipose mesenchymal stem cells (haMSCs) are multipotent adult stem cells of great interest in regenerative medicine or oncology. They present spontaneous calcium oscillations related to cell cycle progression or differentiation but the correlation between these events is still unclear. Indeed, it is difficult to mimic haMSCs spontaneous calcium oscillations with chemical means. Pulsed electric fields (PEFs) can permeabilise plasma and/or organelles membranes depending on the applied pulses and therefore generate cytosolic calcium peaks by recruiting calcium from the external medium or from internal stores. We show that it is possible to mimic haMSCs spontaneous calcium oscillations (same amplitude, duration and shape) using 100 μs PEFs or 10 ns PEFs. We propose a model that explains the experimental situations reported. PEFs can therefore be a flexible tool to manipulate cytosolic calcium concentrations. This tool, that can be switched on and off instantaneously, contrary to chemicals agents, can be very useful to investigate the role of calcium oscillations in cell physiology and/or to manipulate cell fate

A microfluidic biochip for the nanoporation of living cells

International audienceThis paper deals with the development of a microfluidic biochip for the exposure of living cells to nanosecond pulsed electric fields (nsPEF). When exposed to ultra short electric pulses (typical duration of 3-10ns), disturbances on the plasma membrane and on the intra cellular components occur, modifying the behavioral response of cells exposed to drugs or transgene vectors. This phenomenon permits to envision promising therapies. The presented biochip is composed of thick gold electrodes that are designed to deliver a maximum of energy to the biological medium containing cells. The temporal and spectral distributions of the nsPEF are considered for the design of the chip. In order to validate the fabricated biochip ability to orient the pulse towards the cells flowing within the exposition channels, a frequency analysis is provided. High voltage measurements in the time domain are performed to characterize the amplitude and the shape of the nsPEF within the exposition channels and compared to numerical simulations achieved with a 3D Finite-Difference Time-Domain code. We demonstrate that the biochip is adapted for 3 ns and 10 ns pulses and that the nsPEF are homogenously applied to the biological cells regardless their position along the microfluidic channel. Furthermore, biological tests performed on the developed microfluidic biochip permit to prove its capability to permeabilize living cells with nanopulses. To the best of our knowledge, we report here the first successful use of a microfluidic device optimized for the achievement and real time observation of the nanoporation of living cells