Combining Microfluidics, Optogenetics and Calcium Imaging to Study Neuronal Communication In Vitro

International audienceIn this paper we report the combination of microfluidics, optogenetics and calcium imaging as a cheap and convenient platform to study synaptic communication between neuronal populations in vitro. We first show that Calcium Orange indicator is compatible in vitro with a commonly used Channelrhodopsine-2 (ChR2) variant, as standard calcium imaging conditions did not alter significantly the activity of transduced cultures of rodent primary neurons. A fast, robust and scalable process for micro-chip fabrication was developed in parallel to build micro-compartmented cultures. Coupling optical fibers to each micro-compartment allowed for the independent control of ChR2 activation in the different populations without crosstalk. By analyzing the post-stimuli activity across the different populations, we finally show how this platform can be used to evaluate quantitatively the effective connectivity between connected neuronal populations

Respiratory Effects of Sarafotoxins from the Venom of Different Atractaspis Genus Snake Species

Sarafotoxins (SRTX) are endothelin-like peptides extracted from the venom of snakes belonging to the Atractaspididae family. A recent in vivo study on anesthetized and ventilated animals showed that sarafotoxin-b (SRTX-b), extracted from the venom of Atractaspis engaddensis, decreases cardiac output by inducing left ventricular dysfunction while sarafotoxin-m (SRTX-m), extracted from the venom of Atractaspis microlepidota microlepidota, induces right ventricular dysfunction with increased airway pressure. The aim of the present experimental study was to compare the respiratory effects of SRTX-m and SRTX-b. Male Wistar rats were anesthetized, tracheotomized and mechanically ventilated. They received either a 1 LD50 IV bolus of SRTX-b (n = 5) or 1 LD50 of SRTX-m (n = 5). The low-frequency forced oscillation technique was used to measure respiratory impedance. Airway resistance (Raw), parenchymal damping (G) and elastance (H) were determined from impedance data, before and 5 min after SRTX injection. SRTX-m and SRTX-b injections induced acute hypoxia and metabolic acidosis with an increased anion gap. Both toxins markedly increased Raw, G and H, but with a much greater effect of SRTX-b on H, which may have been due to pulmonary edema in addition to bronchoconstriction. Therefore, despite their structural analogy, these two toxins exert different effects on respiratory function. These results emphasize the role of the C-terminal extension in the in vivo effect of these toxins

Peut-on identifier précocement les patients à risque de ventilation mécanique prolongée en réanimation polyvalente ?

Introduction : Les patients hospitalisés en réanimation sont fréquemment ventilés. En cas de ventilation mécanique prolongée (VMP), la trachéotomie est envisagée pour améliorer le confort et le sevrage respiratoire. Repérer les patients à risque de VMP permettrait d anticiper les indications de trachéotomie. L objectif principal de cette étude est de déterminer s il existe des facteurs de risque de VMP en réanimation polyvalente. L objectif secondaire est de construire un score permettant d établir ce risque de VMP. Matériel et méthodes : Nous avons mené une étude descriptive, prospective dans un service universitaire de réanimation polyvalente de seize lits. Tous les patients admis intubés et ventilés plus de 24h ont été inclus. La définition de VMP a été fixée à plus de 10 jours. Les données démographiques, les antécédents médicaux, le motif d admission et d intubation et le score de gravité IGS2 ont été relevés. Nous avons recueilli les critères cliniques et biologiques au 1er et au 5ème jour. Une analyse univariée puis multivariée a été utilisée pour l étude statistique. Un score prédictif de VMP a été établi en utilisant les odds ratio de l analyse multivariée. Une courbe ROC a permis de determiner le meilleur seuil graphiquement. Résultats : Entre Novembre 2010 et Octobre 2011,165 patients ont été inclus. L âge moyen était de 58+-19 ans, l IGS2 était à 49+-17, les motifs d admission étaient médicaux pour 52,7% et chirurgicaux pour 47,3% des patients. 93 patients (56,4%) ont reçu une VM de 10 jours ou moins, 72 patients (43,6%) étaient ventilés plus de 10 jours. L analyse multivariée à l admission a identifié 5 facteurs de risque indépendants de VMP: une pathologie restrictive (Odds-ratio(OR)=2,76 ; Intervalle de confiance à 95% (IC95)[1,11-6,91]; p=0,03), un alcoolisme non sevré (OR=2,29; IC95[1,07-5,38]; p=0,03), une admission pour polytraumatisme (OR=4,82; IC95[1,64-14,13]; p=0,004), une épuration extra-rénale en urgence (OR=2,57; IC95[1,12-5,93]; p=0,03) et un rapport PaO2/FiO2 < 200 (OR=2,21; IC95[1,11-4,44]; p=0,02). A J5, l analyse multivariée a identifié 2 autres facteurs: la persistance d une sédation intraveineuse (OR=2,77; IC95 [1,21-6,36]; p=0,02) et un nombre de défaillance d organe supérieur à 3 (OR=2,48; IC95[1,13-5,47]; p=0,02). Un score de risque supérieur à 3 prédit une VMP avec une sensibilité de 75%; IC95[63-84], spécificité de 76%; IC95[66-84], valeurs prédictives positive de 71%; IC95[60-81] et négative de 80%; IC95[70-87]. Les rapports de vraisemblance positif et négatif sont respectivement de 3,17; IC95[2,7-3,8] et 0,33; IC95[0,2-0,6]. L aire sous la courbe ROC est à 0,812. Conclusion : Nous identifions 7 facteurs de risque indépendants de VMP. Ces éléments nouveaux pourraient nous permettre de repérer précocement ces patients grâce au score de risque élaboré. Celui-ci doit être validé dans une deuxième cohorte propectiveIntroduction : Patients admitted to the ICU usually receive mechanical ventilation. If prolonged mechanical ventilation (PMV) is expected, tracheostomy is generally proposed in order to improve respiratory weaning. Early recognition of these patients could help physicians to anticipate indications of tracheostomy. The objective of this study is to determine risk factors of PMV and build a predicitive risk score of PMV. Methods : After approval by the local ethics committee, a prospective observational study was performed in the general 16 beds medico-surgical ICU of an university hospital. All patients admitted to the ICU who underwent mechanical ventilation more than 24h were included. Patients who needed more than one reintubation due to a long lasting ventilatory weaning were excluded. VMP was an intubation requirement for more than 10 days. Demographic data, comorbidities, reason for admission and intubation, SAPS2 score were recorded. Clinical and biological data were also collected on admission and on day 5. Univariate analysis was performed to determine variables associated with PMV. They were entered in a logistic regression to find independent risk factors of PMV. A score was created using multivariate analysis odds ratio. A ROC curve was plotted to determine the best threshold for predictive risk of PMV. Results : Between November 2010 and October 2011, 446 patients were admitted to this ICU and 165 were included. Mean age was 58+-19 years, mean SAPS2 was 49+-17, reason for admission was medical : 52.7% and surgical : 47.3%. Ninety three patients (56.4%) underwent mechanical ventilation for 10 days or less, whereas 72 patients (43.6%) were ventilated more than 10 days. Logistic regression on admission revealed 5 independent risk factors of PMV: restrictive pulmonary disease (Odds-ratio(OR)=2.76; 95% confidence interval (95CI) [1.11-6.91]; p=0.03), chronic alcoholism (OR=2.29; 95CI [1.07-5.38]; p=0.03), admission for polytrauma (OR=4.82; 95CI [1.64-14.13]; p=0.004), renal replacement therapy (OR=2.57; 95CI [1.12-5.93]; p=0.03), PaO2/FiO2<200 (OR=2.21; 95CI [1.11-4.44]; p=0.02) and 2 independent factors on day 5: ongoing sedation (OR=2.48; 95CI [1.13-5.47]; p=0.024) and multiple organ failure (OR=2.77; 95CI [1.20-6.36]; p=0.016). The score was then constructed from 0 to 20. A risk score higher than 3 on day 5 predicts PMV with a sensitivity of 75%; 95CI [63-84], specificity of 76%; 95CI [66-84], positive predictive value of 71%; 95CI [60-81] and negative predictive value of 80%; 95CI [70-87]. Positive and negative likehood ratios are respectively 3.17; 95CI [2.7-3.8] and 0.33; 95CI [0.2-0.6]. Area under the ROC curve is 0.812; 95CI [0.74-0.87]; p<0.0001. Conclusions : Almost half of the admitted patients received PMV. Seven independent risk factors of PMV were found. The easy score could help physicians to early identify patients at risk of PMV. However, this score has to be validated in a large prospective cohort.AMIENS-BU Santé (800212102) / SudocSudocFranceF

Magnetic fluidized bed for solid phase extraction in microfluidic systems

International audienceFluidization, a process in which a granular solid phase behaves like a fluid under the influence of an imposed upward fluid flow, is routinely used in many chemical and biological engineering applications. It brings, to applications involving fluid–solid exchanges, advantages such as high surface to volume ratio, constant mixing, low flow resistance, continuous operation and high heat transfer. We present here the physics of a new miniaturized, microfluidic fluidized bed, in which gravity is replaced by a magnetic field created by an external permanent magnet, and the solid phase is composed of magnetic microbeads with diameters ranging from 1 to 5 μm. These beads can be functionalized with different ligands, catalysts or enzymes, in order to use the fluidized bed as a continuous purification column or bioreactor. It allows flow-through operations at flow rates ranging from 100 nL min −1 up to 5 μL min −1 at low driving pressures (<100 mbar) with intimate liquid/solid contact and a continuous recirculation of beads for enhanced target capture efficiencies. The physics of the system presents significant differences as compared to conventional fluidized beds, which are studied here. The effects of magnetic field profile, flow chamber shape and magnetic bead dipolar interactions on flow regimes are investigated, and the different regimes of operation are described. Qualitative rules to obtain optimal operation are deduced. Finally, an exemplary use as a platform for immunocapture is provided, presenting a limit of detection of 0.2 ng mL −1 for 200 μL volume samples

On-chip conductometric detection of short DNA sequences via electro-hydrodynamic aggregation

International audienceFluorescence measurement is the main technology for post-amplification DNA detection in automated systems. Direct electrical reading of DNA concentration in solution could be an interesting alternative to go toward more miniaturized or less expensive devices, in particular in the pathogen detection field. Here we present the detection of short bacterial biomarkers with a direct impedancemetric measurement, within solutions of amplified and elongated DNA sequences in a microchannel. This technology relies on the electrohydrodynamic instability occurring in solutions of long charged macromolecules in a strong electric field. This instability specifically induces the aggregation of long DNAs and triggers conductivity variations that can be monitored by on-contact conductometry. An innovative isothermal amplification and elongation strategy was developed, combining SDA and HRCA reactions, in order to yield long DNAs suitable to be detected by the above principle, from a dilute initial DNA target. In contrast with previous label-free detection methods, this new strategy is very robust to matrix effects, thanks to the unique molecular weight dependence of the instability, coupled with this specific DNA amplification strategy. We demonstrate the detection of a 1 pM gene sequence specific to Staphylococcus aureus, in a portable system

Modular microfluidic system for on-chip extraction, preconcentration and detection of the cytokine biomarker IL-6 in biofluid

International audienceAbstract The cytokine interleukin 6 (IL-6) is involved in the pathogenesis of different inflammatory diseases, including cancer, and its monitoring could help diagnosis, prognosis of relapse-free survival and recurrence. Here, we report an innovative microfluidic approach that uses the fluidization of magnetic beads to specifically extract, preconcentrate and fluorescently detect IL-6 directly on-chip. We assess how the physical properties of the beads can be tuned to improve assay performance by enhancing mass transport, reduce non-specific binding and multiply the detection signal threefold by transitioning between packed and fluidization states. With the integration of a full ELISA protocol in a single microfluidic chamber, we show a twofold reduction in LOD compared to conventional methods along with a large dynamic range (10 pg/mL to 2 ng/mL). We additionally demonstrate its application to IL-6 detection in undiluted serum samples

Microfluidic: an innovative tool for efficient cell sorting.

International audienceAt first mostly dedicated to molecular analysis, microfluidic systems are rapidly expanding their range of applications towards cell biology, thanks to their ability to control the mechanical, biological and fluidic environment at the scale of the cells. A number of new concepts based on microfluidics were indeed proposed in the last ten years for cell sorting. For many of these concepts, progress remains to be done regarding automation, standardization, or throughput, but it is now clear that microfluidics will have a major contribution to the field, from fundamental research to point-of-care diagnosis. We present here an overview of cells sorting in microfluidics, with an emphasis on circulating tumor cells. Sorting principles are classified in two main categories, methods based on physical properties of the cells, such as size, deformability, electric or optical properties, and methods based on biomolecular properties, notably specific surface antigens. We document potential applications, discuss the main advantages and limitations of different approaches, and tentatively outline the main remaining challenges in this fast evolving field

High-throughput extraction on a dynamic solid phase for low-abundance biomarker isolation from biological samples

Abstract Liquid biopsy, in particular circulating tumor DNA (ctDNA) analysis, has paved the way for a new noninvasive approach to cancer diagnosis, treatment selection and follow-up. As a crucial step in the analysis, the extraction of the genetic material from a complex matrix needs to meet specific requirements such as high specificity and low loss of target. Here, we developed a new generation of microfluidic fluidized beds (FBs) that enable the efficient extraction and preconcentration of specific ctDNA sequences from human serum with flow rates up to 15 µL/min. We first demonstrated that implementation of a vibration system inducing flow rate fluctuations combined with a mixture of different bead sizes significantly enhanced bead homogeneity, thereby increasing capture efficiency. Taking advantage of this new generation of high-throughput magnetic FBs, we then developed a new method to selectively capture a double-stranded (dsDNA) BRAF mutated DNA sequence in complex matrices such as patient serum. Finally, as proof of concept, ligation chain reaction (LCR) assays were performed to specifically amplify a mutated BRAF sequence, allowing the detection of concentrations as low as 6 × 104 copies/µL of the mutated DNA sequence in serum

Droplet Microfluidic and Magnetic Particles Platform for Cancer Typing

International audienceAnalyses 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

Chromatin immunoprecipitation in microfluidic droplets: towards fast and cheap analyses

International audienceGenetic 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