A critical early step in drug discovery is the screening of a chemical library. Typically, promising compounds are identified in a primary screen and then more fully characterized in a dose–response analysis with 7–10 data points per compound. Here, we describe a robust microfluidic approach that increases the number of data points to approximately 10,000 per compound. The system exploits Taylor–Aris dispersion to create concentration gradients, which are then segmented into picoliter microreactors by droplet-based microfluidics. The large number of data points results in IC50 values that are highly precise (± 2.40% at 95% confidence) and highly reproducible (CV = 2.45%, n = 16). In addition, the high resolution of the data reveals complex dose–response relationships unambiguously. We used this system to screen a chemical library of 704 compounds against protein tyrosine phosphatase 1B, a diabetes, obesity, and cancer target. We identified a number of novel inhibitors, the most potent being sodium cefsulodine, which has an IC50 of 27 ± 0.83 μM.

The response of cells to forces is essential for tissue morphogenesis and homeostasis. This response has been extensively investigated in interphase cells, but it remains unclear how forces affect dividing cells. We used a combination of micro-manipulation tools on human dividing cells to address the role of physical parameters of the micro-environment in controlling the cell division axis, a key element of tissue morphogenesis. We found that forces applied on the cell body direct spindle orientation during mitosis. We further show that external constraints induce a polarization of dynamic subcortical actin structures that correlate with spindle movements. We propose that cells divide according to cues provided by their mechanical micro-environment, aligning daughter cells with the external force field.

This chapter describes a method to study cells migrating in micro-channels, a confining environment of well-defined geometry. This assay is a complement to more complex 3D migration systems and provides several advantages even if it does not recapitulate the full complexity of 3D migration. Important parameters such as degree of adhesion, degree of confinement, mechanical properties, and geometry can be varied independently of each other. The device is fully compatible with almost any type of light microscopy and the simple geometry makes automated analysis very easy to perform, which allows screening strategy. The chapters is divided into five parts describing the design of different types of migration chambers, the fabrication of a mold by photolithography, the assembly of the chamber, the loading of cells, and finally the imaging on live or fixed cells.

In the present work, we have measured the messenger RNA expression of specific genes both from total RNA and cells encapsulated in droplets. The microfluidic chip introduced includes the following functionalities: RNA/cell encapsulation, lysis, reverse transcription and real-time polymerase chain reaction. We have shown that simplex and duplex gene expression measurements can be carried out over a population of 100 purified RNA samples encapsulated simultaneously in 2 nl droplets in less than 2 h. An analysis of 100 samples containing one to three cells has shown excellent consistency with standard techniques regarding average values. The cell-to-cell distributions of the E-cadherin expression suggest fluctuations on the order of 80% in the number of transcripts, which is highly consistent with the general findings from the literature. A mathematical model has also been introduced to strengthen the interpretation of our results. The present work paves the way for the systematic acquisition of such information in biological and biomedical studies.

PURPOSE:
The ability of remotely tagging tissues in a controlled and three-dimensional manner during preoperative imaging could greatly help surgeons to identify targets for resection. The authors' objective is to selectively and noninvasively deposit markers under image guidance for such internal tattooing.
METHODS:
This study describes the production of new ultrasound-inducible droplets carrying large payloads of fluorescent markers and the in vivo proof of concept of their remote and controlled deposition via focused ultrasound. The droplets are monodispersed multiple emulsions produced in a microfluidic system, consisting of aqueous fluorescein in perfluorocarbon in water. Their conversion (either by vaporization or cavitation) is performed remotely using a clinical ultrasonic imaging probe.
RESULTS:
When submitted to 5 MHz imaging pulses, the droplets vaporize in vitro at 1.4 MPa peak-negative pressure and eject their content. After several seconds, a brightly fluorescent spot (0.5 mm diameter) is observed at the focus of the transducer. Experiments in the chorioallantoique membrane of chicken eggs and chicken embryo demonstrate that the spot is stable and is easily seen by naked eye.
CONCLUSIONS:
These ultrasound-inducible multiple emulsions could be used to deliver large amounts of contrast agents, chemotherapy, and genetic materials in vivo using a conventional ultrasound scanner.

We present a novel millifluidic droplet analyser (MDA) for precisely monitoring the dynamics of microbial populations over multiple generations in numerous (>103) aqueous emulsion droplets (~100 nL). As a first application, we measure the growth rate of a bacterial strain and determine the minimal inhibitory concentration (MIC) for the antibiotic cefotaxime by incubating bacteria in a fine gradient of antibiotic concentrations. The detection of cell activity is based on the automated detection of an epifluorescent signal that allows the monitoring of microbial populations up to a size of ~106 cells. We believe that this device is helpful for the study of population dynamic consequences of microbe-environment interactions and of individual cell differences. Moreover, the fluidic machine may improve clinical tests, as it simplifies, automates and miniaturizes the screening of numerous microbial populations that grow and evolve in compartments with a finely tuned composition.

The polymerization of actin in filaments generates forces that play a pivotal role in many cellular processes. We introduce a novel technique to determine the force-velocity relation when a few independent anchored filaments grow between magnetic colloidal particles. When a magnetic field is applied, the colloidal particles assemble into chains under controlled loading or spacing. As the filaments elongate, the beads separate, allowing the force-velocity curve to be precisely measured. In the widely accepted Brownian ratchet model, the transduced force is associated with the slowing down of the on-rate polymerization. Unexpectedly, in our experiments, filaments are shown to grow at the same rate as when they are free in solution. However, as they elongate, filaments are more confined in the interspace between beads. Higher repulsive forces result from this higher confinement, which is associated with a lower entropy. In this mechanism, the production of force is not controlled by the polymerization rate, but is a consequence of the restriction of filaments' orientational fluctuations at their attachment point.

We present a new family of microfluidic chips hot embossed from a commercial fluorinated thermoplastic polymer (Dyneon THV). This material shares most of the properties of fluoro polymers (very low surface energy and resistance to chemicals), but is easier to process due to its relatively low melting point. Finally, as an elastic material it also allows easy world to chip connections. Fluoropolymer films can be imprinted by hot embossing from PDMS molds prepared by soft lithography. Chips are then sealed by an original technique (termed Monolithic-Adhesive-Bonding), using two different grades of fluoropolymer to obtain uniform mechanical, chemical and surface properties. This fabrication process is well adapted to rapid prototyping, but it also has potential for low cost industrial production, since it does not require any curing or etching step. We prepared microfluidic devices with micrometre resolution features, that are optically transparent, and that provide good resistance to pressure (up to 50 kPa). We demonstrated the transport of water droplets in fluorinated oil, and fluorescence detection of DNA within the droplets. No measurable interaction of the droplets with the channels wall was observed, alleviating the need for surface treatment previously necessary for droplet applications in microfluidic chips. These chips can also handle harsh organic solvents. For instance, we demonstrated the formation of chloroform droplets in fluorinated oil, expanding the potential for on chip microchemistry.

We report on the development of a simple and easy to use microchip dedicated to allergy diagnosis. This microchip combines both the advantages of homogeneous immunoassays i.e. species diffusion and heterogeneous immunoassays i.e. easy separation and preconcentration steps. In vitro allergy diagnosis is based on specific Immunoglobulin E (IgE) quantitation, in that way we have developed and integrated magnetic core-shell nanoparticles (MCSNPs) as an IgE capture nanoplatform in a microdevice taking benefit from both their magnetic and colloidal properties. Integrating such immunosupport allows to perform the target analyte (IgE) capture in the colloidal phase thus increasing the analyte capture kinetics since both immunological partners are diffusing during the immune reaction. This colloidal approach improves 1000 times the analyte capture kinetics compared to conventional methods. Moreover, based on the MCSNPs' magnetic properties and on the magnetic chamber we have previously developed the MCSNPs and therefore the target can be confined and preconcentrated within the microdevice prior to the detection step. The MCSNPs preconcentration factor achieved was about 35?000 and allows to reach high sensitivity thus avoiding catalytic amplification during the detection step. The developed microchip offers many advantages: the analytical procedure was fully integrated on-chip, analyses were performed in short assay time (20 min), the sample and reagents consumption was reduced to few microlitres (5 µL) while a low limit of detection can be achieved (about 1 ng mL(-1)).

Pour croître une tumeur doit « faire sa place » au sein d’un tissu : c’est tout un jeu de forces mécaniques qui s’exercent alors entre la tumeur et le tissu sain et dont le perdant pourrait être la tumeur. L’application d’une pression même faible sur un tissu tumoral bloque son expansion et lorsque l’on cesse d’exercer cette pression, la croissance tumorale repart. La pression stoppe la prolifération des cellules, mais pas de façon homogène : les cellules au centre de leur amas cellulaire ne se divisent plus, alors que celles en périphérie continuent. Dans l’organisme, seules les tumeurs capables de soutenir cette pression pourraient continuer à proliférer. L’environnement mécanique pourrait un jour devenir un outil à prendre en compte dans le diagnostic.

Ribosomal (r-) RNA adopts a well-defined structure within the ribosome, but the role of r-proteins in stabilizing this structure is poorly understood. To address this issue, we use optical tweezers to unfold RNA fragments in the presence or absence of r-proteins. Here, we focus on Escherichia coli r-protein L20, whose globular C-terminal domain (L20C) recognizes an irregular stem in domain II of 23S rRNA. L20C also binds its own mRNA and represses its translation; binding occurs at two different sites—i.e., a pseudoknot and an irregular stem. We find that L20C makes rRNA and mRNA fragments encompassing its binding sites more resistant to mechanical unfolding. The regions of increased resistance correspond within two base pairs to the binding sites identified by conventional methods. While stabilizing specific RNA structures, L20C does not accelerate their formation from alternate conformations—i.e., it acts as a clamp but not as a chaperone. In the ribosome, L20C contacts only one side of its target stem but interacts with both strands, explaining its clamping effect. Other r-proteins bind rRNA similarly, suggesting that several rRNA structures are stabilized by “one-side” clamping.

In cells, DNA is constantly twisted, bent and stretched by numerous proteins mediating genome transactions. Understanding these essential biological processes requires in-depth knowledge of how DNA complies to mechanical stress. Two important physical features of DNA, helical structure and sequence, are not incorporated in current descriptions of DNA elasticity. Here we connect well-defined force–extension measurements with a new model for DNA elasticity: the twistable worm-like chain, in which DNA is considered a helical, elastic entity that complies to tension by extending and twisting. In addition, we reveal hitherto unnoticed stick–slip dynamics during DNA overstretching at ~65?pN, caused by the loss of base-pairing interactions. An equilibrium thermodynamic model solely based on DNA sequence and elasticity is presented, which captures the full complexity of this transition. These results offer deep quantitative insight in the physical properties of DNA and present a new standard description of DNA mechanics.

A striking feature of the alpha-hemolysin channel-a prime candidate for biotechnological applications-is the dependence of its ionic conductance on the magnitude and direction of the applied bias. Through a combination of lipid bilayer single-channel recording and molecular dynamics (MD) simulations, we characterized the current-voltage relationship of alpha-hemolysin for all alkali chloride salts at neutral pH. The rectification of the ionic current was found to depend on the type of cations and increase from Li(+) to Cs(+). Analysis of the MD trajectories yielded a simple quantitative model that related the ionic current to the electrostatic potential, the concentration and effective mobility of ions in the channel. MD simulations reveal that the major contribution to the current asymmetry and rectification properties originates from the cationic contribution to the current that is significantly reduced in a cationic dependent way when the membrane polarity is reversed. The variation of chloride current was found to be less important. We report that the differential affinity of cations for the charged residues positioned at the channel's end modulates the number of ions inside the channel stem thus affecting the current properties. Through direct comparison of simulation and experiment, this study evaluates the accuracy of the MD method for prediction of the asymmetric, voltage dependent conductances of a membrane channel.

Currently, most microfluidic devices are fabricated with embedded micro-channels and other elements in a close form with outward connections. Although much functionality has been demonstrated and a large number of applications have been developed, they are not easy for routine operation in biology laboratories where most in vitro cell processing still relies on the use of culture dishes, glass slides, multi-well plates, tubes, pipettes, etc. We report here an open access device which consists of an array of isolated micro-channels plated on a large culture surface, each of them having tiny nozzles for localized drug delivery. In a diffusion dominant regime, steady gradients of molecule concentration could be obtained and varied by changing the flow rate inside the micro-channels. As assay examples, cell staining and drug-induced cell apoptosis were demonstrated, showing fast cell responses in close proximity of the nozzles.

We describe a method to induce by light a reversible switch from a continuous two-phase laminar flow to a droplet generating regime, in microfluidic devices with a usual water-in-oil flow focusing geometry. It consists in adding a photosensitive surfactant to the aqueous phase to modulate using light the interfacial energy between flowing liquids and the microfluidic substrate. We show that UV irradiation induces liquid fragmentation into monodisperse water microdroplets and that many cycles of reversible and rapid switches (<2 s) between continuous laminar flows and stable droplet regimes can be realized. By spatially controlling the application of the light stimulus, we also demonstrate the first spatially resolved remote induction of droplet generation.

Replacement of calcination procedures used during catalyst preparation, by a plasma treatment, was studied over a Co-ferrierite (Co-FER) catalyst. The catalyst was tested in the NOx selective catalytic reduction reaction. A combination of UV–Vis spectroscopy and TG analysis revealed the presence of ammonium ions on the untreated and plasma Co-FER samples but not on the calcined one. Therefore, it can be concluded that the plasma treatment was not able to replace the thermal calcination step. The evaluation of catalyst behaviour was performed both under temperature programmed surface reaction (TPSR) and under steady-state conditions at different temperatures. NO oxidation tests showed that, during TPSR runs, calcined catalyst produces more NO2 than plasma catalyst. NOx consumption during TPSR of plasma catalyst confirms that precursors used on the ion-exchange procedure are still present on the catalyst even after the plasma treatment, reacting with NO to produce R-NOx, N2O and N2. Concerning deNOx tests using ethanol as reducing agent, TPSR tests showed higher NOx conversions over untreated and plasma catalysts due to the presence of ammonium and acetate precursors on these catalysts. Untreated, plasma and calcined catalysts present the same NOx and COx conversions in isothermal tests.

Plasma-enhanced chemical vapour deposition (PECVD) was used to prepare thin films of cobalt oxide. Cobalt oxide-based (CoO and Co3O4) catalysts were chosen due to their efficiency in mineralisation of organic pollutants achieved by catalytic ozonation. In this work, two types of PECVD processes were used for the production of cobalt oxide thin films. In the first one, a solution of nitrate salt of cobalt was sprayed into a RF low pressure plasma discharge (40 MHz, 600 Pa, 200 W) to obtain CoxOy layers. In the second MOPECVD (metal organic plasma-enhanced chemical vapour deposition) process, cobalt oxide thin films were deposited using a capacitive coupled external electrodes RF plasma reactor (13.56 MHz, 100 Pa, 200 W) with cobalt carbonyl Co2(CO)8 dissolved in hexene as precursor sprayed in a gas carrier (argon and oxygen). In the case of coatings produced from a solution of cobalt nitrate salt, a layer of 1 µm of Co3O4 in crystalline form was obtained after annealing. Considering the thin films obtained from cobalt carbonyl precursor, analyses confirmed the presence of cobalt oxide in a polymeric layer on the surface of the substrate. XRD investigation showed the presence of a crystalline phase of Co3O4 (crystallite size of about 40 nm).

Since the late 1970s, approaches have been proposed to replace conventional gas chromatography apparatus with silicon-based microfabricated separation systems. Performances are expected to be improved with miniaturization owing to the reduction of diffusion distances and better thermal management. However, one of the main challenges consists in the collective and reproducible fabrication of efficient microelectromechanical system (MEMS) gas chromatography (GC) columns. Indeed, usual coating processes or classical packing with particulate matters are not compatible with the requirements of collective MEMS production in clean room facilities. A new strategy based on the rerouting of conventional microfabrication techniques and widely used in electronics for metals and dielectrics deposition is presented. The originality lies in the sputtering techniques employed for the deposition of the stationary phase. The potential of these novel sputtered stationary phases is demonstrated with silica sputtering applied to the separation of light hydrocarbons and natural gases. If kinetic characteristics of the sputtered open tubular columns were acceptable with 2500 theoretical plates per meter, the limited retention and resolution of light hydrocarbons led us to consider semipacked sputtered columns with rectangular pillars allowing also significant reduction of typical diffusion distances. In that case separations were greatly improved because retention increased and efficiency was close to 5000 theoretical plates per meter.

Two molecularly imprinted silicas (MISs) were synthesized and used as selective sorbents for the extraction of nitroaromatic explosives in post-blast samples. The synthesis of the MISs was carried out with phenyltrimethoxysilane as monomer, 2,4-dinitrotoluene (2,4-DNT) as template and triethoxysilane as cross-linker by a sol-gel approach in two molar ratios: 1/4/20 and 1/4/30 (template/monomer/cross-linker). Non-imprinted silica sorbents were also prepared following the same procedures without introducing the template. An optimized procedure dedicated to the selective treatment of aqueous samples was developed for both MISs for the simultaneous extraction of the template and other nitroaromatic compounds commonly used as explosives. The capacity of the MISs was measured by the extraction of increasing amounts of 2,4-DNT in pure water and is higher than 3.2 mg/g of sorbent for each MIS. For the first time, four nitroaromatic compounds were selectively extracted and determined simultaneously with extraction recoveries higher than 79%. The potential of these sorbents was then highlighted by their use for the clean-up of post-blast samples (motor oil, post-mortem blood, calcined fragments, etc.). The results were compared to those obtained using a conventional sorbent, thus demonstrating the interest of the use of these MISs as selective sorbents.

The scanning electrochemical microscope (SECM) in the lithographic mode is used to assess quantitatively, from both theoretical and experimental points of view, the kinetics of irreversible transformation of electroactive molecular moieties immobilized on a surface as self-assembled monolayers (SAMs). The SECM tip allows the generation of an etchant that transforms the surface locally and irreversibly. The resulting surface patterning is detectable by different surface analyses. The quantification of the surface transformation kinetics is deduced from the evolution of the pattern dimensions with the etching time. The special case of slow etching kinetics is presented; it is predicted that the pattern evolution follows the expansion of the etchant at the substrate surface. The case of a chemically unstable etchant is considered. The model is then tested by inspecting the slow reductive patterning of a perfluorinated SAM. Good agreement is found with different independent SECM interrogation modes, depending on the insulating or conducting nature of the covered substrate. The surface transformation measurements are also compared to the reduction of solutions of perfluoroalkanes. The three-orders-of-magnitude-slower electron transfer observed at the immobilized molecules likely describes the large reorganization associated with the generation of a perfluoroalkyl-centered radical anion.