Publications

Antibodies that trigger the complement system play a pivotal role in the immune defense against pathogenic bacteria and offer potential therapeutic avenues for combating antibiotic-resistant bacterial infections, a rising global concern. To gain a deeper understanding of the key parameters regulating complement activation by monoclonal antibodies, we developed a novel bioassay for quantifying classical complement activation at the monoclonal antibody level, and employed this assay to characterize rare complement-activating antibacterial antibodies on the single-antibody level in postimmunization murine antibody repertoires. We characterized monoclonal antibodies from various antibody iso-types against specific pathogenic bacteria (Bordetella pertussis and Neisseria meningitidis) to broaden the scope of our findings. We demonstrated activation of the classical pathway by individual IgM- and IgG-secreting cells, that is, mono-clonal IgM and IgG2a/2b/3 subclasses. Additionally, we could observe different epitope density requirements for efficient C1q binding depending on antibody isotype, which is in agreement with previously proposed molecular mechanisms. In short, we found that antibody density most crucially regulated C1q recruitment by monoclonal IgG isotypes, but not IgM isotypes. This study provides additional insights into important parameters for classical complement initiation by monoclonal antibodies, a knowledge that might inform antibody screening and vaccination efforts.

http://doi.org/10.1002/eji.202451228

Oil and water can only be mixed by dispersing droplets of one fluid in the other. When two droplets approach one another, the thin film that separates them invariably becomes unstable, causing the droplets to coalesce. The only known way to avoid this instability is through addition of a third component, typically a surfactant, which stabilizes the thin film at its equilibrium thickness. We report the observation that a thin fluid film of oil separating two water droplets can lead to an adhesive interaction between the droplets. Moreover, this interaction prevents their coalescence over timescales of several weeks, without the use of any surfactant or solvent

https://www.science.org/stoken/author-tokens/ST-1810/full

SARS-CoV-2 mRNA vaccination generates protective B cell responses targeting the SARS-CoV-2 spike glycoprotein. Whereas anti-spike memory B cell responses are long lasting, the anti-spike humoral antibody response progressively wanes, making booster vaccinations necessary for maintaining protective immunity. Here, we qualitatively investigated the plasmablast responses by measuring from single cells within hours of sampling the affinity of their secreted antibody for the SARS-CoV-2 spike receptor binding domain (RBD) in cohorts of BNT162b2-vaccinated naive and COVID-19–recovered individuals. Using a droplet microfluidic and imaging approach, we analyzed more than 4,000 single IgG-secreting cells, revealing high interindividual variability in affinity for RBD, with variations over 4 logs. High-affinity plasmablasts were induced by BNT162b2 vaccination against Hu-1 and Omicron RBD but disappeared quickly thereafter, whereas low-affinity plasmablasts represented more than 65% of the plasmablast response at all time points. Our droplet-based method thus proves efficient at fast and qualitative immune monitoring and should be helpful for optimization of vaccination protocols.

https://insight.jci.org/articles/view/166602

Rheology of concentrated protein solutions is crucial for the understanding of macromolecular crowding dynamics as well as the formulation of protein therapeutics. The cost and scarcity of most protein samples prevents wide-scale rheological studies as conventional viscosity measurement methods require large sample volume. There is a growing need for a precise and robust viscosity measurement tool that minimizes consumption and simplifies the handling of highly concentrated protein solutions. This objective is achieved by combining microfluidics and microrheology: we developed a specific microsystem to study the viscosity of aqueous solutions at high concentrations. The PDMS chip allows in situ production, storing and monitoring of water-in-oil nanoliter droplets. We perform precise viscosity measurements inside individual droplets by particle-tracking microrheology of fluorescent probes. Pervaporation of water through a PDMS membrane induces aqueous droplet shrinking, concentrating the sample up to 150 times, thus allowing viscosity measurements along an extended concentration range in just one experiment. The methodology is precisely validated by studying the viscosity of sucrose solutions. Two model proteins are also studied with sample consumption reduced to as little as 1 μL of diluted solution, showcasing the viability of our approach for the study of biopharmaceuticals.

https://pubs.rsc.org/en/content/articlelanding/2023/lc/d3lc00073g

Increasing evidence suggests that natural killer (NK) cells are composed of distinct functional subsets. This multifunctional role has made them an attractive choice for anticancer immunotherapy. A functional NK cell repertoire is generated through cellular education, resulting in a heterogeneous NK cell population with distinct capabilities responding to different stimuli. The application of a high-throughput droplet-based microfluidic platform allows monitoring of NK cell-target cell interactions at the single-cell level and in real-time. A variable response of single NK cells toward different target cells is observed, and a distinct population of NK cells (serial killers) capable of inducing multiple target lysis is identified. By assessing the cytotoxic dynamics, it is shown that single umbilical cord blood-derived CD34+ hematopoietic progenitor (HPC)-NK cells display superior antitumor cytotoxicity. With an integrated analysis of cytotoxicity and cytokine secretion, it is shown that target cell interactions augment cytotoxic as well as secretory behavior of NK cells. By providing an integrated assessment of NK cell functions by microfluidics, this study paves the way to further functionally characterize NK cells ultimately aimed to improve cancer immunotherapy.

https://doi.org/10.1002/adbi.202200207

The major therapeutic goal for immune thrombocytopenia (ITP) is to restore normal platelet counts using drugs to promote platelet production or by interfering with mechanisms responsible for platelet destruction. 80% of patients possess anti-integrin αIIbβ3 (GPIIbIIIa) IgG autoantibodies causing platelet opsonization and phagocytosis. The spleen is considered the primary site of autoantibody production by autoreactive B cells and platelet destruction. The immediate failure in ~50% of patients to recover a normal platelet count after anti-CD20 Rituximab-mediated B cell depletion and splenectomy suggest that autoreactive, rituximab-resistant, IgG-secreting B cells (IgG-SC) reside in other anatomical compartments. We analyzed >3,300 single IgG-SC from spleen, bone marrow and/or blood of 27 patients with ITP revealing high inter-individual variability in affinity for GPIIbIIIa with variations over 3 logs. IgG-SC dissemination and range of affinities were however similar per patient. Longitudinal analysis of autoreactive IgG-SC upon treatment with anti-CD38 mAb daratumumab demonstrated variable outcomes, from complete remission to failure with persistence of high-affinity anti-GPIIbIIIa IgG-SC in the bone marrow. This study demonstrates the existence and dissemination of high-affinity autoreactive plasma cells in multiple anatomical compartments of patients with ITP that may cause the failure of current therapies.

https://doi.org/10.1172/JCI153580

Stabilizing layers of colloidal dispersions or emulsions to obtain homogeneous films is a real challenge. We describe here a new kind of instability in drying films of emulsions: during evaporation of the internal phase, cracks appear between the droplets that create aggregates according to a regular pattern. We show that this pattern only appears if the emulsion is adhesive, i.e., if droplets stick together. The pattern exhibits a characteristic length which depends on the adhesion strength and film thickness. These experimental results support a model where this instability is due to the gel structure and elastic properties of adhesive emulsions. Understanding this phenomenon will allow us to get a homogeneous film or to control it to get structured materials.

https://doi.org/10.1021/acs.langmuir.1c00246

Aluminium salts such as aluminium chlorohydrate (ACH) are the active ingredients of antiperspirant products. Their mechanism of action involves a temporary and superfcial plugging of eccrine sweat pores at the skin surface. We developed a microfuidic system that allows the real time observation of the interactions between sweat and ACH in conditions mimicking physiological sweat fow and pore dimensions. Using artifcial sweat containing bovine serum albumin as a model protein, we performed experiments under fowing conditions to demonstrate that pore clogging results from the aggregation of proteins by aluminium polycations at specifc location in the sweat pore. Combining microfuidic experiments, confocal microscopy and numerical models helps to better understand the physical chemistry and mechanisms involved in pore plugging. The results show that plugging starts from the walls of sweat pores before expanding into the centre of the channel. The simulations aid in explaining the infuence of ACH concentration as well as the impact of fow conditions on the localization of the plug. Altogether, these results outline the potential of both microfuidic confocal observations and numerical simulations at the single sweat pore level to understand why aluminium polycations are so efcient for sweat channel plugging.

Completing a Swiss-cheese theorem of Lieb and Lebowitz (LL), we prove that any population of spheres with power-law radius distribution can completely fill 3D Euclidean space if the exponent is such that 2.8 ≤ d_f < 3. This sufficient condition extends considerably the known part of the ensemble of space-filling populations of polydisperse spheres. The self-similar spatial arrangement of the polydisperse spheres related to the theorem is discussed using a numerical example with d_f = 2.875. By calculating the small-angle scattering structure factor of the resulting packing, we found it to present several crystalline peaks indicating some regularity. This is significantly different from the featureless structure factor of an Apollonian packing which represents total disorder. We thereby argue that the LL algorithm for filling space with spheres is fundamentally different from Apollonian constructions.

Antibodies with antibacterial activity need to bind to the bacterial surface with affinity, specificity, and sufficient density to induce efficient elimination. To characterize the antibacterial antibody repertoire, we developed an in-droplet bioassay with single-antibody resolution. The assay not only allowed us to identify whether the secreted antibodies recognized a bacterial surface antigen, but also to estimate the apparent dissociation constant (KD app) of the interaction and the density of the recognized epitope on the bacteria. Herein, we found substantial differences within the KD app/epitope density profiles in mice immunized with various species of heat-killed bacteria. The experiments further revealed a high cross-reactivity of the secreted IgG repertoires, binding to even unrelated bacteria with high affinity. This application confirmed the ability to quantify the anti-bacterial antibody repertoire and the utility of the developed bioassay to study the interplay between bacteria and the humoral response.

https://doi.org/10.1038/s42003-020-01296-3

The removal of ultrathin amorphous polymer films in contact with an aqueous gelled solution containing small amounts of good solvent is addressed by means of specular and off-specular neutron reflectometry. The distribution of heavy water and benzyl alcohol is revealed inside Laropal A81, often employed as a protective varnish layer for Culture Heritage in the restoration of easel paintings. The swelling kinetics, interface roughness, and film morphologies were recorded as a function of temperature and increasing benzyl alcohol concentration in the dispersion of Pemulen TR-2, a hydrophobically modified acrylic acid copolymer. The addition of small amounts of good solvent results in the appearance of water-filled cavities inside the varnish, which grow with time. It is shown that while increasing the solvent concentration greatly enhances the hole growth kinetics, an increase in temperature above the glass transition temperature does not have such a big effect on the kinetics.

We have discovered the existence of extremely polydisperse High Internal-Phase-Ratio Emulsions (HIPEs) in which the internal-phase droplets, present at 95% volume fraction, remain spherical and organize themselves in the available space according to Apollonian packing rules. Such Apollonian emulsions are obtained from dispersing oil dropwise in water in the presence of very little surfactant, and allowing them to evolve at rest for at least a week. The packing structure of the droplets was confirmed through size distribution measurements that evolved spontaneously towards power laws with the known Apollonian exponents, as well as comparison of the structure factors of aged HIPEs measured by Small-Angle X-ray Scattering with that of a numerically simulated Random Apollonian Packing. Thanks to the perfect sphericity of the droplets, Apollonian emulsions were found to display Newtonian flow even at such extremely high volume fraction. We argue that these fascinating space-filling assemblies of spherical droplets are a result of coalescence and fragmentation processes obeying simple geometrical rules of conserving total volume and sphericity, and minimizing the elastic energy associated with interactions of neighbouring droplets.

We characterize the motility of athermal swimming droplets within the framework of a persistent random walk. Just like active colloids, their trajectories can be modeled with a constant velocity $V$ and a slow angular diffusion, but the random changes in direction are not thermally driven. Instead, $V$ is determined by the interfacial tension gradient along the droplet surface, while reorientation of the surfactant gradient leads to changes in direction with a persistence time $\tau$. We show that the origin of locomotion is the difference in the critical micellar concentration in the front and the back of the droplet, $\Delta \mathrm{CMC}$. Tuning this parameter by salt controls $V$ from 3 to 15 diameters $d/s$. Surfactant concentration has little effect on speed, but leads to a dramatic decrease in $\tau$ over 4 orders of magnitude. The corresponding range of the persistence length $â„“=V\tau$ extends beyond the realm of synthetic or living swimmers, in which $V$ is limited by fuel consumption and $\tau$ is set by thermal fluctuations or biological activity, respectively. Our tunable swimmers are ideal candidates for the study of the departure from equilibrium to high levels of activity. We show that their collective behavior exhibits the formation of active clusters of a well-defined size.

Measuring the structure factor, S(q), of a dispersion of particles by small-angle X-ray scattering provides a unique method to investigate the spatial arrangement of colloidal particles. However, it is impossible to find the exact location of the particles from S(q) because some information is inherently lacking in the measured signal. The two standard ways to analyse an experimental S(q) are then to compare it either with structure factors computed from simulated systems or with analytical ones calculated from approximated systems. However, such approaches may prove inadequate for dispersions of variously polydisperse particles. While Vrij, Bloom and Stell established a mean-field approach that could yield fairly accurate approximations for experimental S(q), this solution has remained underused because of its mathematical complexity. In the present work, the complete Percus–Yevick solution for general polydisperse hard-sphere systems is derived in a concise form that is straightforward to use. The form of the solution has been simplified enough to provide experimentalists with ready solutions of several commonly encountered particle-radius distributions in real systems (Schulz, truncated normal and inverse Gaussian). The approach is also illustrated with a case study of the exponential radius distribution. Finally, the application of the proposed solution to the power-law radius distribution is discussed in detail by comparing the calculations with experimentally measured S(q) for an Apollonian packing of spherical droplets recently reported in high-internal-phase-ratio emulsions.

Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8–10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells.

https://www.nature.com/articles/s41596-020-0354-0

One of the major goals of vaccination is to prepare the body to rapidly secrete specific Abs during an infection. Assessment of the vaccine quality is often difficult to perform, as simple measurements like Ab titer only partly correlate with protection. Similarly, these simple measurements are not always sensitive to changes in the preceding immunization scheme. Therefore, we introduce in this paper a new, to our knowledge, method to assay the quality of immunization schemes for mice: shortly after a recall with pure Ag, we analyze the frequencies of IgG-secreting cells (IgG-SCs) in the spleen, as well as for each cells, the Ag affinity of the secreted Abs. We observed that after recall, appearance of the IgG-SCs within the spleen of immunized mice was fast (<24 h) and this early response was free of naive IgG-SCs. We further confirmed that our phenotypic analysis of IgG-SCs after recall strongly correlated with the different employed immunization schemes. Additionally, a phenotypic comparison of IgG-SCs presented in the spleen during immunization or after recall revealed similarities but also significant differences. The developed approach introduced a novel (to our knowledge), quantitative, and functional highly resolved alternative to study the quality of immunizations.

DOI: https://doi.org/10.4049/jimmunol.2000112

Affinity maturation is a complex dynamical process allowing the immune system to generate antibodies capable of recognizing antigens. We introduce a model for the evolution of the distribution of affinities across the antibody population in germinal centers. The model is amenable to detailed mathematical analysis, and gives insight on the mechanisms through which antigen availability controls the rate of maturation and the expansion of the antibody population. It is also capable, upon maximum-likelihood inference of the parameters, to reproduce accurately the distributions of affinities of IgG-secreting cells we measure in mice immunized against Tetanus Toxoid under largely varying conditions (antigen dosage, delay between injections). Both model and experiments show that the average population affinity depends non-monotonically on the antigen dosage. We show that combining quantitative modelling and statistical inference is a concrete way to investigate biological processes underlying affinity maturation (such as selection permissiveness), hardly accessible through measurements.

DOI: https://doi.org/10.7554/eLife.55678

Freezing of alum-based vaccines drastically alters their colloidal composition and leads to irreversible cluster formation. The loss of stability is well described, but the impact of frost damage on the functionality of the induced and secreted antibody repertoire has not been studied in detail. We therefore applied our single-cell measurement platform to extract the frequencies of Immunoglobulin G-secreting cells in combination with individual secretion rates and affinities. We showed that, frost-damaged or not, the tested vaccine was able to generate similar frequencies of total and antigen-affine IgG-secreting cells. Additionally, the frost-damaged vaccine stimulated a similar T-cell cytokine secretion pattern when compared to the regularly stored vaccine. However, frost-damaged vaccines induced no efficient affinity maturation and a complete collapse of the affinity distribution was observed. This study unveiled the impact of frost-damage to alum-based vaccines on the induced secreted antibody repertoire, and illustrated the power of functional single-antibody analysis.

https://doi.org/10.1016/j.vaccine.2020.05.066

Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100–1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450–900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility.

https://doi.org/10.1038/s41587-020-0466-7

Cells can rapidly adapt to changing environments through nongenetic processes; however, the metabolic cost of such adaptation has never been considered. Here we demonstrate metabolic coupling in a remarkable, rapid adaptation process (1 in 1,000 cells adapt per hour) by simultaneously measuring metabolism and division of thousands of individual Saccharomyces cerevisiae cells using a droplet microfluidic system: droplets containing single cells are immobilized in a two-dimensional (2D) array, with osmotically induced changes in droplet volume being used to measure cell metabolism, while simultaneously imaging the cells to measure division. Following a severe challenge, most cells, while not dividing, continue to metabolize, displaying a remarkably wide diversity of metabolic trajectories from which adaptation events can be anticipated. Adaptation requires a characteristic amount of energy, indicating that it is an active process. The demonstration that metabolic trajectories predict a priori adaptation events provides evidence of tight energetic coupling between metabolism and regulatory reorganization in adaptation. This process allows S. cerevisiae to adapt on a physiological timescale, but related phenomena may also be important in other processes, such as cellular differentiation, cellular reprogramming, and the emergence of drug resistance in cancer.

https://doi.org/10.1073/pnas.1913767117

We have discovered the existence of polydisperse high internal-phase-ratio emulsions (HIPE) in which the internal-phase droplets, present at 95% volume fraction, remain spherical and organise themselves according to Apollonian packing rules. These polydisperse HIPEs are formed by emulsifying oil dropwise in a surfactant-poor aqueous continuous phase. After stirring has ceased, their droplet size distributions begin to evolve spontaneously and continuously through coalescence towards well-defined power laws with the Apollonian exponent. Small-angle X-ray Scattering performed on aged HIPEs demonstrate that the droplet packing structure agrees with that of a numerically simulated random Apollonian packing. We argue that when such concentrated emulsions are allowed to evolve, the coalescing droplets must obey volume and sphericity conservation. This leads to a mechanism that differs from typical coalescence in dilute emulsions.

The removal of ultrathin amorphous polymer films in contact with nonsolvent/solvent binary mixtures is addressed by means of neutron reflectometry and atomic force microscopy. The high resolution of neutron scattering makes it possible to resolve the distribution profiles of heavy water and benzyl alcohol inside Laropal (R) A81, often employed as a protective varnish layer for Culture Heritage in restoration of easel paintings. The swelling kinetics and distribution profiles were recorded as a function of time and increasing benzyl alcohol concentration in water. The varnish film swells by penetration of the good solvent. At higher concentrations water-filled cavities appear inside the varnish and grow with time. Contrary to homogeneous dissolution dewetting is observed at late stages of exposure to the liquid which leads to the Breakup of the film. The high resolution measurements are compared to bulk behaviour characterized by the ternary phase diagram and the Flory-Huggins interaction parameters are calculated and used to predict the swelling and solvent partition in the films. Distinct differences of the thin film to bulk behaviour are found. The expectations made previously for the behaviour of solvent/non-solvent mixtures on the removal of thin layers in the restoration of easel paintings should be revised in view of surface interactions.

Monte Carlo simulations, fully constrained by experimental parameters, are found to agree well with a measured phase diagram of aqueous dispersions of nanoparticles with a moderate size polydispersity over a broad range of salt concentrations, c(s), and volume fractions, phi. Upon increasing phi, the colloids freeze first into coexisting compact solids then into a body centered cubic phase (bcc) before they melt into a glass forming liquid. The surprising stability of the bcc solid at high phi and c(s) is explained by the interaction (charge) polydispersity and vibrational entropy.

The flow of two immiscible liquids or fluids in bounded systems where confinement geometry varies can lead to drop or bubble formation. This phenomenon has been reported in the context of oil recovery and named snap-off, or exploited for making emulsions, and then foams, by using microfluidic systems, namely, microchannel emulsification or step emulsification. We report a comprehensive experimental investigation of such an emulsification process occurring at the end of a glass rectangular tube filled with oil and immersed in a water bath. This allows us to clearly visualize the breakup event of the dispersed phase liquid finger at the capillary's end. Below a critical flow rate, the drop size varies slowly with the flow rate and it is linked to the pinching time of the dispersed phase. A semiempirical law that gives the resulting drop size as a function of fluid and geometrical properties is proposed. However, this feature is altered for an aspect ratio of the rectangular tube below 2.5 where the forming drop hinders the counterflow of the continuous phase leading to larger drops. Then, above a critical flow rate, or capillary number that weakly depends on the viscosity ratio of the two liquids, the neck adopts a quasistatic shape well accounted for by a model based on a Hele-Shaw flow. In that case, drop formation is driven by gravity and a transition from a dripping regime to a jetting regime is observed at higher flow rates. Monodisperse foam can also be formed by injecting air. While the overall dynamics of bubble formation shares similarities with an incompressible fluid, the bubble size and the critical capillary number do not follow the same scaling laws.

Convective dispersion of solutes is inherent to flow in channels because of the nonuniformity of the velocity profile. When diffusion is negligible, for large particles for example, the trajectory of particles can be solely described by a kinematic approach. Here, we investigate such a phenomenon for micrometer-size beads flowing in a circular pipe. We show that the presence of large bubbles, namely in the case of a segmented flow, either prevents the convective dispersion or leads to the accumulation of particles at the rear of the bubble moving in front. The destabilization of the initially homogeneous suspension occurs when liquid inertia comes into play. Indeed, for moderate Reynolds number of the particles, particles move away from the wall, thus exploring different flow lines that finally impact the axial dispersion features. Moreover, since the bubbles impose an axial boundary condition of the mean velocity, a net flux of particles directed along the flow direction is built up above a critical particle Reynolds number. This work is motivated by the understanding of the flow behavior of biological samples, and especially in the context of cell encapsulation.

Droplet-based microfluidics, using water-in-oil emulsion droplets as microreactors, is becoming a widespread method for performing assays and especially in the cell biology field. Making a simple and highly portable system for creating emulsion droplets would help to continue the popularization of such a technique. Also, the ability to emulsify all the samples would strengthen this compartimenlization technique to handle samples with limited volume. Here, we propose a strategy of droplet formation that combines a classical flow-focusing microfluidic chip, which could be commercially available, with a standard laboratory adjustable micropipette. The micropipette is used as a negative pressure generator for controlling liquid flows. In that way, emulsification does neither require any electrical power supply nor a cumbersome device and functions with small liquid volumes. Droplet formation can be easily and safely performed in places with limited space, opening a wide range of applications especially in biological laboratory environments with higher level of safety regulations, i.e., BSL-3/4. Fortunately, the present methodology that involves small fluid volumes, and thus possible time dependent flow conditions, allows to minimize dead volume while keeping drops’ size homogeneous. A physical characterization of droplet production and a model that describes the emulsion features, in terms of drop size and size distribution, are proposed for rationalizing the performances of the micropipette-powered emulsification process.

The osmotic pressure of polyelectrolyte solutions as a function of concentration has been calculated by Monte Carlo simulations of a spherical cell model and by molecular dynamics simulations with periodic boundary conditions. The results for the coarse-grained polyelectrolyte model are in good agreement with experimental results for sodium polyacrylate and the cell model is validated by the bulk simulations. The cell model offers an alternative perspective on osmotic pressure and also forms a direct link to even simpler models in the form of the Poisson-Boltzmann approximation applied to cylindrical and spherical geometries. As a result, the non-monotonic behaviour of the osmotic coefficient seen in simulated salt-free solutions is shown not to rely on a transition between a dilute and semi-dilute regime, as is often suggested when the polyion is modelled as a linear flexible chain. The non-monotonic behaviour is better described as the combination of a finite-size effect and a double-layer effect. Parameters that represent the linear nature of the polyion, including an alternative to monomer concentration, make it possible to display a generalised behaviour of equivalent chains, at least at low concentrations. At high concentrations, local interactions become significant and the exact details of the model become important. The effects of added salt are also discussed and one conclusion is that the empirical additivity rule, treating the contributions from the polyelectrolyte and any salt separately, is a reasonable approximation, which justifies the study of salt-free solutions.

Some bacteria can act as catalysts to oxidize (or reduce) organic or inorganic matter with the potential of generating electrical current. Despite their high value for sustainable energy, organic compound production and bioremediation, a tool to probe the natural biodiversity and to select most efficient microbes is still lacking. Compartmentalized cell culture is an ideal strategy for achieving such a goal but the appropriate compartment allowing cell growth and electron exchange must be tailored. Here, we develop a conductive composite hydrogel made of a double network of alginate and carbon nanotubes. Homogeneous mixing of carbon nanotubes within the polyelectrolyte is obtained by a surfactant assisted dispersion followed by a desorption step for triggering electrical conductivity. Dripping the mixture in a gelling bath through simple extrusion or a double one allows the formation of either plain hydrogel beads or liquid core hydrogel capsules. The process is shown to be compatible with the bacterial culture (Geobacter sulfurreducens). Bacteria can indeed colonize the outer wall of plain beads or the inner wall of the conductive capsules’ shell that function as an anode from which electrons produced by the cells are collected.

Adjustment of plasmid copy number resulting from the balance between positive and negative impacts
of borne synthetic genes, plays a critical role in the global efficiency of multistep metabolic engineering.
Differential expression of co-expressed engineered genes is frequently observed depending on growth
phases, metabolic status and triggered adjustments of plasmid copy numbers, constituting a dynamic
process contributing to minimize global engineering burden. A yeast model involving plasmid based
expression of phosphoribulokinase (PRKp), a key enzyme for the reconstruction of synthetic Calvin cycle,
was designed to gain further insights into such a mechanism. A conditional PRK expression cassette was
cloned either onto a low (ARS-CEN based) or a high (2-micron origin based) copy number plasmid using
complementation of a trp1 genomic mutation as constant positive selection. Evolution of plasmid copy
numbers, PRKp expressions, and cell growth rates were dynamically monitored following gene derepression
through external doxycycline concentration shifts. In the absence of RubisCO encoding
gene permitting metabolic recycling, PRKp expression that led to depletion of ribulose phosphate, a
critical metabolite for aromatic amino-acids biosynthesis, and accumulation of the dead-end diphosphate
product contribute to toxicity. Triggered copy number adjustment was found to be a dynamic
process depending both on plasmid types and levels of PRK induction. With the ARS-CEN plasmid, cell
growth was abruptly affected only when level PRKp expression exceeded a threshold value. In contrast, a
proportional relationship was observed with the 2-micron plasmid consistent with large copy number
adjustments. Micro-compartment partitioning of bulk cultures by embedding individual cells into inverse
culture medium/oil droplets, revealed the presence of slow and fast growing subpopulations that
differ in relative proportions for low and high copy number plasmids.

https://doi.org/10.1016/j.resmic.2018.06.002

Application of droplet-based microfluidics for the screening of microbial libraries is one of the important ongoing developments in functional genomics/metagenomics. In this article, we propose a new method that can be employed for high-throughput profiling of cell growth. It consists of light-driven labelling droplets that contain growing cells directly in a microfluidics observation chamber, followed by recovery of the labelled cells. This method is based on intracellular expression of green-to-red switchable fluorescent proteins. The proof of concept is established here for two commonly used biological models, E. coli and S. cerevisiae. Growth of cells in droplets was monitored under a microscope and, depending on the targeted phenotype, the fluorescence of selected droplets was switched from a “green” to a “red” state. Red fluorescent cells from labelled droplets were then successfully detected, sorted with the Fluorescence Activated Cell Sorting machine and recovered. Finally, the application of this method for different kind of screenings, in particular of metagenomic libraries, is discussed and this idea is validated by the analysis of a model mini-library.

https://doi.org/10.1016/j.mimet.2018.03.001

Studies of the dynamics of the antibody-mediated immune response have been hampered by the absence of quantitative, high-throughput systems to analyze individual antibody-secreting cells. Here we describe a simple microfluidic system, DropMap, in which single cells are compartmentalized in tens of thousands of 40-pL droplets and analyzed in two-dimensional droplet arrays using a fluorescence relocation-based immunoassay. Using DropMap, we characterized antibody-secreting cells in mice immunized with tetanus toxoid (TT) over a 7-week protocol, simultaneously analyzing the secretion rate and affinity of IgG from over 0.5 million individual cells enriched from spleen and bone marrow. Immunization resulted in dramatic increases in the range of both single-cell secretion rates and affinities, which spanned at maximum 3 and 4 logs, respectively. We observed differences over time in dynamics of secretion rate and affinity within and between anatomical compartments. This system will not only enable immune monitoring and optimization of immunization and vaccination protocols but also potentiate antibody screening

doi:10.1038/nbt.3964

access to a view-only version  http://rdcu.be/vKeI

We report on the impact of surface hydrophobization on the structure of aqueous silica dispersions and how this structure resists drying stress. Hydrophilic silica particles were hydrophobized directly in water using a range of organosilane precursors, with a precise control of the grafting density. The resulting nanostructure was precisely analyzed by a combination of small-angle X-ray scattering (SAXS) and cryo-microscopy (cryo-TEM). Then, the dispersion was progressively concentrated by drying, and the evolution of the nanostructures as a function of the grafting density was followed by SAXS. At the fundamental level, because the hydrophobic character of the silica surfaces could be varied continuously through a precise control of the grafting density, we were able to observe how the hydrophobic interactions change particles interactions and aggregates structures. Practically, this opened a new route to tailor the final structure, the residual porosity, and the damp-proof properties of the fully dried silica. For example, regardless of the nature of the hydrophobic precursor, a grafting density of 1 grafter per nm² optimized the interparticle interactions in solution in view to maximize the residual porosity in the dried material (0.9 cm³/g) and reduced the water uptake to less than 4% in weight compared to the typical value of 13% for hydrophilic particles (at T = 25 °C and relative humidity = 80%).

DOI: 10.1021/acs.langmuir.6b04505

Aluminium salts are widely used to control sweating for personal hygiene purposes. Their mechanism of
action as antiperspirants was previously thought to be a superficial plugging of eccrine sweat pores by
the aluminium hydroxide gel. Here we present a microfluidic T junction device that mimics sweat ducts,
and is designed for the real time study of interactions between sweat and ACH (Aluminium Chloro
Hydrate) under conditions that lead to plug formation. We used this device to image and measure the
diffusion of aluminium polycationic species in sweat counter flow. We report the results of small angle
X-ray scattering experiments performed to determine the structure and composition of the plug, using
BSA (Bovine Serum Albumin) as a model of sweat proteins. Our results show that pore occlusion occurs
as a result of the aggregation of sweat proteins by aluminium polycations. Mapping of the device shows
that this aggregation is initiated in the T junction at the location where the flow of aluminium
polycations joins the flow of BSA. The mechanism involves two stages: (1) a nucleation stage in which
aggregates of protein and polycations bind to the wall of the sweat duct and form a tenuous
membrane, which extends across the junction; (2) a growth stage in which this membrane collects
proteins that are carried by hydrodynamic flow in the sweat channel and polycations that diffuse into
this channel. These results could open up perspectives to find new antiperspirant agents with an
improved efficacy.

DOI: 10.1039/c6sm02510b

We report on a measurement of forces between particles adsorbed at a water–oil interface in the presence of an oil-soluble polymer. The cationic polymer interacts electrostatically with the negatively charged particles, thereby modulating the particle contact angle and the magnitude of capillary attraction between the particles. However, polymer adsorption to the interface also generates an increase in the apparent interfacial viscosity over several orders of magnitude in a time span of a few hours. We have designed an experiment in which repeated motion trajectories are measured on pairs of particles. The experiment gives an independent quantification of the interfacial drag coefficient (10–7–10–4 Ns/m) and of the interparticle capillary forces (0.1–10 pN). We observed that the attractive capillary force depends on the amount of polymer in the oil phase and on the particle pair. However, the attraction appears to be independent of the surface rheology, with changes over a wide range of apparent viscosity values due to aging. Given the direction (attraction), the range (∼μm), and the distance dependence (∼1/S5) of the observed interparticle force, we interpret the force as being caused by quadrupolar deformations of the fluid–fluid interface induced by particle surface roughness. The results suggest that capillary forces are equilibrated in the early stages of interface aging and thereafter do not change anymore, even though strong changes in surface rheology still occur. The described experimental approach is powerful for studying dissipative as well as conservative forces of micro- and nanoparticles at fluid–fluid interfaces for systems out of equilibrium.

DOI: 10.1021/acs.langmuir.6b03910

Liquid core capsules having a hydrogel membrane are becoming a versatile tool for three- dimensional culture of micro-organisms and mammalian cells. Making sub-millimeter capsules at a high rate, via the breakup of a compound jet in air, opens the way to high throughput screen- ing applications. However, a control of the capsule size monodispersity, especially required for quantitative bioassays, was still lacking. Here, we report how the understanding of the underlying hydrodynamic instabilities that occur during the process can lead to calibrated core-shell biore- actors. This implies: i) to damp the shear layer instability that develops inside the injector arising from the co-annular flow configuration of liquid phases having contrasted viscoelastic properties; ii) to control the capillary instability of the compound jet by superposing a harmonic perturbation to the shell flow; iii) to avoid coalescence of drop during jet fragmentation as well as during drop flight towards the gelling bath, and iv) to ensure a proper engulfment of the compound drops into the gelling bath for building a closed hydrogel shell. We end up with the creation of numerous identical compartments in which cells are able to form mutlicellular aggregates, namely spheroids. In addition, we implement an intermediate composite hydrogel layer, composed of alginate and collagen, allowing cell adhesion and thus the formation of epithelia or monolayers of cells.
 

We obtained osmotic pressure data of lysozyme solutions, describing their physical states over a wide
concentration range, using osmotic stress for pressures between 0.05 bar and about 40 bar and volume
fractions between 0.01 and 0.61. The osmotic pressure vs. volume fraction data consist of a dilute, gas-phase
regime, a transition regime with a high-compressibility plateau, and a concentrated regime where the system is
nearly incompressible. The first two regimes are shifted towards a higher protein volume fraction upon
decreasing the strength or the range of electrostatic interactions. We describe this shift and the overall shape of
the experimental data in these two regimes through a model accounting for a steric repulsion, a short-range
van der Waals attraction and a screened electrostatic repulsion. The transition is caused by crystallization, as
shown by small-angle X-ray scattering. We verified that our data points correspond to thermodynamic equilibria,
and thus that they consist of the reference experimental counterpart of a thermodynamic equation of state.

Monte Carlo simulations and coarse-grained modelling have been used to analyze
Histatin 5, which is an unstructured short cationic salivary peptide known to have anticandidical
properties. The calculated scattering functions have been compared with
intensity curves and the distance distribution function P(r) obtained from SAXS, at
both high and low salt concentrations. The aim is to achieve a molecular understanding
and a physico-chemical insight of the obtained SAXS results and to gain information of
conformational changes of Histatin 5 due to altering salt content, charge distribution,
and net charge. From a modelling perspective, the accuracy of the electrostatic interaction
is of special interest. The used coarse-grained model is based on the primitive
model in which charged hard spheres differing in charge and in size represent the ionic
particles, and the solvent only enters the model through its relative permittivity. The
Hamiltonian of the model comprises three different contributions: (i) excluded volumes,
(ii) electrostatic, and (iii) van der Waals interactions. Even though the model can be
considered as gross omitting atomistic details, a great correspondence is obtained with
experimental results.

DOI 10.1002/prot.25025

Liquid jets ultimately break up into droplets through an instability driven by surface tension. For highly viscous liquids, drops are connected by cylindrical liquid filaments whose radii linearly decrease with time, thus forming drops on a string structure. For a jet composed of two aqueous phases made in air by coaxial extrusion, we observe that, for moderate Weber and capillary numbers, drops slow down with different velocities, leading to drop coalescence. The origin of the traffic collision is linked to the spatial feature of the capillary instability where capillary and viscous forces acting on the drops evolve along the jet and ultimately amplify small velocity fluctuations. The emergence of such fluctuations is related to the unstable nature of the annular coflow of liquids having contrasting viscoelastic properties. From a practical point of view, flow and actuation conditions can be adjusted to inhibit drop collision and thus drop coalescence. These findings allow then the fabrication of monodisperse submillimeter core-shell objects based on the fragmentation of compound jets made of polymer solutions that find applications for three-dimensional cell culture.
 

The fabrication of alginate hydrogel microparticles with embedded liposomes and magnetic nanoparticles for radiofrequency controlled release of encapsulated chemical cargo was considered. An extractive gelation process was implemented in a microfluidic device, which enabled the production of uniform composite microparticles of dimensions comparable to those of blood cells (between 5 and 10 mm). The critical parameters that control the extractive gelation process were systematically explored and feasible values that provide microgel particles of a defined size and morphology were identified. First, the initial water-in-oil droplet is formed in a flow-focusing junction whose size is controlled by the flow-rate of the oil phase. Then, the train of droplets is sandwiched between two streams of oil containing calcium ions. In that way, a flux of water molecules from the droplets towards the continuous phase as well as a transport of calcium ions towards the disperse phase are initiated. The final microparticle properties were thus found to be sensitive to three elementary sub-processes: (i) the initial droplet size; (ii) the extraction of water into the oil phase, which was controlled by the volume of the oil phase and its initial moisture content; and (iii) the kinetics of ionic cross-linking of the alginate matrix, which was controlled by the varying calcium concentration. The size and morphology of the final composite microgels were fully characterized.
 

We describe an interfacial rheometry technique based on pairs of micrometer-sized magnetic particles
at a fluid–fluid interface. The particles are repeatedly attracted and repelled by well-controlled magnetic
dipole–dipole forces, so-called interfacial rheometry by intra-pair magnetophoresis (IPM). From the
forces (pN), displacements (µm) and velocities (µm s-1) of the particles we are able to quantify the
interfacial drag coefficient of particles within a few seconds and over very long timescales. The use of
local dipole–dipole forces makes the system insensitive to fluid flow and suited for simultaneously
recording many particles in parallel over a long period of time. We apply IPM to study the timedependent
adsorption of an oil-soluble amino-modified silicone polymer at a water–oil interface using
carboxylated magnetic particles. At low polymer concentration the carboxylated particles remain on the
water side of the water–oil interface, while at high polymer concentrations the particles transit into the
oil phase. Both conditions show a drag coefficient that does not depend on time. However, at intermediate
polymer concentrations data show an increase of the interfacial drag coefficient as a function
of time, with an increase over more than three orders of magnitude (10^-7 to 10^-4 N s m1), pointing to
a strong polymer-polymer interaction at the interface. The time-dependence of the interfacial drag
appears to be highly sensitive to the polymer concentration and to the ionic strength of the aqueous
phase. We foresee that IPM will be a very convenient technique to study fluid–fluid interfaces for a
broad range of materials systems.

DOI: 10.1039/c5sm02917a

A modified version of the Gibbs-ensemble Monte-Carlo method reveals how polydisperse charged colloidal particles can build complex colloidal crystals. It provides general rules that are applicable to this fractionated crystallization that stems from size segregation. It explains the spontaneous formation of complex crystals with very large unit-cells in suspensions of nanoparticles with a broad size distribution.

DOI: 10.1039/C5FD00145E

We report small-angle x-ray scattering experiments on aqueous dispersions of colloidal silica with a broad monomodal size distribution (polydispersity, 14%; size, 8 nm). Over a range of volume fractions, the silica particles segregate to build first one, then two distinct sets of colloidal crystals. These dispersions thus demonstrate fractional crystallization and multiple-phase (bcc, Laves AB2, liquid) coexistence. Their remarkable ability to build complex crystal structures from a polydisperse population originates from the intermediate-range nature of interparticle forces, and it suggests routes for designing self-assembling colloidal crystals from the bottom up.

http://dx.doi.org/10.1103/PhysRevLett.116.208001

Physics (viewpoint), at http://physics.aps.org/articles/v9/57

Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypic analyses are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals, cells were sampled and individually compartmentalized in 100 nL drops. Growth through 15 generations was monitored using a fluorescent protein reporter. Amplification of heritable changes–via growth–over multiple generations yields phenotypically distinct clusters reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during 30 days of starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence of heritable phenotypes. Mutations corresponding to each phenotypic class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods with special utility for microbiology and microbial population biology.

http://dx.doi.org/10.1371/journal.pone.0152395

We present the MilliDrop Analyzer (MDA), a droplet-based millifluidic system for digital antimicrobial susceptibility testing (D-AST), which enables us to determine minimum inhibitory concentrations (MICs) precisely and accurately. The MilliDrop technology was validated by using resazurin for fluorescence readout, for comparison with standard methodology, and for conducting reproducibility studies. In this first assessment, the susceptibility of a reference Gramnegative strain Escherichia coli ATCC 25922 to gentamicin, chloramphenicol, and nalidixic acid were tested by the MDA, VITEK®2, and broth microdilution as a reference standard. We measured the susceptibility of clinically relevant Grampositive strains of Staphylococcus aureus to vancomycin, including vancomycin-intermediate S. aureus (VISA), heterogeneous vancomycin-intermediate S. aureus (hVISA), and vancomycin-susceptible S. aureus (VSSA) strains. The MDA provided results which were much more accurate than those of VITEK®2 and standard broth microdilution. The enhanced accuracy enabled us to reliably discriminate between VSSA and hVISA strains.

doi:10.​1007/​s10096-015-2554-z

This work focuses on the creation of a new biocompatible reactor allowing the encapsulaion and the study of electroactive bacteria. Made by co-extrusion, this millimeter bioreactor is a liquid core capsule with a conducting hydrogel membrane. To create such an object, we formulate a composite hydrogel of alginate/carbon nanotubes in two steps. The first step is rapid and creates the hydrogel matrix by diffusion of divalent ions inside the alginate/carbon nanotubes mix. The second step is slower and permits the dialysis of the surfactant used to stabilize the nanotubes. During this last step, the carbone nanotube network percolates, creating a conducting network in the hydrogel for sufficient nanotube contents (above 0.5 wt%). This composite material has a macroscopic conductivity around 0.1 S.m^-1. An electrochemical study of this material allows to follow the nanotube connection inside the hydrogel. Bacteria can adhere on this composite hydrogel. Then, we demonstrate that the electroactivity of a biofilm developped on the inner side of the conductive capsule shell can be measured. This new biocompatible and electron-conducting compartment opens the way towards the development of a screening tool for the selection of electroactive bacteria but also brings innovative perspectives in the field of microbial fuel cells fabrication.
 

We investigate, through osmotic pressure measurements, the validity of the single-parameter equation of state (EOS) for solutions of polyethylene glycols in water, by Cohen et al.^1,2 We show that it is physically meaningful and that a reasonable good correspondence between the osmotic pressures for PEG35 in large range of concentrations is obtained. We also take the chain length dependence into account in our analysis, as suggested by Cohen et al. By recalculating the experimental pressures in the paper by Jönsson et al.³ applying the new calibration curve, which is based on the experimental results obtained in this study and the EOS obtained by Cohen et al., there is almost a perfect correspondence between the simulations and the experiments. These results have implications for correctly probing macromolecular interactions in wide range of systems when applying the osmotic stress method.

doi:10.1016/j.polymer.2015.10.056

The dissolution and swelling properties of montmorillonite at different pH have been studied, using small angle X-ray scattering (SAXS), imaging and osmotic stress methods combined with Monte Carlo simulations. The acidity of montmorillonite dispersions has been varied as well as the counterions to the net negatively charged platelets. At low pH, Na montmorillonite dissolves and among other species Al3+is released, hydrated, polymerized and then it replaces the counterions in the clay. This dramaticallychanges the microstructure of Na montmorillonite, which instead of having fully exfoliated platelets, turns into a structure of aggregated platelets, so-called tactoids. Montmorillonite dispersion still has a significant extra-lamellar swelling among the tactoids due to the presence of very small nanoplatelets.

Reinforcing precipitated silica systems have a complex hierarchical structure consisting of a branched
network made of connected clusters composed of small silica beads welded together into larger dense
aggregates. Here, we study the evolution of such structural features during a filtration process. The
typical behaviour is that the cakes formed at constant pressure do not reorganize at local scale during
a filtration experiment. Accordingly, the creep resistance of a precipitated silica network is high.
Overall, there is a percolation threshold, which appears when the branches are pushed into each other.
Once this percolation path is reached, the cake withstands compression over more than a decade of
applied pressure. Beyond, it seemed useful to make predictions of the filtration properties knowing the
typical length scales – small silica beads, dense aggregates, and consolidation behaviour of the cake.
A simple approach introducing the concept of an effective medium approximation into Darcy’s law
was tested. This approach treats the network as a pseudo-continuum of porous medium built at two main
length scales: the size of dense aggregates and a length scale representing the typical distance between
the aggregates. The quality of the fit of experimental filtration rates by this simple model indicates that a
description based on a continuous network made of two material phases is accurate.

http://dx.doi.org/10.1016/j.seppur.2015.10.005

We present the principle of a fast magnetic field enhanced colloidal agglutination assay, which is based on the acceleration of the recognition rate between ligands and receptors induced by magnetic forces. By applying a homogeneous magnetic field of 20 mT for only 7 s, we detect CRP (C-reactive protein) in human serum at a concentration as low as 1 pM for a total cycle time of about 1 min in a prototype analyzer. Such a short measurement time does not impair the performances of the assay when compared to longer experiments. The concentration range dynamic is shown to cover 3 orders of magnitude. An analytical model of agglutination is also successfully fitting our data obtained with a short magnetic pulse.

Montmorillonite is a key ingredient in a number of technical applications. However, little is known regarding the microstructure and the forces between silicate platelets. The size of montmorillonite platelets from different natural sources can vary significantly. This has an influence on their swelling behavior in water as well as in salt solutions, particularly when tactoid formation occurs, that is when divalent counterions are present in the system. A tactoid consists of a limited number of platelets aggregated in a parallel arrangement with a constant separation. The tactoid size increases with platelet size and with very small nanoplatelets, ∼30 nm, no tactoids are observed irrespectively of the platelet origin and concentration of divalent ions. The formation and dissociation of tactoids seem to be reversible processes. A large proportion of small nanoplatelets in a mixed-size system affects the tactoid formation, reduces the aggregation number and increases the extra-lamellar swelling in the system.

DOI: 10.1039/C5NR04615G

We present the principle of a fast magnetic field
enhanced colloidal agglutination assay, which is based on the
acceleration of the recognition rate between ligands and
receptors induced by magnetic forces. By applying a
homogeneous magnetic field of 20 mT for only 7 s, we detect
CRP (C-reactive protein) in human serum at a concentration as
low as 1 pM for a total cycle time of about 1 min in a prototype
analyzer. Such a short measurement time does not impair the
performances of the assay when compared to longer experiments.
The concentration range dynamic is shown to cover 3 orders of
magnitude. An analytical model of agglutination is also successfully fitting our data obtained with a short magnetic pulse.

The local functionalities of biocompatible objects can be characterized under conditions similar to their operating ones by scanning electrochemical microscopy (SECM). In the case of alginate beads entrapping carbon nanotubes (CNTs), SECM allows evidencing the local conductivity, the organization, and the communication between CNTs. It shows that the CNTs network is active enough to allow long range charge evacuation, enabling the use of alginate/CNT beads as soft 3D electrodes. Direct connection or local interrogation by microelectrodes allows visualizing their communication as a network and eventually studying them individually at the nanoscale.

Microbiology has continuously pushed efforts towards understanding microbial diversity. Technologies and methods have also evolved, from plating, and use of mi- croscopes and cytometers, towards micro-well handling robots and, finally, fluidic- based devices. The aim of this review was to bring microbiologists attention to the outstanding analytical and handling power of millifluidic droplet technologies for analysing and sorting phenotypic diversity in the microbial world. This new format overcomes many limitations of previous approaches. It provides outstanding repro- ducible growth conditions over droplet reservoirs allowing unprecedented sensitive read-out over thousands of colonies over time. The confinement of the millifluidic train within tubes and the implementation of a three phases format excludes any con- tamination issues. The automation and handling of reservoir droplets is inherently facilitated. We show as a proof of principle the efficiency of capturing phenotypic diversity within a bacterial sample submitted to a sub-minimum inhibitory con- centration of antibiotic. The precision offered by the millifluidic format allows the detection of a variety of resistance strategies that compete and coexist. The review finally explores the potential of this approach to address new challenges such as community-based growth of multiple-strain systems.

To address possible cell-to-cell heterogeneity in growth dynamics of isogenic cell popula- tions of Chlamydomonas reinhardtii, we developed a millifluidic drop-based device that not only allows the analysis of populations grown from single cells over periods of a week, but is also able to sort and collect drops of interest, containing viable and healthy cells, which can be used for further experimentation. In this study, we used isogenic algal cells that were first synchronized in mixotrophic growth conditions. We show that these synchronized cells, when placed in droplets and kept in mixotrophic growth conditions, exhibit mostly homoge- neous growth statistics, but with two distinct subpopulations: a major population with a short doubling-time (fast-growers) and a significant subpopulation of slowly dividing cells (slow- growers). These observations suggest that algal cells from an isogenic population may be present in either of two states, a state of restricted division and a state of active division. When isogenic cells were allowed to propagate for about 1000 generations on solid agar plates, they displayed an increased heterogeneity in their growth dynamics. Although we could still identify the original populations of slow- and fast-growers, drops inoculated with a single progenitor cell now displayed a wider diversity of doubling-times. Moreover, popula- tions dividing with the same growth-rate often reached different cell numbers in stationary phase, suggesting that the progenitor cells differed in the number of cell divisions they could undertake. We discuss possible explanations for these cell-to-cell heterogeneities in growth dynamics, such as mutations, differential aging or stochastic variations in metabolites and macromolecules yielding molecular switches, in the light of single-cell heterogeneities that have been reported among isogenic populations of other eu- and prokaryotes.

Two coarsening mechanisms of emulsions are well established: droplet coalescence (fusion of two droplets) and Ostwald ripening (molecular exchange through the continuous phase). Here a third mechanism is identified, contact ripening, which operates through molecular exchange upon droplets collisions. A contrast manipulated small-angle neutron scattering experiment was performed to isolate contact ripening from coalescence and Ostwald ripening. A kinetic study was conducted, using dynamic light scattering and monodisperse nanoemulsions, to obtain the exchange key parameters. Decreasing the concentration or adding ionic repulsions between droplets hinders contact ripening by decreasing the collision frequency. Using long surfactant chains and well-hydrated heads inhibits contact ripening by hindering fluctuations in the film. Contact ripening can be controlled by these parameters, which is essential for both emulsion formulation and delivery of hydrophobic ingredients.

DOI: 10.1002/anie.201407858

We examine the internal structure of milk casein micelles using the contrast variation method in Small-
Angle Neutron Scattering (SANS). Experiments were performed with casein dispersions of different
origins (i.e., milk powder or fresh milk) and extended to very low q-values (
9  10^-4 °A-1), thus making
it possible to precisely determine the apparent gyration radius Rg at each contrast. From the variation of
I(q / 0) with contrast, we determine the distribution of composition of all the particles in the
dispersions. As expected, most of these particles are micelles, made of casein and calcium phosphate,
with a narrow distribution in compositions. These micelles always coexist with a very small fraction of fat
droplets, with sizes in the range of 20–400 nm. For the dispersions prepared from fresh milk, which
were purified under particularly stringent conditions, the number ratio of fat droplets to casein micelles
is as low as 1 to 106. In that case, we are able to subtract from the total intensity the contribution of the
fat droplets and in this way obtain the contribution of the micelles only. We then analyze the variation of
this contribution with contrast using the approach pioneered by H. B. Stuhrmann. We model the casein
micelle as a core–shell spherical object, in which the local scattering length density is determined by the
ratio of calcium phosphate nanoclusters to proteins. We find that models in which the shell has a lower
concentration of calcium phosphate than the core give a better agreement than models in which the
shell has a higher density than the core.

The aim of this work is to produce submillimetric aqueous-core caspules with a thin alginate hydrogel membrane. These biocompatible capsules can be used for biotechnological appli- cations. They are produced by the break-up of a compound jet with an aqueous core and an alginate shell. We showed that the co-flow inside the injector can exhibit an elastic instability in presence of cations. Growth media contain such cations. Alginate is a polyelectrolyte and cations change its viscoelastic properties. We showed that this instability results in a beating of the compound jet. The instability induces a displacement of the core fluid position which leads to an asymmetric velocity relaxation that creates a torque and finally bends the jet. We produced curved jets with a beveled capillary to evidence this effect. In this case, the fluid viscosity imposes the degree of asymmetry. The jet break-up is controlled by a piezoelectric actuator. We measured a decrease of the wave speed on the jet surface (due to law Weber numbers) and amplified speed dispersion. This dispersion is created by the formation of a beads-on-a-string structure and low distances variations between drops. The addition of sur- factants can create a dynamic surface tension at the jet interface that enhances this dispersion. Finally, we managed to produce submillimetric and monodisperse capsules with a size we can tune, a thin membrane and an encapsulation rate above 99%.

This work, in collaboration with Michelin for the fabrication of composite materials, deals with the colloidal stability of the latex of natural rubber (NR). The NR latex is a polydisperse colloidal dispersion of a bio-polymer in an aqueous serum. The particles are stabilized by a complex layer of phospholipids and proteins. This dispersion can be coagulated by a physical way (under shearing), or by a physical-chemicalway (addition of divalent cations or hydrophobic particles). In this thesis, we studied the coagulation of the NR latex by divalent cations with physical-chemical tools (stability diagrams with different cations, aggregation kinetic of particles, rheological measuremments). Then, the surface of NR particles has been characterized by electrophoresis, firstly in the presence surfactants and secondly depending on the particles size. From the results of the first part we suggest that the coagulation with divalent cations is due to a screening effect because of the increase of ionic strength and that divalent cations can bridge the particles together. This allows keeping them in contact. Adhesion between polymer chains near the surface ensures the irrversible cohesion. In a second time, we saw that the adsorption of surfactants changes the particles surface and the coagulation of NR latex with cations. A small difference of surface charge is observed between the biggest and the smallest NR particles. Finally, a similar behavior has been obtained with a synthetic latex in presence of divalent cations. Our results could be used to develop an industrial process based on this coagulation phenomenon.

The actin cytoskeleton has the unique capability of producing pushing forces at the leading edge of motile cells without the implication of molecular motors. This phenomenon has been ex- tensively studied theoretically, and molecular models, including the widely known Brownian ratchet, have been proposed. However, supporting experimental work is lacking, due in part to hardly accessible molecular length scales. We designed an experiment to directly probe the mechanism of force generation in a setup where a population of actin filaments grows against a load applied by magnetic microparticles. The filaments, arranged in stiff bundles by fascin, are constrained to point toward the applied load. In this protrusion-like geometry, we are able to directly measure the velocity of filament elongation and its dependence on force. Using numerical simulations, we provide evidence that our experimental data are consistent with a Brownian ratchet-based model. We further demonstrate the existence of a force regime far below stalling where the mechanical power transduced by the ratcheting filaments to the load is maximal. The actin machinery in migrating cells may tune the number of filaments at the leading edge to work in this force regime.

Capsules having a thin alginate hydrogel membrane and an aqueous core can be obtained by a process that involves a co-extrusion step in air followed by a sol–gel transition of the shell after immersion into a gelling bath. The possibility to encapsulate cells that further grow in these biocompatible compartments, and thus offer a versatile tool for cell culture, led us to investigate the physicochemical properties of the capsules. A cut-off pore size of the semi-permeable membrane is extrapolated from the release of polymers out of the capsule. When polymers cannot diffuse through the membrane, the osmotic pressure mismatch between the core and the surrounding medium triggers an inflation of the capsule. The swelling may reach a steady state that allows the determination of the elastic features of the hydrogel shell. On the other hand, the capsule membrane may rupture and then contract. From this stress-relaxation process, a critical deformation of the hydrogel shell above which plasticity occurs can be deduced. Finally, thanks to the physical nature of the hydrogel, the core content can be released by dissolving the membrane with the help of small electrolytes. The shell life is shown to vary inversely with the ionic strength of the solution.

Microfluidics has proven to be an efficient tool for making fine and calibrated emul- sion droplets. The parallelization of drop makers is required for producing large amounts. Here, we investigate the generation of emulsion drops along a series of shallow microchannels emerging in a deep one, in other words the step-emulsification process. The dynamics of a single drop maker is first characterized as a function of interfacial tension and viscosities of both phases. The characteristic time scale of drop formation, namely, the necking time that finally sets drop size, is shown to be principally governed by the outer phase viscosity to interfacial tension ratio with a minor correction linked to the viscosity ratio. The step emulsification process experi- ences a transition of fragmentation regime where both the necking time and drop size suddenly raise. This transition, that corresponds to a critical period of drop formation and thus defines a maximum production rate of small droplets, is observed to be ruled by the viscosity ratio of the two phases. When drops are produced along an array of microchannels with a cross flow of the continuous phase, a configuration comparable to a one-dimensional membrane having rectangular pores, a drop boundary layer develops along the drop generators. In the small drop regime, the local dynamics of drop formation is shown to be independent on the emulsion cross flow. Moreover, we note that the development of the drop boundary layer is even beneficial to ho- mogenize drop size along the production line. On the other hand, in the large drop regime, drop collision can trigger fragmentation and thus lead to size polydispersity.

The capacity of microfluidic technology to fabricate monodisperse emulsion droplets is well established. Parallelization of  droplet production is a prerequisite for using such an approach for making high quality materials for either fundamental or industrial applications where product quantity matters.  Here, we investigate the emulsification efficiency  of parallelized drop generators based on a flow focusing geometry when incorporating the role of partial wetting in order to make emulsion droplets with a diameter  below 10 microns. Confinement intrinsically encountered  in microsystems intensifies the role played by interfaces between liquids and solids.  We thus take advantage of partial wetting to enhance the maximum confinement accessible due to liquid flow focusing. We  compare the performances brought by partial wetting to more established routes such as  step emulsification. We show that the step configuration and the partial wetting regime are both well suited for being parallelized and thus open the way to the production of fine and calibrated emulsions for further applications. Finally, this new route of emulsification that exploits partial wetting between the fluids and the channel walls opens possibilities to the formation of substantially smaller droplets, as required in many fields of application.

Colloids are used in many applications such as contrast agents in MRI or magnetic immuno-agglutination in diagnosis. To improve their performance, the interaction between particles and biological media – in particular proteins – has been studied for more than 150 years; nevertheless this phenomenon is still not well described.
The first part of this thesis is dedicated to an application of colloids: detection of target proteins. Based on orientation of anisotropic magnetic aggregates, a new method has been developed, which generates a signal of aggregation, directly proportional to the amount of protein in the sample: lowering the detection limit. This technique has been validated on a real system: C-reactive protein.
The second part is dedicated to protein adsorption on colloids. The first objective was the development of a protocol to obtain reliable measurements; adsorption could then be characterized in a model system: bovine serum albumin and silica. Results obtained were thus able to dismiss certain paradoxes and to propose a new model.
Ultimately the studies reveal how proteins, by adsorbing between two particles, aggregate colloids. In sufficient quantities, they can also protect against aggregation, enabling a new method of particle stabilization.

Bacteria play a major role in our environment. Their ubiquity comes from their amazing ability to adapt to environmental conditions. Understand their adaptability can help to cure in fections or to use bacteria for biotechnology. The diversity of phenotypes within a population - different observable characters - and the diversification mechanisms contribute to the bacterial adaptability. In order to study the adaptation of bacteria, we followed the temporal evolution of diversity within populations of Escherichia coli through environmental transition. We get a picture of the diversity with a phenotyping tool in millifluidic allowing parallel acquisition of one thousand the bacteria growth curves. Through three examples, we show the potential of this approach to read the phenotypes within a population and we explore the roles of diversification and Darwinian selection on the dynamics of adaptation observed. The phase variation of the ag43 gene and the exposure to a non lethal concentration of antibiotic illustrate a first phase of adaptation governed by the competition between the phenotypes with the highest apparition probabilities. Finally we study the adaptation of a population in a depleted environment for thirty days. After fifteen days we observe a convergence towards a single phenotype. This determinism is common to all three examples. However, the end of the experiment is unpredictable: the observation is different for each repetition. It corresponds to a second phase of adaptation which is dominated by the rare appearance of beneficial mutations.

En 10 ans le coût du séquençage d’un génome a été divisé par 10 000. Alors qu’il fallait trois ans pour séquencer le génome humain il ne faut plus que 10 jours avec les séquenceurs de dernière génération. Ces progrès technologiques sur la « lecture » du code génétique ont des répercussions sur de nombreux domaines de la biologie.

Lipid nanocapsules (LNCs) and lipid nanoemulsions (LNEs) are biomimetic synthetic nanocarriers. Their in vitro and in vivo performance was evaluated as a function of their size (25, 50 and 100 nm) and the surface PEG chain length. Analysis methods included complement activation test, particle uptake in macrophage and HEK293(beta 3) cells and biodistribution studies with tumor-grafted mice by fluorescence imaging. A particular attention was paid to keep the concentration of each nanocarrier and to the amount of fluorescent dye in comparable conditions between the in vitro and in vivo studies. Under these conditions, no significant differences were found among the three tested particle sizes and the two nanocarrier types. Longer PEG chains on the LNE surface provided better stealth properties, whereas PEG modification on the LNC formulations inhibited the production of stable nanocarriers. Passive accumulation of LNCs and LNEs in different tumor types depended on the degree of tumor vascularization.

Synopsis Dermo-pharmacy and cosmetic industries have utilized nanotechnologies for two decades. Initially proposed as vector systems for encapsulation of actives, they gained interest in increasing cutaneous bioavailability. Here, we assay the benefits of self-assembled nanoemulsions bearing ligands for targeting specific skin cells. Nanoemulsions, small droplets ranging typically from 20nm to 150nm, possess key properties for further use in cosmetics: long-term stability, optical transparency, extended range of textures and versatility. We investigated this nanoemulsion system and show ability to encapsulate a range of cosmetic actives with various physicochemical properties. Furthermore, this nanoemulsion presents a low cytotoxicity and is capable of directly targeting skin cells through simple addition of specific ligand in a one-step production protocol. This is of interest for increasing bioavailability of actives encapsulated into nanoemulsion droplets which may have penetrated the skin barrier to specific skin cell. Taken together, these chemical and in vitro observations suggest follow-up with in vivo models. Resume L'utilisation des nanotechnologies en cosmetique et en dermo-pharmacie a commencee il y a vingt ans. Initialement simples vecteurs pour l'encapsulation d'actifs, de nombreux systemes nanoparticulaires ont ete developpes afin d'ameliorer la biodisponibilte cutanee des actifs. Dans notre etude nous proposons un systeme de nanoemulsion pour le ciblage specifique de certaines cellules cutanees, dont les gouttelettes presentent des ligands auto-assembles en surface. Les nanoemulsions, dont la taille des gouttelettes varie entre 20 et 150nm, offrent de nombreux avantages dans leur utilisation en cosmetique: une stabilite prolongee, une transparence optique, une texture modifiable et une polyvalence d'emploi. Nous avons effectivement demontre la possibilite d'utiliser ces nanoemulsions afin d'encapsuler des actifs ayant des proprietes physicochimiques differentes, sans affecter la stabilite du systeme. De plus, ces nanoemulsions presentent une cytotoxicite minime et sont capables de cibler specifiquement certaines cellules cutanees par simple ajout de ligands specifiques lors de leur production en une etape. Ces nanoemulsions offrent un interet majeur dans l'amelioration de la biodisponibilite des actifs encapsules en concentrant les gouttelettes ayant traversees la barriere cutanee sur les cellules cibles. Les caracteristiques physico-chimiques ainsi que les observations in vitro realisees dans cette etude encouragent a tester ces nanoemulsions sur modele in vivo.

Deciphering the multifactorial determinants of tumor progression requires standardized high-throughput preparation of 3D in vitro cellular assays. We present a simple microfluidic method based on the encapsulation and growth of cells inside permeable, elastic, hollow microspheres. We show that this approach enables mass production of size-controlled multicellular spheroids. Due to their geometry and elasticity, these microcapsules can uniquely serve as quantitative mechanical sensors to measure the pressure exerted by the expanding spheroid. By monitoring the growth of individual encapsulated spheroids after confluence, we dissect the dynamics of pressure buildup toward a steady-state value, consistent with the concept of homeostatic pressure. In turn, these confining conditions are observed to increase the cellular density and affect the cellular organization of the spheroid. Postconfluent spheroids exhibit a necrotic core cemented by a blend of extracellular material and surrounded by a rim of proliferating hypermotile cells. By performing invasion assays in a collagen matrix, we report that peripheral cells readily escape preconfined spheroids and cell–cell cohesivity is maintained for freely growing spheroids, suggesting that mechanical cues from the surrounding microenvironment may trigger cell invasion from a growing tumor. Overall, our technology offers a unique avenue to produce in vitro cell-based assays useful for developing new anticancer therapies and to investigate the interplay between mechanics and growth in tumor evolution.

This work focuses on the study of physico-chemical properties of millimetric capsules having a liquid core and a thin alginate membrane.
First, we caracterized the diffusion of molecules through the hydrogel shell and estimated the cut-off radius of the semi-permeable membrane. Moreover, the leakage of small hydrophilic solutes can be limited by adding a hydrophobic shell between the aqueous core and the alginate membrane. We demonstrated that the characteristic time of transport is determined by the geometry of the system (hydrophobic shell thickness and capsule diameter)
and by the solubility and diffusion coefficient of the solute in the oil phase. Furthermore, swelling of a capsule under an osmotic pressure difference allowed us to identify the viscoelastic and viscoplastic regimes
of the membrane under stretching. We deduced the elastic modulus of the membrane which decreases above the plastic threshold. However, when capsules are compressed, the hydrogel is concentrated due to the irreversible evacuation of water. This leads to rigidification of its structure.
Then, we focused on the flow of oil-core capsules dispersed in water. There is a friction between capsules that we can control by adding surfactant. Thus we studied its role in the flow of capsules in the constriction of an hourglass. We showed that the flow is constant and does not depend on friction. It is established by the size of the constriction. Indeed, the flow is only determined by the hydrodynamic in fluid regime in
the hourglass' neck.

Dry skin concerns many individuals, conferring discomfort and even impacting the lives of those severely affected. However, in most cases it can be adequately managed with current moisturizing products.¹

Glycerol is one humectant commonly employed due to its high hygroscopic and hyperosmotic properties. It is used in cosmetics as a moisturizer and in pharmaceutical formulations as an active compound.² Being naturally present in the skin, glycerol was quickly identified for its role in skin hydration, similar to natural moisturizing factors (NMF). A byproduct of triglyceride lipolysis, it is transported from the dermis through keratinocytes by a transmembrane water/glycerol transporting protein, aquaporin 3.^{3, 4}

Glycerol also protects skin against irritant dermatitis and accelerates the recovery of irritated skin.^{5, 6} One in vivo study by Flurh et al.⁷ assumes that the stimulus for barrier repair occurs through water flux due to the hygroscopic properties of glycerol. Likewise, most studies about skin barrier recovery are performed with relatively high concentrations of glycerol, e.g., from 5% to 50%, often dispersed in an emulsion.

The question arises as to whether glycerol acts solely through its hygroscopic properties or implies more specific mechanisms. Indeed, diglycerol and triglycerol, with higher humectant activity, have demonstrated low improvements in skin dryness, compared with glycerol.⁸ Also, one in vivo attempt has been made to characterize the effects of low glycerol doses, e.g. from 1% up to 10%, on water transport through the skin after acute chemical irritation by sodium lauryl sulfate (SLS).⁹ The present article therefore summarizes and clarifies the benefit of glycerol on irritated skin.

Lipid nanoparticles, based on nanoemulsion templates, are interesting candidates for contrast agent and active ingredient transport and delivery due to their small size, biodegradable nature and high versatility. An optimized nanosystem for drug delivery should present an important encapsulation ratio, as well as controlled release kinetics. In this context, the influence of the lipid nanoparticle core composition on the encapsulation and release behavior of the Nile Red fluorophore, used as a model molecule of intermediate lipophilicity, is explored. For that purpose, the lipid nanoparticle physical state, the encapsulated molecule localization, and the encapsulation/release behavior are studied. Careful characterization of lipid nanoparticles is performed by DSC and 1H NMR analysis.Nile Red localization is evaluated by complementary fluorescence spectroscopy and 1H NMR techniques. The encapsulation of Nile Red is governed and limited by its solubility in the lipids. A double localization of the fluorophore at the membrane and in the particle core is observed, resulting in a two-phase release kinetics: a quick burst release, followed by a slow prolonged release. Adding wax to the formulation increases the lipid nanoparticle internal viscosity while favoring Nile Red localization in the core, which results in slower release kinetics.

Emulsions have been studied for a long time because of the richness of their related fundamental physicochemical phenomena and owing to their wide industrial applications. The development of microfluidics offers new opportunities to investigate emulsion features and behaviours. This review relates the use of microfluidic tools for probing the interfacial properties of emulsion droplets and the two main mechanisms of destabilisation, namely the coalescence of adjacent drops and the molecular transfer between neighbouring drops.

The incorporation of mineral particles into a polymer matrix leads to extra properties of the polymeric material that are linked to the homogeneity of the mixture. A fine homogeneity can be obtained by using a latex route where the two components are first dispersed in water and then dried. Here, we show a novel way to make an elastomeric composite material via a bulk destabilization of a binary colloidal dispersion. The mixture is composed of a natural rubber latex, which is naturally stabilized, and carbon black particles that are dispersed in water with the help of surfactants. We observe either a transition towards a weak gel, which is a reversible state, or a transition towards an irreversible elastic solid. These structural transitions are mainly controlled by the surfactant concentration that tunes the interaction between natural rubber and carbon black particles, the latter bridging polymer droplets together. We also show that the weak gel can become an irreversible elastic solid under a high shear. This shearinduced heteroaggregation phenomenon exhibits the features of a thermally activated process.

The search for novel organic molecules with biological activity on human, animal, and plant physiological systems has passed through many phases over the centuries. From first steps testing single molecules on whole living systems to fully automated high-throughput screening (HTS) testing tens or hundreds of thousands of molecules per day on purified protein targets, the search has become ever more complex (1, 2). However, the increase in success has not been proportional to the effort and expense entailed. In particular, when considering the screening of “small molecules” (molecular mass <1,000 Da), the results of contemporary HTS have been plagued with problems of false positives, false negatives (3), and the abnormal behavior of certain molecules resulting from their physicochemical properties rather than  their biological activity (4). In PNAS, Miller et al. (5) describe a significant evolution of current HTS technology that increases the confidence in the detection of truly active

Microorganisms are widely used to generate valuable products, and their efficiency is a major industrial focus. Bioreactors are typically composed of billions of cells, and available measurements only reflect the overall performance of the population. However, cells do not equally contribute, and process optimization would therefore benefit from monitoring this intrapopulation diversity. Such monitoring has so far remained difficult because of the inability to probe concentration changes at the single-cell level. Here, we unlock this limitation by taking advantage of the osmotically driven water flux between a droplet containing a living cell toward surrounding empty droplets, within a concentrated inverse emulsion. With proper formulation, excreted products are far more soluble within the continuous hydrophobic phase compared to initial nutrients (carbohydrates and salts). Fast diffusion of products induces an osmotic mismatch, which further relaxes due to slower diffusion of water through hydrophobic interfaces. By measuring droplet volume variations, we can deduce the metabolic activity down to isolated single cells. As a proof of concept, we present the first direct measurement of the maintenance energy of individual yeast cells. This method does not require any added probes and can in principle apply to any osmotically sensitive bioactivity, opening new routes for screening, and sorting large libraries of microorganisms and biomolecules.

Water drops dispersed in chloroform and stabilized with phospholipids become adhesive if a bad solvent for lipids, such as silicone oil, is added to the continuous phase. In this way, two sticking drops are separated by a bilayer of phospholipids. By using microfluidic technologies, we probe the stability and properties of such membranes likewise encountered in foams or vesicles. We first establish the stability diagram of adhering drop pairs as a function of the continuous phase composition. We found two regimes of destabilization of the bilayer. The first one concerns a competition between the dynamics of adhesion and the transport of surfactants toward the interfaces that leads to a dilute surfactant coverage. The second one corresponds to a dense surface coverage where the lifetime distribution of the bilayer exponentially decreases as a signature of a nucleation process. In the stable regime, we observe the propagation of adhesion among a concentrated collection of drops. This is another remarkable illustration of the suction consequence when two close deformable objects are pulled apart. Moreover, the present experimental strategy offers a novel way to study the phase diagrams of bilayers from a single phospholipid to a mixture of phospholipids. Indeed, we detect phase transitions at a liquid−liquid interface that are ruled by the amount of bad solvent. Finally, we probe the transport of water molecules through the bilayer and show that its permeability is linked to the adhesion energy that reflects its fluidity.

Superparamagnetic colloids are used in many studies to apply a force or a torque on biological systems such as cells or DNA molecules. The physical process by which magnetization appears in those beads, allowing them to exert forces, is still poorly understood. In a first part, with an original experimental setup based on optical anisotropy measurements, we show that the apparently magnetically isotropic
beads can orient themselves in a magnetic field. Equilibrium and out of equilibrium measurements allowed ,to develop a model explaining how a few big grains in the beads are responsible for the whole coupling. The robustness of this model lies in its generalization to several sizes of beads and its consistency with the properties of the ferrofluid used to make them. In a second part, the reaction of a few actin filaments to the pressure applied by superparamagnetic colloids is studied. This setup represents a direct way to probe the physical mechanism underlying the production of force by actin filaments polymerization in cellular motility. Controlling the filaments’ organization allows to study two ideal ideal situations. We experimentally show that in the former situation the polymerization velocity is independent of the applied force. This is no longer true in the latter situation where the filaments are verys ti. Thus, we experimentally show for the first time the eect of force on actin filaments polymerization. A Brownian ratchet based numerical model with no load sharing can describe the production of force in that case.

L'utilisation des antibiotiques est un procédé curatif très courant (parfois trop), utilisé contre les infections microbiennes. La désinfection d'agents pathogènes est précédée d'études portant sur l'action des antibiotiques à l'égard des bactéries. Les micro-organismes possèdent naturellement une résistance à un sous dosage en antibiotique. Il existe une concentration minimale en antibiotique, relative à chaque espèce microbienne, à partir de laquelle il est possible d'inhiber les organismes. Cette concentration seuil est appelée la Concentration Minimale Inhibitrice (CMI), c'est un critère essentiel à la détermination de la posologie d'un antibiotique.En ce qui concerne la détermination de la CMI d'un antibiotique sur un organisme, les méthodes de mesures se révèlent fastidieuses à mettre en place, et possèdent une faible résolution. Ces méthodes ont très peu évolué depuis leur création, seules des machineries automatisées ont pu faciliter les manipulations. L'utilisation de ces outils révèle que la CMI d'un antibiotique sur un organisme, dépend de la densité initiale de la population bactérienne. Mais surtout que cette dépendance n'est pas linéaire.

Au cours de ma thèse, nous avons développé un procédé expérimental permettant de mesurer la CMI d'un antibiotique. Le dispositif est fondé sur l'utilisation de la milli-fluidique pour concevoir des gouttes d'émulsion, qui sont les échantillons de mesures gérés de manière automatisée. L'utilisation de cette méthodologie a été réservée à la conception d'un gradient de concentration en antibiotique. Chaque échantillon, d'un train d'un millier de gouttes, contenant une quantité en antibiotique différente de sa voisine.Ce procédé donne la faculté de simplifier l'échantillonnage par rapport aux méthodes usuelles. Il permet de contrôler de manière précise l'environnement direct des cellules, et également d'identifier chaque échantillon. Donnant ainsi la capacité d'observer la dynamique de croissance dans les gouttes, et l'influence des paramètres environnementaux. La machine ainsi utilisée a permis d'affirmer et de compléter les informations de dépendance non linéaire entre la CMI et la densité initiale de cellules pour un couple antibiotique/bactérie.

Nous avons mis au point un système expérimental modèle d'adhésion spécifique entre deux surfaces fluides sur lesquelles diffusent des protéines de type ligand-récepteur. Nous avons notamment voulu relier la mesure d'une énergie d'adhésion macroscopique à son moteur microscopique, la formation de liens spécifiques. Pour mesurer une énergie macroscopique d'adhésion, nous avons utilisé des gouttelettes d'émulsion. Lorsqu'elles adhèrent, les gouttelettes se déforment. Le gain de surface permet d'accéder à l'énergie d'adhésion. Comme moteur de l'adhésion spécifique, nous avons utilisé le couple modèle streptavidine/biotine. Les gouttelettes d'émulsion sont fonctionnalisées avec ces protéines ligantes. Amenées par gravité en présence de bicouches lipidiques supportées fluides dont une fraction des lipides porte les protéines réceptrices, les gouttelettes adhèrent spécifiquement aux bicouches lipidiques. Nous avons mené une étude statique de cette adhésion. Grâce à différentes techniques de fluorescence (épifluorescence, FCS (Fluorescence Correlation Spectroscopy), dosages en retour) nous avons quantifié le nombre de protéines mises en jeu dans nos patches adhésifs afin de le comparer à l'énergie de déformation de la goutte. Nous avons pu observer que l'énergie d'adhésion est proportionnelle à l'énergie d'un lien. Par ailleurs, des mesures en RICM (Reflection Iterference Contrast Microscopy) nous ont permis d'étudier la dynamique de croissance du patch adhésif. Nous avons pu mettre en évidence une croissance diffusive et quasistatique de la zone de contact. En jouant sur les quantités de protéines mises en jeu ainsi que sur leur nature (longueur des liens), nous avons exploré les différentes situations statiques et dynamiques de l'adhésion dans notre système modèle. Nous avons notamment mis en évidence les effets de compressibilité des films adhésifs qui apportent de nouvelles clés pour envisager l'adhésion cellulaire.

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.

Lipid nanoparticles (LNP) have been designed based on low cost and human-use approved excipients, and manufactured by an easy, robust, and up-scalable process. Fluid colloidal dispersions or gel viscous formulations of highly stable nanoparticles (more than 12 month stability is achieved for some formulations) can be obtained. Their physicochemical properties are studied by Dynamic Light Scattering, Differential Scanning Calorimetry, and NMR. The results picture nanoparticles with a non-crystalline core, which viscosity can be finely tuned by the lipid composition and the temperature. A design of experiments has been used to investigate the limits of the system colloidal stability. The impact of core and surfactant weight fractions have been explored both experimentally and using the design of experiments. The versatility of this physicochemical system could open the way to a wide range of future pharmaceutical applications.

Liquid pearls designate liquid core capsules having a thin elastic shell.

Water-in-oil emulsion drops are formed and stabilized with phospholipids which can adhere and form a bilayer. Using microfluidics, adhesive drop pairs are then trapped and submitted to an ac electric field. We observe three distinct states as a function of the adhesion energy and the electric field intensity. The pair can be either stable, though slightly deformed, or unzip and separate, or coalesce. The frontiers between the different states directly reflect vesicle detachment forces and electroporation theories. The experimental approach that we propose for probing liquid interface wetting between monolayers allows us to finely tuned the tension in the bilayer and gives access to bilayer unzipping.

We introduce an approach for forming bilayer polymer membranes by adhesion of amphiphile-laden interfaces. This adhesion is induced by a reduction of solvent quality for the amphiphilic diblock copolymers through selective evaporation of good solvent in the solvent mixture. By combining this membrane formation mechanism with a double-emulsion-templated approach for vesicle formation, we fabricate monodisperse polymersomes that exhibit excellent membrane uniformity, and structural stability, using a method that has high encapsulation efficiency. Moreover, we also show that the technique is versatile and can be applied to different block copolymers. The ability to direct the assembly of amphiphiles into a membrane creates new opportunities to engineer the structures of vesicles on the level  of the individual bilayer leaflets.

The phase inversion that undergoes an emulsion while being sheared is a sudden phenomenon that is still puzzling. In this Letter, we report an experimental investigation on propagative coalescence by using a microfluidic device where a calibrated two-dimensional emulsion is created and destabilized. The velocity of propagation and the probability of the coalescence are reported as a function of the size and the spatial distribution of the drops, respectively. We then discuss the efficiency of this novel scenario of phase inversion and suggest that inversion can be favored by the existence of a drop size distribution.

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.

Practical and theoretical considerations that apply when aiming to formulate by ultrasonication very small nanoemulsions (particle diameter up to 150 nm) with very high stability are presented and discussed. The droplet size evolution during sonication can be described by a monoexponential function of the sonication time, the characteristic time scale depending essentially on the applied power. A unique master curve is obtained when plotting the mean diameter size evolution as a function of sonication energy. We then show that Ostwald ripening remains the main destabilization mechanism whereas coalescence can be easily prevented due to the nanometric size of droplets. The incorporation of “trapped species” within the droplet interior is able to counteract Ostwald ripening, and this concept can be extended to the membrane compartment. We finally clarify that nanoemulsions are not thermodynamically stable systems, even in the case where their composition lies very close to the demixing line of a thermodynamically stable microemulsion domain. However, as exemplified in the present work, nanoemulsion systems can present very long-term kinetic stability.

Les travaux accomplis au cours de cette thèse au Laboratoire de Colloïdes et Matériaux Divisés résultent d'une volonté de développer et d'utiliser des outils physico-chimiques pour la compréhension de problématiques biologiques. Nous avons choisi comme objet d'étude ce champignon unicellulaire extraordinaire qu'est la levure Saccharomyces cerevisiae. En particulier nous nous sommes intéressés à la fermentation alcoolique à travers la mesure d'un des paramètres du métabolisme : l'énergie de maintenance. Les résultats obtenus s'articulent autour de deux réalisations principales. *** Développement d'un outil de mesure original : Nous avons mis au point un système physico-chimique qui permet de mesurer la polymérisation ou la dépolymérisation au sein de microréservoirs de quelques dizaines de picolitres . Le principe repose sur l'osmose, c'est-à-dire la di usion, à travers une membrane semi-perméable, de solvant entre des réservoirs qui possèdent des potentiels chimiques di érents. Pour réaliser ce système nous avons fabriqué un réseau de gouttes d'émulsion inverse qui constituent des microréservoirs dans lesquels nous avons encapsulé les levures. Les gouttes ont été organisées en un réseau bi-dimensionnel qui nous a permis de suivre des cinétiques de croissance sur un grand nombre d'échantillons (haut débit). Nous avons alors élaboré une nouvelle formulation de l'émulsion afin de rendre les gouttes adhésives entre elles. Ceci nous a permis d'imposer les dimensions et les propriétés de transport à l'intérieur des membranes semi-perméables ainsi formées entre les gouttes en contact. Nous avons alors démontré qu'en imposant des potentiels chimiques constants dans le réseau de gouttes, nous pouvions suivre, à travers l'évolution de leur volumes, la cinétique de processus de polymérisation (création de biomasse dans le cas de la levure) et de dépolymérisation (dans le cas de la lyse enzymatique) qui ont lieu dans des réservoirs. *** Mesure directe de l'énergie de maintenance : Nous avons utilisé notre outil pour mesurer la consommation du glucose par S. cerevisiae lors de la fermentation alcoolique en milieu anaérobie. Nous avons ainsi distingué deux comportements suivant que les cellules se divisent ou qu'elles sont en phase stationnaire. Nous avons développé un modèle qui nous a permis d'expliquer les comportements observés en fonction des paramètres expérimentaux (concentration en sucre, transport) et du métabolisme de la levure. Nous nous sommes alors rendu compte qu'en imposant le réactif limitant nous pouvions mesurer l'énergie de maintenance, c'est-à-dire l'énergie qui n'est pas utilisée pour la division cellulaire. Nous avons enfin mesuré le taux de consommation de glucose pour trois souches différentes. Nous avons ainsi montrer qu'il était possible d'identifier des souches selon leur capacité à métaboliser le glucose. Nous nous sommes alors interrogés sur la possibilité de cribler à haut débit des souches capables de métaboliser des sucres différents. Aussi, nous pouvons envisager, pour augmenter la taille de la librairie à trier, une approche 3D. Les gouttes sont collectées dans une seringue dans laquelle le mûrissement entre les gouttes se produit. Elles sont alors réinjectées dans un microsystème contenant une centaine de zones de tri. Avec un débit d'injection des gouttes de 10kHz, sachant qu'une goutte sur 10 contient une levure, on peut alors cribler une librairie de 108 individus différents en moins de 30 minutes. *** Au total : Le principe de mûrissement des gouttes développé dans cette thèse peut s'appliquer à tout processus qui se traduit par une modification du nombre d'espèces osmotiquement actives. On peut donc envisager de l'utiliser pour un champ vaste d'applications telle que la recherche de nouvelles enzymes de protéolyse. Enfin, nous aimerions attirer l'attention du lecteur sur l'aspect statistique de cette méthode. Elle offre la possibilité d'étudier des processus de sécrétion ou d'assimilation de plusieurs milliers d'individus isolés. Ainsi, des questions telles que la variabilité entre individus vont pouvoir être abordées.

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Les tests de diagnostic ont pour but de détecter et de quantifier un ou plusieurs analytes en milieu complexe. Depuis les années 80, leur évolution s'est concentrée sur la détection de très faibles quantités d'analytes. Actuellement, cette ligne directrice subsiste mais en parallèle les développements se portent sur de nouveaux objectifs comme la mise au point de tests simples, rapides, transportables et sensibles afin de répondre au mieux aux attentes des nouveaux marchés : le point of care et les pays émergents. Au cours de ce travail, nous nous sommes confrontés à ces nouvelles problématiques. Nous avons choisi d'améliorer la sensibilité du test d'agglutination magnétique précédemment développé au laboratoire en raison de sa simplicité (test homogène) et de sa rapidité (manipulation de billes magnétiques). Nous avons montré que deux paramètres conditionnaient le seuil de détection de ce test à savoir le bruit (variabilité du signal sans antigène) et le signal émis par les billes. Le premier facteur dépend principalement du nombre de billes et le deuxième nous a amené à travailler avec de grandes billes nécessitant une nouvelle méthode de détection, la cytométrie en flux. Le gain en sensibilité n'a pas été clairement démontré en raison de la très faible réactivité des billes utilisées (500 fois moins réactives que les billes "habituelles"). Une chose est sûre, de grandes billes améliorent le signal, la cinétique et la thermodynamique au détriment d'une plus faible mobilité des objets sous champ. Sous couvert d'une bonne réactivité, nous nous sommes aperçus que la première étape de notre test d'agglutination devenait thermodynamiquement et cinétiquement limitante aux faibles concentrations en analyte. Dans le cadre du projet européen DetectHIV, nous avons essayé d'une part de franchir ces barrières à travers un système microfluidique et d'autre part de mettre au point un test complètement intégré. De nouvelles technologies ont vu le jour à l'EPFL (plug magnétique) et à DTU (cytométrie sur puce). Le système développé par l'EPFL est thermodynamiquement favorable, valide l'idée de préconcentration mais nécessiterait un nouveau dimensionnement fluidique pour réellement supplanter les tests en volume. L'intégration, quant à elle, a permis d'acquérir un certain savoir faire dans le domaine de la microfluidique, d'identifier les verrous technologiques et de développer un nouveau mode de circulation des fluides. Pour conclure, ce travail a permis de cerner l'évolution possible des tests d'agglutination magnétique. La sensibilité du test originel c'est-à-dire en volume couplé à une détection turbidimétrique est fixée par le bruit de l'appareil de mesure. Ce paramètre étant peu optimisable, seule une augmentation de la taille des billes permettrait d'augmenter significativement la sensibilité du test (un facteur 10 en théorie). Au delà, une détection individuelle des objets via la cytométrie est nécessaire. D'une part, elle permet de travailler avec de gros objets afin d'amplifier grandement le signal et d'autre part, elle permet de s'affranchir du bruit de l'appareil de mesure. Le bruit mesuré est uniquement dû à la comptabilisation des populations, bruit inhérent au test de diagnostic. Avec cette configuration, un gain de sensibilité passe par une diminution du non-spécifique (relatif au comptage des populations) et par une augmentation de la réactivité des "grosses" billes. En dessous de la dizaine de femtomoles, la formation de complexe n'est plus assurée et devient longue, un changement de format fluidique est alors nécessaire. Le nouveau format fluidique testé, la microfluidique, s'avère très prometteur à condition d'intégrer une méthode de détection (cytométrique ou autre) et d'augmenter les débits.

The invention describes a method for isolating one or more genetic elements encoding a gene product having a desired activity, comprising the steps of: (a) compartmentalising genetic elements into microcapsules; and (b) sorting the genetic elements which express the gene product having the desired activity; wherein at least one step is under microfluidic control. The invention enables the in vitro evolution of nucleic acids and proteins by repeated mutagenesis and iterative applications of the method of the invention.

Gratiam enim amicos si simulatione dixisse tum quos quos se tum ut habuisset dumtaxat fit se plerumque posset Coluntur ut.

Lipid nanoparticles (LNP) coated by a poly(oxyethylene) polymer have been manufactured from low cost and human use-approved materials, by an easy, robust, and up-scalable process. The incorporation in the formulation of maleimide-grafted surfactants allows the functionalization of the lipid cargos by targeting ligands such as the cRGD peptide binding to avb3 integrin, a well-known angiogenesis biomarker. LNP are able to encapsulate efficiently lipophilic molecules such as a fluorescent dye, allowing their in vivo track- ing using fluorescence imaging. In vitro study on HEK293(b3) cells over-expressing the avb3 integrins demonstrates the functionalization, specific targeting, and internalization of cRGD-functionalized LNP in comparison with LNP-cRAD or LNP-OH used as negative controls. Following their intravenous injection in Nude mice, LNP-cRGD can accumulate actively in slow-growing HEK293(b3) cancer xenografts, leading to tumor over skin fluorescence ratio of 1.53 ± 0.07 (n = 3) 24 h after injection. In another fast-growing tumor model (TS/A-pc), tumor over skin fluorescence ratio is improved (2.60 ± 0.48, n = 3), but specificity between the different LNP functionalizations is no more observed. The different results obtained for the two tumor models are discussed in terms of active cRGD targeting and/or passive nanoparticle accumu- lation due to the Enhanced Permeability and Retention effect.

Lipid nanocapsules (LNCs) were modified by adsorbing sequentially dextran sulfate (DS) and chitosan (CS) on their surface by the layer-by-layer (LBL) approach. Tangential flow filtration (TFF) was used in intermediate purifications of the LNC dispersion during the LBL process. The surface modification was based on electrostatic interactions between the coating polyelectrolytes (PEs) and the LNCs. Therefore, a cationic surfactant, lipochitosan (LC), was synthesised by coupling stearic anhydride on chitosan, and the surface of LNCs was first modified by this LC by the post-insertion technique. The PEs could be successfully adsorbed on the LNC surface as verified by alternating zeta potential and increase in size. To present a therapeutic application, fondaparinux sodium (FP), a heparin-like synthetic pentasaccharide, was introduced on the LNC surface instead of DS.

We report an experimental investigation on the formation of liquid-core capsules having a thin hydrogel elastic membrane, named ‘liquid pearls’. These fish-egg-like structures are initially made of a millimetric liquid drop, aqueous or not, coated with an aqueous liquid film containing sodium alginate that gels once the double drop enters a calcium chloride bath. The creation of such pearls with a micrometer-thick membrane requires the suppression of mixing until gelling takes place. Here, we show that superimposing a two-dimensional surfactant precipitation at the interface confers a transient rigidity that can damp the shear-induced instability at impact. Based on this, pearls containing almost any type of liquid can be created. This opens the possibility to use such structures as a new tool for screening microorganism survival and growth in various three-dimensional environments.

Grâce à la microfluidique, la stabilité et les propriétés de deux types d'émulsions ont pu être sondées. Il s'agit d' émulsions non adhésives et d'émulsions adhésives. Les points importants des différentes thématiques abordées sont résumés dans les paragraphes suivants. – La déstabilisation hydrodynamique d'une émulsion : La fusion de gouttes proches est favorisée par leur séparation. En réalité les gouttes sont localement rapprochées car une dépression se crée lors de leur éloignement. Cela les oblige à former localement des bourgeons qui les rendent plus proches. – La déstabilisation d'une émulsion par un champ électrique : L'état de coalescence temporaire a été mis en évidence. Un diagramme de coalescence de paires a été établi. Pour des trains de gouttes, le diagramme de stabilité coïncide avec celui de paires. La seule différence est observée pour des temps d'excitation plus courts que le temps de relaxation des charges. Dans ce cas, la "coalescence partielle" est remplacée par une simple coalescence. – La caractérisation d'émulsions inverses adhésives : La technique pour former une émulsion inverse très adhésive, c'est de la former très peu adhésive et d'évaporer le bon solvant. Les bicouches artificielles formées avec ces émulsions miment presque toutes les propriétés des membranes biologique. Suivant la quantité de mauvais solvant, elles peuvent se présenter sous forme gaz, fluide ou gel. – La perméabilité des bicouches artificielles : Avec un choc osmotique, la perméabilité à l'eau de ces bicouches a été mis en évidence. Elles sont faiblement perméables aux ions. Avec un champ électrique, leur perméabilité aux ions est possible grâce à la formation de pores à travers elles. Un diagramme d'électroporation s'en est suivi. Pour les deux moteurs de perméabilité, le constat principal est la variation notable de la perméabilité instantanée lors d'une transition de phase dans les bicouches. Ces résultats sont très intéressants et montrent à quel point les émulsions en générale regorge encore de secrets. Pour les émulsions adhésives, les comportements que nous avons observés sur les trains de gouttes sont déjà très surprenants. Nous pouvons nous attendre à des comportements encore plus surprenants dans le cas d'émulsions macroscopiques. En outre, ces effets sont généralement "contre-intuitifs" : la déstabilisation d'une émulsion dense est facilitée par sa décompression ! la séparation des phases huileuses et aqueuses d'une émulsion de pétrole est optimale pour une application d'un champ électrique pas très élevé (un champ électrique élevé entraîne la "coalescence partielle" des gouttes)... Tous ces comportements "contre intuitifs" peuvent être très utiles pour des applications futures dans le domaine des matériaux. En ce qui concerne les émulsions adhésives, les résultats établis ici sont très prometteurs. Elles permettent de mimer les membranes biologiques. Grâce à la perméabilité des membranes artificielles formées, nous pouvons imaginer des gouttes, très concentrées en ions, qui seraient des pompes à solvant : elle permettraient, par exemple, de changer des concentrations de microréacteurs... En ce qui concerne l'électroporation, les expériences peuvent encore être peaufinées. La prochaine étape sera certainement de contrôler directement le potentiel des gouttes, par des pointes par exemple. Cela éviterait, en effet, leur séparation. Dans ce cas, il est clair que le mécanisme d'électroporation pourrait avoir une réelle valeur ajoutée car les réservoirs seront figés et des échanges pourraient se faire. On pourrait espérer ainsi réaliser des expériences d'insertion de molécules dans des gouttes, par électroporation, comme cela existe actuellement pour des vésicules.

Fluorescence is a very promising radioactive-free technique for functional imaging in small animals and, in the future, in humans. However, most commercial near-infrared dyes display poor optical properties, such as low fluorescence quantum yields and short fluorescence lifetimes. In this paper, we explore whether the encapsulation of infrared cyanine dyes within the core of lipid nanoparticles (LNPs) could improve their optical properties. Lipophilic dialkylcarbocyanines DiD and DiR are loaded very efficiently in 30-35-nm-diam lipid droplets stabilized in water by surfactants. No significant fluorescence autoquenching is observed up to 53 dyes per particle. Encapsulated in LNP, which are stable for more than one year at room temperature in HBS buffer (HEPES 0.02 M, EDTA 0.01 M, pH 5.5), DiD and DiR display far improved fluorescence quantum yields Phi (respectively, 0.38 and 0.25) and longer fluorescence lifetimes tau (respectively, 1.8 and 1.1 ns) in comparison to their hydrophilic counterparts Cy5 (Phi = 0.28, tau = 1.0 ns) and Cy7 (Phi = 0.13, tau = 0.57 ns). Moreover, dye-loaded LNPs are able to accumulate passively in various subcutaneous tumors in mice, thanks to the enhanced permeability and retention effect. These new fluorescent nanoparticles therefore appear as very promising labels for in vivo fluorescence imaging.

We study the thermal expansion of chains formed by self-assembly of magnetic colloidal particles in a magnetic field. Using video microscopy, complete positional data of all the particles of the chains is obtained. By changing the ionic strength of the solution and the applied magnetic field, the interaction potential can be tuned. We analyze the thermal expansion of the chain using a simple model of a one-dimensional anharmonic crystal of finite size.

Biotechnological applications of emulsions, such as micro-reactors or drug carriers, demand accurate characterization techniques, able to measure the size and biochemical content of the droplets at the individual level. Since no available characterization technique completely fulfills these needs, we extended the use of flow cytometry, which was originally developed for cell studies, to the straightforward and quantitative characterization of micron-sized emulsions. Our method determines the size of soybean oil droplets from flow cytometric measurements of forward scattering and side scattering intensities combined with the theoretical scattered intensities exactly derived from Mie theory and numerically integrated with respect to the optical setup of the instrument. We evaluate the accuracy of our method by comparing the size distribution obtained for a monodisperse emulsion sample to the corresponding distribution measured with a commercial instrument. Applied to emulsion droplets functionalized with fluorescent streptavidin, our method allows for monitoring of the rate of grafted molecules on interfaces with a precision never obtained before.

By using microfluidic chips, we investigate the stability regarding coalescence of droplet pairs under an electric field as a function of drop separation and ac field intensity. Three different regimes are found: stable, coalescence, and partial merging. From this, we identify the two breaking scenarios of a one dimensional train of droplets: in one case the coalescence front propagates; in the other case, in which pairs belong to the partial merging regime, the coalescence front can become heterogeneous. From these findings, we can propose a destruction mechanism for a macroscopic emulsion, which includes the packing condition for which total and immediate destruction is effective.

In 1994 Leal Calderon et al. (Phys. Rev. Lett. 72, 2959 (1994)) introduced the magnetic chaining technique to directly probe the force-distance profile between colloidal particles. In this paper, we revisit this approach in two ways. First, we describe a new experimental design which allows us to utilize sample volumes as low as a few microliters, involving femtomoles of surface active macromolecules. Secondly, we extensively describe the characterization and preparation of the magnetic colloids, and we give a quantitative evaluation of performance and resolution of the technique in terms of force and interparticle separation.

We have produced emulsion droplets of controlled size and composition coated by ligands, and studied the adhesion of these drops on a solid substrate coated by receptors and polymers. Using transmission, RICM and fluorescence microscopy we assess the size, contact angle and ligand density for each drop. We first show that non-specific interactions significantly enhance the proteins density within the adhesive patch. Then we show that binding within the patch is partially inhibited in good agreement with the hypothesis of an absence of translational diffusion. We confirm that the density of specific bonds sets the adhesive energy and therefore the final contact angle, and finally show that specific binding in our system is always associated with the existence of a positive line tension, which linearly increases with the density of receptors. These experiments describe a new scenario for specific wetting which raises the importance of the coupling between non-specific interactions and specific binding.

Chirality is an important element of biology, chemistry and physics. Once symmetry is broken and a handedness is established, biochemical pathways are set. In DNA, the double helix arises from the existence of two competing length scales, one set by the distance between monomers in the sugar backbone, and the other set by the stacking of the base pairs1. Here we use a colloidal system to explore a simple forcing route to chiral structures. To do so we have designed magnetic colloids that, depending on both their shape and induced magnetization, self-assemble with controlled helicity. We model the two length scales with asymmetric colloidal dumbbells linked by a magnetic belt at their waist. In the presence of a magnetic field the belts assemble into a chain and the steric constraints imposed by the asymmetric spheres force the chain to coil. We show that if the size ratio between the spheres is large enough, a single helicity is adopted, right or left. The realization of chiral colloidal clusters opens up a new link between colloidal science and chemistry. These colloidal clusters may also find use as mesopolymers, as optical and light-activated structures2, and as models for enantiomeric separation.

We discuss the theory of ligand receptor reactions between two freely rotating colloids in close proximity to one other. Such reactions, limited by rotational diffusion, arise in magnetic bead suspensions where the beads are driven into close contact by an applied magnetic field as they align in chainlike structures. By a combination of reaction-diffusion theory, numerical simulations, and heuristic arguments, we compute the time required for a reaction to occur in a number of experimentally relevant situations. We find in all cases that the time required for a reaction to occur is larger than the characteristic rotation time of the diffusion motion tau_rot. When the colloids carry one ligand only and a number n of receptors, we find that the reaction time is, in units of tau_rot, a function simply of n and of the relative surface alpha occupied by one reaction patch alpha = pi r_C²/(4pi r²), where r_C is the ligand receptor capture radius and r is the radius of the colloid.

Silicon biomineralization is a widespread mechanism found in several kingdoms that concerns both unicellular and multicellular organisms. As a result of genomic and molecular tools, diatoms have emerged as a good model for biomineralization studies and have provided most of the current knowledge on this process. However, the number of techniques available to study its dynamics at the cellular level is still rather limited. Here, new probes were developed specifically to label the pre-existing or the newly synthesized silica frustule of several diatoms species. It is shown that the LysoTracker Yellow HCK-123, which can be used to visualize silica frustules with common filter sets, presents an enhanced signal-to-noise ratio and allows details of the frustules to be imaged without of the use of ionophores. It is also demonstrated that methoxysilane derivatives can be coupled to fluorescein-5-isothiocyanate (FITC) to preferentially label the silica components of living cells. The coupling of labeling procedures might help to address the challenging question of the process of frustule exocytosis.

The destabilization process of an emulsion under flow is investigated in a microfluidic device. The experimental approach enables us to generate a periodic train of droplet pairs, and thus to isolate and analyze the basic step of the destabilization, namely, the coalescence of two droplets which collide. We demonstrate a counterintuitive phenomenon: coalescence occurs during the separation phase and not during the impact. Separation induces the formation of two facing nipples in the contact area that hastens the connection of the interfaces prior to fusion. Moreover, droplet pairs initially stabilized by surfactants can be destabilized by forcing the separation. Finally, we note that the fusion mechanism is responsible for a cascade of coalescence events in a compact system of droplets where the separation is driven by surface tension.

Recent advances in micro-machining allow very small cargos, such as single red blood cells, to be moved by outfitting them with tails made of micrometre-sized paramagnetic particles yoked together by polymer bridges. When a time-varying magnetic field is applied to such a filament, it bends from side to side and propels itself through the fluid, dragging the load behind it. Here, experimental data and a mathematical model are presented showing the dependence of the swimming speed and direction of the magnetic micro-swimmer upon tunable parameters, such as the field strength and frequency and the filament length. The propulsion of the filament arises from the propagation of bending waves between free and tethered ends: here we show that this gives the microswimmer a gait that is intermediate between a eukaryotic cell and a waggled elastic rod. Finally, we extract from the model design principles for constructing the fastest swimming micro-swimmer.

We introduce a general methodology based on magnetic colloids to study the recognition kinetics of tethered biomolecules. Access to the full kinetics of the reaction is provided by an explicit measure of the time evolution of the reactant densities. Binding between a single ligand and its complementary receptor is here limited by the colloidal rotational diffusion. It occurs within a binding distance that can be extracted by a reaction-diffusion theory that properly accounts for the rotational Brownian dynamics. Our reaction geometry allows us to probe a large diversity of bioadhesive molecules and tethers, thus providing a quantitative guidance for designing more efficient reactive biomimetic surfaces, as required for diagnostic, therapeutic, and tissue engineering techniques.

Several biological mechanisms use polymerization of actin filaments as mechanical motor. The chemical energy released with the addition of a monomer in the filament is converted into mechanical work thereby generating a force. Filaments are regulated by actin binding proteins, and form structures that possess different mechanical and elastical properties, and also different functions in the biological processes. Our experimental setup permits to study the link between the mechanical properties and the mechanisms that produce a force. The polymerization of actin filaments is directly initiated on the surface of magnetic particles. Within a magnetic field, particles organize themselves in a chain due to dipole-dipole interactions, and a compressive force is induced on the polymerizing filaments. Polymerization pushes the particles apart over time and as a function of the applied force, the velocity of the particles gap slows down. While following the evolution of the distance between particles, we obtain information on the force-velocity profile and the mechanical properties of the filaments.

Nanomedecine represents the application field of nanotechnology, and more especially
nanoparticles, to medicine. This new domain is promising in particular for the detection and
treatment of infectious, genetic and cancer pathologies. We have developed new
biocompatible nanoparticles, and evaluated their potential as nanocarriers for therapeutic or
diagnostic agents thanks to in vivo fluorescence imaging. These particles are oil in water
nanoemulsions, which diameter is less than an hundred nanometers, stabilized by a mixture of
phospholipids and stealth agents. The encapsulation of organic dyes with absorbing and
emitting wavelengths suitable for in vivo fluorescence imaging, allows the follow up of the
nanoemulsions after their injection in mice models. Fluorescent stealth nanoemulsions can
also be functionalized in order to covalently link onto their surface targeting ligands, such as
the cRGD peptide. Indeed, cRGD functionalized nanoemulsions exhibit in vitro specific
adhesion with cells expressing αvβ3 integrine, an angiogenic biomarker. The particle uptake,
either passive and/or cRGD-mediated, in different tumor models bear by Nude mice, is
observed using non invasive fluorescence imaging.

Le travail de recherche exposé dans cette thèse, montre dans la premier partie la mise en oeuvre de la méthode d'émulsification pour la préparation de petits agrégats de forme anisotrope monodisperse et de forme géométrique bien définie et on a montré que la maîtrise des paramètres de la formulation permet la sélection et la production d'un maximum d'agrégats de formes données (dimère, trimère, tétramère). On a montré la faisabilité et la possibilité d'assembler tête-tête les différents types d'agrégats de façon organisée et directionnelle, grâce à la fonctionnalisation des têtes des agrégats, avec des fonctions réactives utilisant la force d'adhésion spécifique (couple biotine/streptavidine). En deuxième partie, on a fait intervenir des forces magnétiques pour forcer et accélérer le processus d'assemblage par rapport à des forces spécifiques qui sont basées sur les effets de diffusion ; on a montré la formation de chaînes colloïdales de structure variable avec la mise en évidence des effets de relaxation sur les chaînes, rotation et torsion, qui dépendent de l'intensité de champ appliqué et de la morphologie des particules (Janus avec une partie magnétique, doublets avec un anneau). Enfin, nous avons conçu des doublets asymétriques avec des anneaux magnétiques qui engendrent la formation de chaînes chirales sous forme d'hélice. Et on a mis en évidence l'analogie entre les colloïdes et les macromolécules sur le plan structural par un simple auto-assemblage.

e domaine de l’évolution dirigée regroupe des approches technologiques s’inspirant, en laboratoire et à l’échelle moléculaire, de l’évolution Darwinienne. Grâce à des algorithmes moléculaires stochastiques, on peut ainsi améliorer des protéines existantes ou même créer de nouvelles protéines. Dans une première partie, les différentes façons de générer de la diversité génétique sont introduites, ainsi que notre contribution au développement d’une technologie de mutagenèse dirigée très haut débit (MM). En particulier, deux variants deMMsont présentés :MMC, une méthode utilisant de très larges banques d’oligonucléotides synthétisés en parallèle sur des puces à ADN et MMS, une méthode de recombinaison in vitro circulaire simple et efficace. Dans une deuxième partie, après avoir rappellé les différentes modalités de tri des protéines améliorées, on présente notre contribution au domaine naissant et prometteur du criblage haut-débit en émulsion d’activité catalytique . On décrit les premières étapes de la validation d’une méthode de criblage bidimensionnel d’une émulsion inverse cristallisable. Appliquée à l’évolution moléculaire, cette nouvelle technologie pourrait permettre d’augmenter de plusieurs ordres de grandeur le débit des meilleures techniques actuelles par cytométrie de flux. Sont également évoquées, dans une troisième partie, d’autres applications de cette technique de criblage à plat d’émulsions cristallisées dans le domaine du diagnostic et pour l’analyse du cycle cellulaire

We describe a six-step method for making colloidal clusters of 2, 3, or 4 silica particles with a radius of 1.2 um. This method, originally described by Manoharan et al. (Manoharan, V. N.; Elsesser, M. T.; Pine, D. J. Science 2003, 301, 483), is based on the encapsulation of silica spheres in emulsion droplets. The originality of our work lies in the preparation of monodisperse emulsions, which allows us to obtain some high yields of small aggregates over a wide range of conditions. Using optical microscopy and disk centrifugation, we show that the relative fractions of 2, 3, and 4 particle aggregates are controlled by the emulsification conditions, particularly the concentration of silica in the dispersed phase. Our best yields are obtained using low to moderate shear rates, a highly viscous continuous phase, and intermediate amounts of silica. The sedimentation of the colloidal solution into a gradient of concentration leads to aqueous suspensions of identical clusters. Since the overall process can easily be scaled up, large quantities of identical clusters may be prepared, which should allow the thermodynamic properties of these new colloidal objects to be measured for the first time. These nonspherical particles could serve as building blocks for more complex assemblies, such as colloidal crystals which could find applications as photonic materials.

We report on frequency and field-dependent complexsusceptibility, , measurements of a magnetic colloidal system consisting of 200 nm spherical beads, containingmaghemite (γFe₂O₃) nanoparticles. The relaxation properties of both the magnetic colloid and a free suspension of the γFe₂O₃ particles, are investigated over the frequency range 200 Hz–1 MHz. Under a polarizing field H, an absorption peak is detected in the component at frequencies f_max between 1.1 and 1.7 kHz. We show that this absorption peak can be attributed to the Néel relaxation of the inner maghemitenanoparticles. It is also shown that the general trend for the value of f_max and the amplitude of both and is to increase with increasing H. Furthermore, the relation between and and their dependence on frequency, ω/2π, is investigated by means of the magnetic analogue of the Cole–Cole plot and a measure of the Cole–Cole distribution parameter α_s is determined.

We have studied the effect of shear on the stability of suspensions made of non-Brownian solid particles. We demonstrate the existence of an irreversible transition where the solid particles aggregate at remarkably low volume fractions (Phi~0.1). This shear-induced aggregation is dramatic and exhibits a very sudden change in the viscosity, which increases sharply after a shear-dependent induction time. We show that this induction time is related exponentially to the shear rate, reflecting the importance of the hydrodynamic forces in reducing the repulsive energy barrier that prevents the particles from aggregating.

Following a novel realization of low-Reynolds-number swimming (Dreyfus et al., Nature, vol. 436, 2005, p. 862), in which self-assembled filaments of paramagnetic micron-sized beads are tethered to red blood cells and then induced to swim under crossed uniform and oscillating magnetic fields, the dynamics of magnetoelastic filaments is studied. The filament is modelled as a slender elastica driven by a magnetic body torque. The model is applied to experiments of Goubault et al. (Phys.Rev. Lett., vol. 91, 2003, art. 260802) to predict the lifetimes of metastable static filament conformations that are known to form under uniform fields. A second experimental swimming scenario, complementary to that of Dreyfus et al. (2005), is described: filaments are capable of swimming even if not tethered to red blood cells. Yet, if both ends of the filament are left free and the material and magnetic parameters are uniform along its length then application of an oscillating transverse field can only generate homogeneous torques, and net translation is prohibited by symmetry. It is shown that fore–aft symmetry is broken when variation of the bending stiffness along the filament is accounted for by including elastic defects, which produces results consistent with the swimming phenomenology. 

When ligands and receptors are both attached on surfaces, because of the restriction of configurational freedom, their recognition kinetics may be substantially reduced as compared with freely diffusing species. In nature, this reduction may influence the efficiency of the capture and adhesion of circulating cells. Here we show that similar consequences are observed for colloids grafted with biomolecules that are used as probes for diagnostics. We exploit Brownian magnetic colloids that self-assemble into linear chains to show also that the resulting one-dimensional confinement considerably accelerates the recognition rate between grafted receptors and their ligands. We propose that because confinement significantly augments the colliding frequency, it also causes a large increase in the attempt frequency of the recognition. This work gives the basis of a rapid, homogeneous, and highly sensitive bioanalysis method.

A new general methodology based on magnetic colloids to study the recognition kinetics of tethered biomolecules is introduced here. By tuning the architecture of the tethers that link molecules to surfaces, we explain the large diversity of observed association times. We thus studied the association kinetics between a streptavidin and a biotin grafted on two opposite surfaces. We also considered the case of cellular adhesion molecules, the cadherins, for which we measured an association kinetic constant smaller than that of the strepatividin-biotin interaction.

We explored the ability of emulsion droplets to bind specifically solid model substrates and we study the relationship between drop composition and the contact angle. We developed a in-sisu method to graft covalently biotin and streptavidin on the O/W emulsion interface. We characterized the size distribution and the surface density of the streptavidine by flow cytometry. We showed that the contact angle and the adhesion energy grow linearly with the biotin density on the glass coverslip, and we observed a different patterning (disk, ring) of the streptavidin within the adhesion patch, depending on the PEG density on the surface. This result suggest a 2D phase transition of the proteins.

Ce manuscrit présente l'étude de la stabilité et des propriétés d'encapsulation et de relargage des émulsions doubles cristallisables. Notre démarche a été de remplacer l'huile liquide classiquement utilisée pour les émulsions doubles par une huile solide à température ambiante. Une barrière répulsive stérique assure la stabilité de ces objets au repos. Sous écoulement, les objets s'agrègent : l'énergie fournie par le cisaillement réduit la barrière répulsive alors comparable à kT et facilement franchissable. Ceci se traduit par une dépendance exponentielle du temps caractéristique d'agrégation avec le taux de cisaillement. Sous régime iso-osmotique, la nature solide de la membrane ralentit les cinétiques de libération par diffusion des espèces initialement encapsulées. Sous régime hypo-osmotique, le matériau solide libère rapidement l'espèce encapsulée. Les processus mis en jeu et les différents paramètres permettant de contrôler les cinétiques de libération ont été identifiés.

The invention describes a method for the identification of compounds which bind to a target component of a biochemical system or modulate the activity of the target, comprising the steps of: a) compartmentalising the compounds into microcapsules together with the target, such that only a subset of the repertoire is represented in multiple copies in any one microcapsule; and b) identifying the compound which binds to or modulates the activity of the target; wherein at least one step is performed under microfluidic control. The invention enables the screening of large repertoires of molecules which can serve as leads for drug development.

We demonstrate here the wet chemical synthesis of cobalt doped γ-Fe₂O₃ nanoparticles and the subsequent effect on magnetic properties with the variation in dopant concentration. It is observed that cobalt can be homogeneously doped into the γ-Fe₂O₃ lattice up to 5 mol% without any appreciable change in the particle size (~6 nm). Further increase in cobalt concentration (10 mol% here) resulted in an increase in particle size (~9 nm) due to possible adsorption of a cobalt layer on the surface of γ-Fe₂O₃ nanoparticles rather than complete doping in the iron oxide lattice. The ac susceptibility measurements revealed an increase in blocking temperature (T_B) with percentage variation in cobalt doping (2–10%), indicating substitution of Fe³⁺ ions by Co²⁺ ions in the γ-Fe₂O₃ lattice. The dc magnetization measurements showed an increase in saturation magnetization only up to 5%, beyond which it significantly diminished. The reduction in saturation magnetization is attributed to the contribution from surface anisotropy in cobalt coated γ-Fe₂O₃ nanoparticles.

We study the coupled rotational diffusion in a two-particle chain on the basis of a Smoluchowski equation and calculate time correlation functions that are measurable in an experiment. This approach might be used to explore hydrodynamic interactions in the limit where lubrication theory is valid.

An object consisting of three spheres, linked like the spokes on a wheel, can undergo a net rotational movement when the relative positions of the spheres proceed through a four-step cycle. This rotational motion is the analogue of the two-hinged swimmer originally proposed by Purcell (1977), which has served as a prototype for mechanical implementations of swimming. We also note that the rotational motion analysed here may be useful in the design of micromachines and has similarities to molecular-scale rotational motors that have been identified recently.

Superparamagnetic particles offer a new way to probe the kinetics of adhesive processes. Two different scenarios of physical adhesion are studied. The thermal activation of van der Waals adhesion is well described by an Arrhenius model. In contrast, it is necessary to go beyond the Arrhenius description to understand the thermal activation of bridging between colloidal particles by a polymer at equilibrium adsorbance. We show that polymer bridging requires some removal of adsorbed polymer and is strongly influenced by the proximity of a glass transition within the adsorbed polymer.

We perform oil coating of hydrophobic solid surfaces via aqueous media, from emulsions, and under the presence of a shear flow. The principle of such coating is based on the use of a system at the limit of aggregation to give rise to adhesion, with asymmetrical interfaces (oil droplet/water and solid surface/water) in order to favor the oil/surface adhesion in comparison to the oil/oil adhesion. This way, droplets stick to the solid substrate, whereas they are stable and homogeneously dispersed in the bulk. We have realized coatings from two systems of emulsions made of a mixture of hydroxy-terminated silicone oil and classical silicone oil and a mixture of sunflower oil and mineral oil. The kinetics of the coating is described by a Langmuir model where the adhesion between the oil particle and the surface is modeled as a first-order reaction. The resulting coatings are formed of oil droplets uniformly covering the solid surface. The coating density can vary with the nature of the experimental systems.

In this letter, we investigate the mechanism of formation of a recently discovered new type of colloid, irreversible flexible chains of magnetic particles. The chain formation mechanism is based on magnetically induced bridging by adsorbed polymers, andweinvestigate here the associated phase diagrams, considering both thermodynamic and kinetic aspects. This phase diagram is the consequence of a balance between entropic repulsion between polymer layers at the particles surfaces, depletion forces pushing the particles together, and a short-range attractive force developing when polymers can bridge two particles. We end up with a very simple protocol allowing the formation of long, extremely regular chains, which can find numerous applications in chemistry and biology. The perspectives for the development of a new field of “macrocolloidal chemistry” are discussed.

Silica particles are dispersed in the continuous phase of bitumen-in-water emulsions. The mixture remains dispersed in quiescent storage conditions. However, rapid destabilization occurs once a shear is applied. Observations under the microscope reveal that the bitumen droplets form a colloidal gel and coalesce upon application of a shear. We follow the kinetic evolution of the emulsions viscosity at constant shear rate remains initially constant and exhibits a dramatic increase after a finite time, t. We study the influence of various parameters on the evolution of t: bitumen droplet size and volume fraction, silica diameter and concentration, shear rate, etc.

Oils containing both fluorescent semiconductor and magnetic oxide nanoparticles are used to produce oil in water emulsions. This technique produces oil droplets with homogeneous fluorescence and high magnetic nanoparticle concentrations. The optical properties of the oil droplets are studied as a function of the droplet sizes for various concentrations of fluorescent and magnetic nanoparticles. For all concentrations tested, we find a linear variation of the droplet fluorescent intensity as a function of the droplet volume. For a given size and a given quantum dot (QD) concentration, the droplet fluorescence intensity drops sharply as a function of the magnetic nanoparticle concentration.Weshow that this decrease is due mainly to the strong absorption cross section of the magnetic nanoparticles and to a lesser extent to the dynamicandstatic quenching of theQDfluorescence.Therole of the iron oxide nanoparticle localization in the droplet (surface versus volume) is also discussed.

The manuscript is devoted to two applications of chains of magnetic particles.
The first chapter reports the design of force sensors between colloids. Various experiments are designed and performed to characterise the particles under investigation. Our experimental setup and signal processing techniques are described in detail. Finally, we compare this technique to previous ones.
The second chapter describes the design and fabrication of the first microscopic artificial swimmers. Flexible magnetic filaments are created by aligning magnetic particles under a magnetic field and gluing them together by means of DNA strands. Their behaviour under a magnetic field is investigated. Subjected to an oscillating magnetic field, the filaments experience deformations so that a wave propagates along them, thereby generating a propulsive force, which we demonstrate through the motion of a spermatomorphic swimmer. Theoretical and numerical predictions are validated experimentally.

A composition comprising in a continuous aqueous phase a monodisperse lipid phase comprising one or more crystallizable lipids, one or more active principles and a least one dispersed phase stabilizing compound comprising two fatty acid chains and a polyethylene glycol chain, is new. - Independent claims are included for a methods of preparing the claimed composition by: - (a) introducing the active principle(s) into the crystallizable lipid; - (b) dispersing the lipid phase in the aqueous phase in the presence of an emulsifier to form an emulsion; - (c) subjecting the emulsion to shear to form a monodisperse emulsion or where the lipid phase comprises an internal aqueous phase, by: - (i) dispersing an aqueous solution comprising the active principles into the lipid (which may contain one or more active principles) in a melted state in the presence of a lipophilic surfactant; - (ii) subjecting the emulsion obtained to shear to render it monodisperse; - (iii) incorporating the monodisperse emulsion in an aqueous phase in the presence of a stabilizer to form a double emulsion; and - (iv) subjecting the double emulsion to shear to produce a monodisperse double emulsion

Producing monodisperse biodegradable microspheres (I), comprising preparing an emulsion comprising at least one polymer phase containing an active agent (A) and at least one aqueous phase, the ratio of viscosities of the organic and aqueous phases being 0.1-10, subjecting the emulsion to controlled laminar shear, removing the solvent from the polymer phase, and recovering the obtained microspheres, is new.

Assembling colloidal particles in the form of irreversibly organized chains bearing species-recognition sites different from the ligands involved in organizing the particles, is new. Independent claims are also included for: (1) producing an assembly by assembling colloidal particles in the form of linear objects and contacting the objects with an agent capable of bridging them irreversibly; (2) producing an assembly by mixing and/or grafting colloidal particles with a bridging agent or bridging agent precursor, assembling the particles in the form of linear objects, and initiating bridging between the particles; (3) detecting, analysis, purification, identification, separation or ex-vivo monitoring of at least one species using an assembly; (4) a surface element bearing an assembly; (5) a hybridization array comprising the surface element of (4); (6) a microfluidic channel or cell comprising an assembly.

We present a new technique to measure the mechanical properties of small biomolecules. This technique uses long range repulsive colloidal forces together with magnetic attraction as a force probing tool. The biomolecules are grafted between superparamagnetic particles, which are regularly spaced within long chains maintained by an external magnetic field. Varying the magnetic field results in compression or extension of the molecules between the particles. In order to demonstrate this technique we use, as a size controlled model molecule, a short double stranded DNA (151 base pairs) for which the force-extension law is determined and found in agreement with existing predictions.

Microorganisms such as bacteria and many eukaryotic cells propel themselves with hair-like structures known as flagella, which can exhibit a variety of structures and movement patterns1. For example, bacterial flagella are helically shaped2 and driven at their bases by a reversible rotary engine3, which rotates the attached flagellum to give a motion similar to that of a corkscrew. In contrast, eukaryotic cells use flagella that resemble elastic rods4 and exhibit a beating motion: internally generated stresses give rise to a series of bends that propagate towards the tip5–7. In contrast to this variety of swimming strategies encountered in nature, a controlled swimming motion of artificial micrometresized structures has not yet been realized. Here we show that a linear chain of colloidal magnetic particles linked by DNA and attached to a red blood cell can act as a flexible artificial flagellum. The filament aligns with an external uniformmagnetic field and is readily actuated by oscillating a transverse field. We find that the actuation induces a beating pattern that propels the structure, and that the external fields can be adjusted to control the velocity and the direction of motion.

This manuscript introduces the study of self-assembled magnetic latexes. First, we present a method to obtain magnetic filaments made of magnetic particles and linkers. These objects provide a new micromechanical approach to probe bending rigidity at the molecular scale. Under a magnetic field, they adopt a hairpin configuration. Measuring the hairpin curvature as a function of the field intensity and the linker length allows us to deduce the linker bending rigidity. Second, these filaments can be self-organized in a microchannel to provide a new matrix for cell sorting. Control of the spacing between filaments, anchoring, and functionnalization with specific antibodies are described, together with the feasability for performing cell chromatography with the system. These new objects introduce a wide field of very promising biomedical applications e.g. for lab-on-chip devices.

Detecting and/or quantifying (M1) at least one analyte (A) in a liquid comprising using colloidal magnetic particles (B), functionalized on the surface with at least one ligand (L) specific for (A), is new. M1 comprises: (a) combining test sample and (B); (b) applying a magnetic field strong enough to structure (B) as chains; (c) maintaining the field for long enough to allow coupling/association of (I) with at least two (L), present on two adjacent particles in the chain; (d) turning of the field; and (e) detecting presence/absence of (I), and optionally its concentration, from the presence/absence of permanent chains of (B). An Independent claim is also included for a reagent kit containing (B).

Food emulsion comprising a primary water-in-oil emulsion dispersed in an aqueous phase is stabilized with a natural lipophilic surfactant system present in the primary emulsion. An Independent claim is also included for a process for producing the emulsion by dispersing an internal aqueous phase in an oil phase containing the surfactant system and dispersing the resulting water-in-oil emulsion in an external aqueous phase.

The invention concerns a method for the controlled release of an active principle contained in a multiple water-in-oil-in-water emulsion characterised in that: said multiple emulsion comprises an invert emulsion Ei with a aqueous phase A1 containing at least a hydrophilic active principle, said emulsion Ei being dispersed in the form of direct emulsion droplets Ed, in a continuous aqueous phase A2, with the two emulsions Ed and Ei stabilised by at least a surfactant, present in their respective continuous phases; and said multiple emulsion is brought in the presence of an effective amount of an agent for transforming it into a direct emulsion and induce the release of the active principle, contained in the aqueous phase A1 of emulsion Ei, in the aqueous phase A2.

The invention concerns composite nanospheres having a diameter ranging between about 50 and 1000 nm plus or minus 5%, preferably between about 100 and 500 nm plus or minus 5% and advantageously between 100 and 200 nm plus or minus 5%, and comprising an essentially liquid core consisting of an organic phase and inorganic nanoparticles, distributed inside the organic phase, and a skin consisting of at least a hydrophilic polymer derived from the polymerisation of at least one water soluble monomer, in particular N-alkylacrylamide or a N-N-dialkylacrylamide; conjugates derived from said nanospheres; their preparation methods and their uses.

The invention concerns composite particles comprising a hydrophobic polymer and inorganic nanoparticles, said particles having a diameter of about 50 to 1000 nm, preferably about 100 to 500 nm, and advantageously from about 100 to 250 nm and comprising a hydrophobic polymer consisting of a polymer matrix and the inorganic nanoparticles stabilised and distributed relative homogeneously inside said matrix. The invention also concerns conjugates derived from said composite particles, their method of preparation and their uses.

The invention concerns a fractionable polydisperse stable double oil-in-water emulsion, consisting of 50 to 95 wt.%, relative to the total weight of the double emulsion, droplets of an invert monodisperse emulsion Ei dispersed in a continuous aqueous phase; the continuous aqueous phase comprising a polysaccharide thickening agent in a proportion of 1 to 10 wt.% relative to the total weight of the continuous aqueous phase; a water-soluble ethylene oxide and propylene oxide block copolymer as surfactant; and an osmotic pressure balancing agent; the emulsion Ei having a viscosity not higher than the viscosity of the continuous aqueous phase and consisting of 50 to 95 wt.%, relative to the total weight of Ei, droplets of an internal aqueous phase dispersed in an oily phase; the internal aqueous phase comprising at least an active hydrophilic substance; the oily phase comprising polyglycerol polyricinoleate as surfactant. The invention further concerns the corresponding monodisperse double emulsion of the same formulation and the method for preparing it starting from the claimed polydisperse double emulsion.

Water-redispersible granules including at least one active material in the form of a hydrophobic liquid, at least one non-ionic surfactant selected from polyoxyalkylene derivatives, and at least one water-soluble compound selected from polyelectrolytes belonging to the weak polyacid family, are disclosed. A method for preparing such granules by preparing an aqueous emulsion of at least one active material, at least one non-ionic surfactant and at least one water-soluble compound, and drying the resulting emulsion, is also disclosed.

The invention concerns a method for separating particles or molecules whereby these particles or molecules are introduced in a separating medium and a moving force is applied to them in said medium. The method is characterised in that the separating medium is a ferrofluid, i.e. a colloidal suspension of magnetic particles and a magnetic field is applied to this ferrofluid generating therein at least an alternation of a zone rich in magnetic particles and a zone poor in magnetic particles, part at least of the region of the ferrofluid in which this alternation is generated is passed through by the particles or molecules to be separated during their migration.

The invention concerns a process for preparing a secondary emulsion consisting of droplets of a phase A, dispersed in a phase B, A being immiscible in B, starting with a polydispersed primary emulsion of identical formulation and consisting of droplets of the said phase A dispersed in the said phase B, the droplets of the secondary emulsion having a diameter in all cases which is less than the diameter of the droplets of the primary emulsion, characterized in that the starting primary emulsion is viscoelastic and in that the said starting primary emulsion is subjected to a controlled shear such that the same maximum shear is applied to the entire emulsion.

A process for the preparation of aqueous dispersions of magnetizable polymer particles with a narrow distribution from aqueous dispersions with a wide distribution. The amount of water in the aqueous dispersion with a wide distribution is adjusted so that the proportion by weight of magnetizable polymer particles is between about 1 and 40% of said dispersion. The surfactant concentration of the dispersion after the water adjustment is increased until two phases are obtained: a so-called liquid phase and a so-called solid phase. After separation of these two phases is accomplished, these steps are repeated as desired. An aqueous dispersion with a narrow size distribution is recovered.