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International audience; Induction time, a measure of how long one will wait for nucleation to occur, is an important parameter in quantifying nucleation kinetics and its underlying mechanisms. Due to the stochastic nature of nucleation, efficient methods for measuring large number of independent induction times are needed to ensure statistical reproducibility. In this work, we present a novel approach for measuring and analyzing induction times in sessile arrays of microdroplets via deliquescence/recrystallization cycling. With the help of a recently developed image analysis protocol, we show that the interfering diffusion-mediated interactions between microdroplets can be eliminated by controlling the relative humidity, thereby ensuring independent nucleation events. Moreover, possible influence of heterogeneities, impurities, and memory effect appear negligible as suggested by our 2-cycle experiment. Further statistical analysis (k-sample Anderson-Darling test) reveals that upon identifying possible outliers, the dimensionless induction times obtained from different datasets (microdroplet lines) obey the same distribution and thus can be pooled together to form a much larger dataset. The pooled dataset showed an excellent fit with the Weibull function, giving a mean supersaturation at nucleation of 1.61 and 1.85 for the 60pL and 4pL microdroplet respectively. This confirms the effect of confinement where smaller systems require higher supersaturations to nucleate. Both the experimental method and the data-treatment procedure presented herein offer promising routes in the study of fundamental aspects of nucleation kinetics, particularly confinement effects, and are adaptable to other salts, pharmaceuticals, or biological crystals of interest.

International audience; Nucleation, the birth of a stable cluster from disorder, is inherently stochastic. Yet up to date, there are no quantitative studies on NaCl nucleation that accounts for its stochastic nature. Here, we report the first stochastic treatment of NaCl-water nucleation kinetics. Using a recently developed microfluidic system and evaporation model, our measured interfacial energies extracted from a modified Poisson distribution of nucleation time show an excellent agreement with theoretical predictions. Furthermore, analysis of nucleation parameters in 0.5 pL, 1.5pL and 5.5 pL microdroplets reveals an interesting interplay between kinetic confinement and shifting of nucleation mechanisms. Overall, our findings highlight the need to treat nucleation stochastically rather deterministically to bridge the gap between theory and experiment.

International audience; Drug efficacy strongly relies on the solid state of the active pharmaceutical ingredient. Classical solid-state screening methods involve different solvent compositions and supersaturations. Moreover, the many repeat experiments needed to address the stochasticity of nucleation make this approach costly. This paper presents a newly developed modular microfluidic platform that provides a universal and flexible plug-and-play tool for crystallisation studies without use of surfactants. By dissolving a powder, our setup generates saturated solutions that can be used for solubility measurements or distributed in microdroplets. Here, we describe solubility measurements performed on different forms, stable and metastable, of pharmaceutical molecules (Irbesartan, Rimonabant and Aripiprazole) in organic and aqueous solvents. In addition, we provide nucleation statistics obtained for Sulfathiazole in water and in acetonitrile. Reporting polymorph screening on Sulfathiazole and statistics for nucleated forms, we find that the cooling rate influences both nucleation and polymorphism results, reflecting the competition between thermodynamics and kinetics. Three unknown forms were discovered, with XRD patterns and Raman spectra that do not match any referenced form. We also demonstrate the limitations of microfluidics for crystallisation by cooling: reducing the crystalliser volume considerably increases nucleation induction time.

International audience; This communication presents a new microfluidics device for solubility measurement based on the generation of saturated solutions starting from powder and compatible with all solvents and molecules. This device is simple and easily incorporated into any laboratory, even those not specialized in microfluidics. We described the method used to measure solubility, applied here to the pharmaceutical molecules paracetamol and gliclazide in different solvents. The device rapidly measures the solubility of a compound at a variety of temperatures ranging from 20 to 60°C using extremely small quantities (here, only 30mg) of material, with a concentration range from mg/mL to hundreds of mg/mL.

International audience; We describe a microfluidic platform for solid phase screening using extremely small quantities of raw materials. Based on our previous setup for solubility measurement that generates saturated solutions directly from powder, the platform requires no solution in excess of that used for the droplet-crystallization experiment. The setup is compatible with most solvents and molecules without using surfactant. Using this microfluidic platform, we first measured the solubility of Sulfathiazole in water, isopropanol, and acetonitrile. Second, we performed a polymorph screening of Sulfathiazole using as little as 30 mg of raw material, for numerous identical cooling crystallization experiments from 80 to 10°C. In the experiments presented, we obtained the 3 usual polymorphs of Sulfathiazole. We show that this economical approach yields reliable information on the probability of nucleation of a given polymorph, useful in pharmaceutical development.

International audience; Seeking to produce microparticles that mimic red blood cells (RBCs), we present a microfluidic method of generating monodispersed hydrogel microparticles of Na-/Ca-alginate with controllable sizes (micrometer range) and mechanical properties. No surfactant is used. Transformation from Na-alginate to Ca-alginate microparticles is realized via ex situ gelation, which proves essential to obtaining desired microparticle properties, such as insolubility in water and RBC-like mechanical properties. For both Na-alginate and Ca-alginate microparticles, a smooth surface and a porous inner structure are observed under a scanning electron microscope. A platform of microgrippers is successfully developed to manipulate the microparticles. The Young’s modulus measured using an atomic force microscope on the surface of Ca-alginate microparticles is of the same order as that of RBCs.

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This review compares droplet-based microfluidic systems used to study crystallization fundamentals in chemistry and biology. An original high-throughput droplet-based microfluidic platform is presented. It uses nanoliter droplets, generates a chemical library, and directly solubilizes powder, thus economizing both material and time. It is compatible with all solvents without the need for surfactant. Its flexibility permits phase diagram determination and crystallization studies (screening and optimizing experiments) and makes it easy to use for nonspecialists in microfluidics. Moreover, it allows concentration measurement via ultraviolet spectroscopy and solid characterization via X-ray diffraction analysis.

International audience; This work presents an experimental and a numerical study to highlight a kinetic explanation to of the Ostwald rule of stages (ORS). To demonstrate this explanation, L-glutamic acid (LGlu) (a monotropic system with two polymorphs) were crystallized in three different scales: Liter scale in a 2L stirred crystallizer, milliliter scale in a 4ml stagnant cell and microliter scale in microfluidic channels. Cooling crystallization experiments were performed in water at different temperature and supersaturation conditions. The LGlu polymorphic system was found to follow the ORS at low temperature (between 5°C and 30°C). However, in similar operating conditions, the stable polymorph crystallized preferentially or exclusively in the stagnant cell and in microfluidics. To explain the ORS in the stirred crystallizer at low temperature, a model based on the kinetic equation was used. By taking into account the Gibbs Thomson effect (Solubility variation with size), the simulations in the nanoscopic scale showed the dissolution of the slow-growing stable phase nuclei in favor of the fast-growing metastable phase nuclei. Consequently, the numerical results showed that the Gibbs Thomson effect is a key factor in polymorph competition and that considering this effect, in certain kinetic and equilibrium conditions, could allow explaining and simulating the ORS.

International audience; Here, we describe a plug-and-play microfluidic platform, suitable for protein crystallization. The droplet factory is designed to generate hundreds of droplets as small as a few nanoliters (2 to 10nL) for screening and optimization of crystallization conditions. Commercially-available microfluidic junctions and tubing are combined to create the appropriate geometry. In addition, a " chemical library " is produced in tubing. The microfluidic geometry for a " crystallization agent-based chemical library " is validated by screening crystallization conditions of lysozyme. The microfluidic geometry for a " ligand-based chemical library " is also explored to co-crystallize the protein QR2 with a ligand, for the purposes of structure-based drug design. This platform mixes aqueous phases (containing the protein and the crystallization agent) and organic phases (containing the ligand), during the droplet generation and circulation without using any surfactant. The droplet composition is controlled by the respective flow-rates of the different solutions, and checked by measuring on-line absorbance. The low volumes involved in the crystallization trials, the speed of execution and the absence of a microfabrication stage make our platform a cheap, easy-to-use and versatile tool for crystallization studies.

International audience; We describe a universal, high-throughput droplet-based microfluidic platform for crystallization. It is suitable for a multitude of applications, due to its flexibility, ease of use, compatibility with all solvents and low cost. The platform offers four modular functions: droplet formation, on-line characterization, incubation and observation. We use it to generate droplet arrays with a concentration gradient in continuous long tubing, without using surfactant. We control droplet properties (size, frequency and spacing) in long tubing by using hydrodynamic empirical relations. We measure droplet chemical composition using both an off-line and a real-time on-line method. Applying this platform to a complicated chemical environment, membrane proteins, we successfully handle crystallization, suggesting that the platform is likely to perform well in other circumstances. We validate the platform for fine-gradient screening and optimization of crystallization conditions. Additional on-line detection methods may well be integrated into this platform in the future, for instance, an on-line diffraction technique. We believe this method could find applications in fields such as fluid interaction engineering, live cell study and enzyme kinetics.

A microfluidic platform was used to address the problems of obtaining diffraction-quality crystals and crystal handling during transfer to the X-ray diffractometer. Crystallization conditions of a protein of pharmaceutical interest were optimized and X-ray data were collected both in situ and ex situ.

Although cocrystals have undergone a recent boost in popularity, limited time is spent on exploring the variety of possible cocrystal phases, which is likely due to the popularity and success of grinding methods. The number of studies where cocrystallization is systematically applied in solution remains limited. In this contribution, we present a rapid method for cocrystal synthesis in solution, which not only provides single crystals suitable for X-ray diffraction structural analysis but also allows one to explore the variety of cocrystal systems (polymorphs and solvates). Applying this approach to caffeine/dicarboxylic acid systems, we discovered 7 cocrystals and solved the structures for 6 of them. Astonishingly, the caffeine/mesaconic acid cocrystal system not only seems to be stoichiometrically diverse but furthermore also shows no less than 4 polymorphs of the 2 : 1 cocrystal.

The nucleation mechanisms behind crystallized products remain mysterious. In this communication, we describe experiments performed using small volumes, microdroplets, to control nucleation and thus product properties. The effect of small-volume systems on nucleation is discussed.

Aim To assess the influence of concentration, temperature and surfactant addition to a sodium hypochlorite solution on its dynamic viscosity and to calculate the corresponding Reynolds number to determine the corresponding flow regimen. Methodology The dynamic viscosity of the irrigant was assessed using a rotational viscometer. Sodium hypochlorite with concentrations ranging from 0.6% to 9.6% was tested at 37 and 22 °C. A wide range of concentrations of three different surfactants was mixed in 2.4% sodium hypochlorite for viscosity measurements. The Reynolds number was calculated under each condition. Data were analysed using two-way anova. Results There was a significant influence of sodium hypochlorite concentration (P

The aim of this review is to provide biocrystallographers who intend to tackle protein-crystallization with the- ory and practical examples. Crystallization involves two separate processes, nucleation and growth, which are rarely com- pletely unconnected. Here we give theoretical background and concrete examples illustrating protein crystallization. We describe the nucleation of a new phase, solid or liquid, and the growth and transformation of existing crystals obtained by primary or secondary nucleation or by seeding. Above all, we believe that a thorough knowledge of the phase diagram is vital to the selection of starting position and path for any crystallization experiment.

Phase diagrams of cocrystals often show a highly unsymmetrical nature. The solvent has an important impact on the overall aspect of these diagrams. In this paper, we show how the solvent affects the composition of the stoichiometric solid phase nucleated. Suitable conditions for nucleation and growth of a single 2:1 caffeine/maleic acid cocrystal are obtained in ethyl acetate, showing comparable solubility toward both caffeine and maleic acid. Through a full kinetic screen, we were able to identify, for the first time, reproducible conditions for the spontaneous crystallization of the 2:1 phase in solution. Furthermore, during the screening experiments, a hithertho unknown form of the 1:1 cocrystal phase was encountered. Structural X-ray diffraction analyses of both the 2:1, as well as the 1:1 polymorphic phases, show an out of plane maleic acid compound. The carboxylic acid groups are oriented in such a manner to promote intermolecular formation of hydrogen bonded synthons.

International audience; We study the formation of nanoliter-sized droplets in a microfluidic system composed of a T-junction in PEEK and tubing in Teflon. This system, practical for a ‘plug and play’ set-up, is designed for droplet-based experiments of crystallization with a statistical approach. Hence the aim is to generate hundreds of droplets identical in size and composition and spatially homogeneous. Therefore, parameters of control are droplet size and frequency. However, the geometry of the T-junction is not perfect and, moreover, its channels are circular, as opposed to the planar geometries with rectangular cross-sections that are usually used. However, based on 3D experiments and 2D simulations, we observe the same regimes of droplet generation in circular channels as in planar geometries, and with the same stability. Therefore, we refer to velocities instead of flow rates to characterize the system. Then we define operating range in terms of droplet size and frequency through empirical relations using total velocity, velocity ratio and capillary number, to ensure homogeneous droplets in channels of 500 µm and 1 mm diameters.

Do the differing properties of materials influence their nucleation mechanisms? We present different experimental approaches to study and control nucleation, and shed light on some of the factors affecting the nucleation process.

We present a simple and easy-to-use microfluidic setup for the crystallization of mineral, organic, and biological materials. After briefly presenting the hydrodynamic properties of the setup, we test and validate it with viscous media by crystallizing a protein in an aqueous solution of PEG. We obtain nucleation data in nanocrystallizers using a droplet-based method and precisely controlling input flows to test different crystallization conditions.

Sarah Price opened a general discussion of the paper by Sven Schroeder: I have been generating the thermodynamically plausible crystal structures of organic molecules for many years, and back in 2004 we did a crystal structure prediction (CSP) study on imidazole1 and found that it was relatively straightforward. Following your paper, we have reclassified the low energy structures according to the tilt within the hydrogen-bonded chain and the relative direction of the chains. Although the observed structure was the global minimum, two other structures with a displacement of otherwise identical layers are very close in energy. Do you think that if imidazole had crystallised in one of these alternative structures it would be distinguishable by NEXAFS? This would be a very sensitive test of whether NEXAFS combined with CSP could be used in characterising crystal structures.

This chapter presents different practical ways to address nucleation stochasticity. The methods use either statistical studies on spontaneous nucleation or local control of nucleation. Techniques developed in our laboratory are described: droplet-based microfluidics, microinjectors in oil, and external electrical or mechanical fields in confined systems. Results of nucleation kinetics obtained on various molecules are presented in terms of metastable zone, critical supersaturation, nucleation rate, induction time, interfacial energy of the critical nucleus, polymorphism, and detection of the critical nucleus. These practical approaches show considerable potential to increase understanding and control of the nucleation mechanism.

Calcium carbonate precipitation at different values of the nominal ionic activity product (IAP) is studied in nanoliter and picoliter droplets at (20 ± 2 °C). Experiments are carried out through direct mixing of equimolar reactant solutions using two different setups: first, droplet-based microfluidics using Teflon capillaries (nanoliter experiments) and second, the microinjection technique under oil (picoliter droplets). Instantaneous precipitation of a metastable CaCO3 phase is initially observed. This phase is stabilized in time by reducing the initial volume of the experiments from the nano- to picoliters range and when the CaCl2/Na2CO3 ratio approaches 1. Further analysis by X-ray diffraction, transmission electron microscopy, and selected area electron diffraction confirms the first nucleated phase is CaCO3·6H2O (ikaite) and in few droplets ikaite plus CaCO3·H2O (monohydrocalcite). No evidence of amorphous calcium carbonate (ACC) is found even in conditions where the IAP exceeds the solubility produ...

International audience; A Scanning Tunneling Microscopy in Break Junction regime and a Mechanically Controllable Break Junction are used to acquire thousands of conductance-elongation curves by stretching until breaking and reconnecting Au junctions. From a robust statistical analysis performed on large sets of experiments , parameters such as lifetime, elongation and occurrence probabilities are extracted. The analysis of results obtained for different stretching speeds of the electrodes indicates that the breaking mechanism of di-and mono-atomic junction is identical, and that the junctions undergo atomic rearrangement during their stretching and at the moment of breaking. PACS. 73.63.Rt electronic transport in nanocontacts – 81.07.Lk fabrication of nanocontacts

The tapping mode in Atomic Force Microscopy (AFM) is a key advance in AFM technology for functionalized surface imaging. In fact, the basic technique of AFM, which is contact AFM, is not well adapted to examine such surfaces due to problems of friction and adhesion. Therefore, the tapping mode of operation was developed to overcome drawbacks of contact mode (Binnig et al, 1986). This mode uses oscillation of the cantilever tip at or near its natural resonant frequency while allowing the cantilever tip to impact the target sample for a minimal amount of time. This intermittent contact lessens the damage done to the soft surface and to the tip, compared to the amount done in contact. Thus, the tapping mode is the method of choice for imaging functionalized surfaces. A functionalized surface is a solid surface where molecules have been adsorbed leading to various patterns, from isolated molecules dispersed on the surface to homogeneous films. The patterns provide ideal platforms for the immobilization or recognition and specific interaction of biomolecules. In the preparation of functionalized surfaces, the first process consists in understanding the mechanism of adsorption. The aim is to yield good orientation and stability of the immobilized molecules, thus leading to high-functionality of the surface for applications, such as biosensors.

The purposes of the present study were to (1) assess the effect of the addition of benzalkonium chloride to sodium hypochlorite on its wetting properties, contact angle, and surface energy; (2) determine the critical micellar concentration of benzalkonium chloride in sodium hypochlorite; and (3) investigate the influence of addition of benzalkonium chloride on the free chlorine level, cytotoxicity, and antiseptic properties of the mixture.Solutions of benzalkonium chloride, with concentrations ranging from 0%-1%, were mixed in 2.4% sodium hypochlorite and tested as follows. The wetting properties were investigated by measuring the contact angle of the solutions on a nondehydrated dentin surface by using the static sessile drop method. The pending drop technique was subsequently used to determine the surface energy of the solutions. The critical micellar concentration of benzalkonium chloride mixed in sodium hypochlorite was calculated from the data. When 2.4% NaOCl was mixed with benzalkonium chloride at the critical micellar concentration, 3 parameters were tested: free chloride content, cytotoxicity, and antibacterial effects against Enterococcus faecalis.The contact angle (P.001) as well as the surface energy (P.001) significantly decreased with increasing benzalkonium chloride concentrations. The critical micellar concentration of benzalkonium chloride in sodium hypochlorite was 0.008%. At this concentration, the addition of benzalkonium chloride had no effect on the free chlorine content, cytotoxicity, or antibacterial efficiency of the mixture.The addition of benzalkonium chloride to sodium hypochlorite at the critical micellar concentration reduced the contact angle by 51.2% and the surface energy by 53.4%, without affecting the free chloride content, cytotoxicity, or antibacterial properties of the mixture.

Microfluidic devices are increasingly used for the screening of crystallization conditions. Their advantage is the generation of droplets, every droplet being an independent crystallizer with volumes in the nanoliter range. This enables a large number of experiments to be carried out under identical conditions necessitating only small quantities of materials. However, classic microfluidic crystallization devices are made of poly(dimethylsiloxane), only compatible with an aqueous medium. In addition, they generally involve very complicated setups, often inaccessible to nonmicrofluidics specialists. In this paper, we overcome these drawbacks, presenting a cheap and universally applicable microfluidic crystallization tool. This thermostatted device makes it possible to study nucleation in both aqueous and organic solvents, rendering microfluidic devices applicable to organic molecules such as APIs, explosives, and metal oxide nanoparticles.

We present an easy-to-use microfluidic set-up, easily transferable to the laboratory and determine an accurate method for metastable zone width measurement. We clearly define a zone in the phase diagram where nucleation is mononuclear. We nucleate a single crystal of metastable phase, which turns out to be stable. This approach holds promise for the control and the study of crystallization processes.

International audience; Conductance-elongation curves of gold nanojunctions exhibit characteristic plateaus just prior to breaking. Conductances of these plateaus are integer multiples of the quantum of conductance. These plateaus occur because, in this size regime, transport is ballistic and the conductance is independent of the length of the nanojunction. We developed a STM-based break junction technique to access to the conductance-elongation curves. In this paper, specific tools adapted for measurements in solution are described. We then report on our results obtained for both metallic and molecular nanowires.

Here we measure lysozyme nucleation kinetics using an easy-to-use and simply constructed microfluidics setup previously described. We confirm that microfluidics is a direct, accurate, and fast method to measure nucleation frequency using only a few milligrams of molecules. Moreover, our microfluidics setup, by diminishing crystallizer volumes, increases the experimental supersaturation range accessible and can be applied to all water-soluble molecules.

Room temperature conductance-elongation curves of gold atomic wires are measured using a Scanning Tunnelling Microscope Break Junction technique. Landauer's conductance plateaus are individually identified and statistically analysed. Both the probabilities to observe, and the lengths of the two last plateaus (at conductance values close to 2e2/h and 4e2/h) are studied. All results converge to show that the occurrence of these two conductance plateaus on a conductance-elongation curve are statistically independent events., 8 pages, 5 figures. Accepted for publication in Nanotechnology

We report the modification of gold and graphite electrodes with commercially available carbon nanotubes for immobilization of Desulfovibrio fructosovorans [NiFe] hydrogenase, for hydrogen evolution or consumption. Multiwalled carbon nanotubes, single-walled carbon nanotubes (SWCNs), and amine-modified and carboxyl-functionalized SWCNs were used and compared throughout. Two separate methods were performed: covalent attachment of oriented hydrogenase by controlled architecture of carbon nanotubes at gold electrodes, and adsorption of hydrogenase at carbon-nanotube-coated pyrolytic graphite electrodes. In the case of self-assembled carbon nanotubes at gold electrodes, hydrogenase orientation based on electrostatic interaction with the electrode surface was found to control the electrocatalytic process for H(2) oxidation. In the case of carbon nanotube coatings on pyrolytic graphite electrodes, catalysis was controlled more by the geometry of the nanotubes than by the orientation of the enzyme. Noticeably, shortened SWCNs were demonstrated to allow direct electron transfer and generate high and quite stable current densities for H(2) oxidation via adsorbed hydrogenase, despite having many carboxylic surface functions that could yield unfavorable hydrogenase orientation for direct electron transfer. This result is attributable to the high degree of oxygenated surface functions in addition to the length of shortened SWCNs that yields highly divided materials.

A label-free two-dimensional colorimetric DNA sensor is reported. This sensor is based on the 2D aggregation of oligonucleotide-modified gold nanoparticle probes induced by the molecular hybridization of single-stranded oligonucleotide probes and their complementary single-stranded DNA targets. To detect the aggregation, we have developed a new detection method based on the selective desorption of nonaggregated nanoparticles. We will show here that this detection method is highly specific and allows the quantification of the DNA targets.

This paper highlights the work of our group on the control and the observation of nucleation with techniques using nanotechnologies. This control is performed either by triggering nucleation in time with an external field or by localising it spatially in a microdroplet. Localisation in time using light irradiation induces nucleation by forming radicals; the use of electric field acts locally on the density of the solution. Localisation in space with a microfluidic device produces hundreds of nanovolume crystallisers where concentration and temperature are easily monitored. Thus, accurate statistical studies lead to the nucleation parameters (metastable zone, nucleation rate and polymorphism). Lastly, confinement with a microdroplet generator permits to reach very high supersaturations in fL to pL volumes allowing nucleation of a single crystal per microdroplet. All these methods clearly enhance nucleation in the metastable zone. Finally, they use small quantities of products offering potentialities for the screening of crystallisation conditions and phases (polymorphism).

Droplet-based nucleation experiments reveal discrepancies in kinetic and thermodynamic factors. Here we examine how the chemical nature of the water–oil interface, and not the type of the device, used in different set-ups by three different groups impacts nucleation rate and explain discrepancies among lysozyme interfacial energies and pre-exponential factors encountered in the literature.

A 2D colorimetric DNA sensor is reported based on the 2D aggregation of oligonucleotide-modified gold nanoparticle probes resulting from the molecular hybridization between these latest and their complementary single stranded DNA targets. To increase their mobility the nanoparticles are adsorbed on a fluid lipid bilayer, itself supported on a substrate. The hybridization between the target and the mobile nanoparticle probes creates links between the nanoparticles resulting in the formation of nanoparticle aggregates in the plane of the substrate. This aggregation is detected using a new method based on the selective desorption of non-aggregated nanoparticles. The addition of dextran sulfate induces the substitution of non-aggregated gold nanoparticles while aggregated ones are stable on the substrate. We show that this detection method is highly specific and allows the detection of DNA mismatches and damages.

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International audience; The occurrence of concentration and temperature gradients in saline microdroplets evaporating directly in air makes them unsuitable for nucleation studies where homogeneous composition is required. This can be addressed by immersing the droplet in oil under regulated humidity and reducing the volume to the picoliter range. However, the evaporation dynamics of such a system is not well understood. In this work, we present evaporation models applicable for arrays of sessile microdroplets with dissolved solute submerged in a thin layer of oil. Our model accounts for the variable diffusion distance due to the presence of the oil film separating the droplet and air, the diffusive interaction of neighboring droplets, as well as the variation of the solution density and water activity due to the evolving solute concentration. Our model shows excellent agreement with experimental data for both pure water and NaCl solution. With this model, we demonstrate that assuming a constant evaporation rate and neglecting the diffusive interactions can lead to severe inaccuracies in the measurement of droplet concentration particularly during nucleation experiments. Given the significance of droplet evaporation in a wide array of scientific and industrial applications, the models and insights presented herein would be of great value to many fields of interest.

Most of the active pharmaceutical ingredients (API) can exist, in the solid state, under several crystalline phases (polymorphs, solvates, cocristals…). These phases have different physicochemical properties, which can affect the drug efficacy and its manufacturing process. In the pharmaceutical industry, a solid-state screening is thus carried out in the early stages of a drug development, to identify the existing phases and choose the one to develop. At this step, only a small amount of raw material is available, reducing the number of experiments. Droplet microfluidics is used to reduce the crystallization volumes to nanoliter scale, and thus the amount of product. This makes it possible to increase the number of experiments, bringing a statistical approach to address the stochasticity of nucleation.This thesis presents the development of a modular microfluidic platform, easy to use and compatible with most solvents, for solid-state screening of active pharmaceutical ingredients and fundamental nucleation studies. It enables to generate saturated solutions and droplets starting from powder and their characterization, to measure solubility, to crystallize API by cooling or non-solvent addition processes and to monitor nucleation in time and as a function of the temperature to establish global statistics on nucleation (all phases included). Lastly, the different phases are identified in situ by Raman spectroscopy to perform phase to phase statistics.; La plupart des principes actifs pharmaceutiques peuvent exister à l’état solide sous différentes phases cristallines (polymorphes, solvates, cocristaux…). Celles-ci ont des propriétés physico-chimiques différentes, pouvant affecter l’efficacité d’un médicament, ainsi que son procédé de fabrication. Dans l’industrie pharmaceutique, un criblage de l’état solide a donc lieu dès les premières étapes du développement d’un médicament, afin d’identifier les phases existantes et choisir celle qui sera développée. A ce stade du développement, seulement une petite quantité de produit est disponible, limitant ainsi le nombre de tests réalisés. La microfluidique à base de gouttes permet de réduire les volumes de cristallisation à quelques nL, et donc les quantités de matière nécessaires. Il est ainsi possible de multiplier les expériences, apportant un point de vue statistique afin de répondre à la stochasticité du phénomène de nucléation.Cette thèse présente le développement d’une plateforme microfluidique modulable utilisant des composants compatibles avec la majorité des solvants et facile d’utilisation, pour le criblage de l’état solide de molécules pharmaceutiques et des études fondamentales sur la nucléation. Elle permet de générer des solutions saturées et des gouttes à partir de poudre et de les caractériser, mesurer des solubilités, cristalliser des principes actifs dans les gouttes par refroidissement ou ajout d’un non-solvant et suivre la nucléation au cours du temps et de la température afin d’établir des statistiques globales de nucléation (toutes phases confondues). Enfin, les différentes phases obtenues sont caractérisées in situ par spectroscopie Raman afin d’établir des statistiques de nucléation phase par phase.

Nucleation is a key step in crystallization processes which is a crucial unit operation in the manufacture and purification of products, occurring in almost all sorts of industries including foods, pharmaceuticals, cosmetics, fine chemicals, ceramics, metallurgy and electronics. Thus, fundamental understanding of its nucleation kinetics is of immense importance yet it remains poorly understood from both experimental and theoretical perspective. With these motivations, this thesis seeks to develop innovative methods in quantifying nucleation kinetics both in industrially-relevant agitated crystallizers and in fundamentally-oriented microfluidic systems. Starting with agitated crystallizers, a protocol for estimating primary nucleation was developed based on laser backscattering which involves extrapolating the nucleation rates to zero agitation. To validate the approach, a multiscale investigation of nucleation kinetic parameters was performed using various techniques in L, mL, and μL scales. This sheds light into the transferability of kinetic data for engineering purposes. To focus on the fundamental aspects of nucleation, an approach to extract nucleation kinetic parameters from evaporative microcrystallizers was developed, using microdroplets at pL scale. This involves the measurement of induction time via deliquescence-efflorescence cycle, the derivation of evaporation model to accurately determine the supersaturation at nucleation, and the use of a modified Poisson distribution to model the stochastic nature of nucleation and extract nucleation kinetic parameters. The combination of these three developments have led to a successful quantification of nucleation kinetic parameters in evaporating microdroplets (i.e; at variable supersaturation), demonstrating a remarkable agreement between theory and experiment.; La nucleation est une etape essentielle dans le processus de cristallisation, qui est notamment utilise pour la fabrication et la purification de produits industriels (pharmaceutiques, cosme tiques, de chimie fine, alimentaires, ce ramiques, de me tallurgie et d'e lectronique). Cependant, il reste encore des questions fondamentales, notamment sur la cine tique de nucle ation qui est cruciale dans ces applications. Cette the se cherche a mieux comprendre la nucle ation des cristaux par le de veloppement de me thodes innovantes pour quantifier cette cine tique de nucle ation : dans des cristallisoirs agite s a grande e chelle (L, mL, μL) qui sont industriellement pertinents, ainsi qu’a petite e chelle (nL, pL) dans des syste mes microfluidiques a base de microgouttelettes qui pre sentent un inte re t fondamental. A l'e chelle du L, la mesure de la re flectance optique couple e a la spectroscopie Raman in situ nous a permis de suivre l'e volution du nombre de particules et de la concentration de la solution. Et par l’extrapolation du de compte des particules jusqu'a une vitesse d'agitation nulle, nous avons extrait la cine tique de nucle ation primaire. A l’e chelle du mL et du μL en syste mes agite s, les parame tres de la cine tique de nucle ation obtenus par l'approche de distribution du temps d'induction re ve lent des e carts de six a sept ordres de grandeur, par rapport a ceux obtenus dans des volumes de l’ordre du L, ce qui les rend inexploitables a l'e chelle industrielle. Toutefois, les valeurs d'e nergie interfaciale effective γeff sont relativement cohe rentes a toutes ces e chelles. Tandis qu’a l’e chelle du nL, en microfluidique a base de microgouttelettes dans des capillaires, l’e nergie interfaciale effective γeff est e leve e. Ceci est lie a la barrie re thermodynamique e leve e pour atteindre la nucle ation. Par conse quent, la sursaturation doit y e tre tre s e leve e pour nucle er, faisant ainsi de la nucle ation homoge ne le me canisme pre dominant. A l’e chelle du pL, la me thode microfluidique est base e sur la ge ne ration de microgouttelettes sessiles sur une surface. Le temps de nucle ation est de tecte par microscopie in situ et analyse d'images lors de cycles de de liquescence-efflorescence, la sursaturation au moment de la nucle ation est de termine e avec pre cision a partir d'un mode le d'e vaporation que j'ai de veloppe , et la nature stochastique de la nucle ation est analyse e a l'aide d'une distribution de Poisson modifie e. Ainsi la combinaison de ces trois de veloppements nous a permis de quantifier les parame tres de la cine tique de nucle ation dans les microgouttelettes en e vaporation, avec une cohe rence entre la the orie et l'expe rience.

This work is included in an ANR project named CUMBA (Cellular Ultrasonic imaging Modality to assess red Blood cell Aggregation in vivo) whose purpose is to predict the aggregation of red blood cells (“RBCs”). The hyperaggregation of RBCs is a hemorheological disorder which leads to microvascular dysfunctions and venous thromboses. Moreover, RBCs may also aggregate during the organ transplantation due to the increase of inflammatory plasmatic macromolecules. The aim of the project CUMBA is to develop an ultrasonic imaging modality permitting in situ monitoring of RBC aggregation for early diagnosis of hyperaggregation. In this context, RBC-like microparticles are essential for both calibration and preliminary tests on the model. To do this, microparticles must be controlled in size and mechanical properties. In addition, the use of surfactants should be avoided for application in the ultrasound imaging device. This thesis focuses on a droplet-based microfluidic method without the addition of surfactants to prepare RBC-like microparticles. The microparticles are obtained by ionic gelation of a natural polymer, sodium alginate (Na-alginate), with calcium chloride to form a gel of calcium alginate (Ca-alginate). Gelation is studied in situ in the microfluidic system or ex situ after the collection of Na-alginate microparticles. The size, the structure and the alginate concentration of the final microparticles are studied according to the operating parameters of the microfluidic system. The microfluidic method developed throughout this thesis allows to control the size and mechanical properties of Ca-alginate microparticles while avoiding their coalescence, despite the absence of surfactants. Thus, monodispersed Ca-alginate microparticles with a Young’s modulus close to that of RBCs are obtained and the feasibility of their use for ultrasonic measurements is also shown. In addition, alternative methods explored in parallel are presented at the end of the thesis., Ce travail de thèse fait partie d’un projet ANR appelé CUMBA (Cellular Ultrasonic imaging Modality to assess red Blood cell Aggregation in vivo) qui vise à prédire l’agrégation de globules rouges (GRs). En effet, l’hyperagrégation de GRs est un trouble hémorhéologique qui cause des dysfonctions microvasculaires et des thromboses veineuses. De plus, l’hyperagrégation de GRs est liée à l’augmentation de macromolécules plasmatiques inflammatoires, comme lors d’une transplantation d’organe. Ainsi, le but du projet CUMBA est de surveiller in situ l’agrégation de GRs grâce à une modalité d’imagerie ultrasonore mise au point pour du diagnostic précoce de l’hyperagrégation. Dans ce contexte, des microparticules mimes de GRs sont indispensables pour la calibration et les premiers tests sur un modèle. Pour cela, les microparticules doivent être contrôlées en taille et en propriétés mécaniques. De plus, l’utilisation de tensioactifs est à éviter pour l’application dans le dispositif d’imagerie ultrasonore.Cette thèse porte sur une méthode microfluidique à base de gouttes et sans ajout de tensioactifs pour préparer les microparticules mimes de GRs. Les microparticules sont obtenues par gélification ionique d’un polymère naturel, l’alginate de sodium (Na-alginate), avec du chlorure de calcium pour former un gel d’alginate de calcium (Ca-alginate). La gélification est étudiée in situ dans le système microfluidique ou ex situ après la collecte de microparticules de Na-alginate. La taille, la structure et la teneur en alginate des microparticules de Na- et Ca-alginate formées sont étudiées en fonction des paramètres opératoires du montage microfluidique. La méthode microfluidique développée tout au long de cette thèse permet de contrôler la taille et les propriétés mécaniques des microparticules de Ca-alginate tout en évitant leur coalescence, malgré l’absence de tensioactifs. Ainsi, des microparticules de Ca-alginate monodisperses et présentant un module de Young proche des GRs sont obtenues et la faisabilité de leur utilisation pour des mesures ultrasonores est également montrée. Par ailleurs, des méthodes alternatives explorées en parallèle sont présentées à la fin de la thèse.

The solid state study of an Active Pharmaceutical Ingredient (API) is a critical step in the development of pharmaceuticals. Depending on the crystallization conditions, a molecule can appear in different forms or phases (polymorphs, solvates, salts, co-crystal, amorphous), each of them having significantly different physico-chemical properties such as solubility, crystal habit, hygroscopicity or stability. Then the purpose is to identify the solid form that provides the most appropriate properties and to determine its crystallization conditions. Therefore, a wide screening is necessary. Moreover, due to the stochasticity of the nucleation phenomenon, a large number of experiments is required in order to obtain reliable data which leads to significant consumption of raw material. In this context High Throughput Screening techniques have been developed. Among them, droplet-based microfluidics seems to be promising. Indeed, it allows the generation of hundreds of monodisperse droplets (isolated from each other by oil), each of them being considered as independent nano-crystallizer (volume of about one hundred nanoliter).This thesis presents the development of a microfluidic platform for solid state study of API. First, this versatile platform allows to start directly from the powder, to dissolve it in different solvents, to estimate solubilities online and to prepare supersaturated solutions. Second, we recrystallize the API in the droplets for different experimental conditions: chemical composition, API concentration and temperature. Finally, the crystals obtained are characterized in situ by Raman microscopy and X-ray diffraction. If it is necessary, crystals can be collected for ex-situ X-ray Diffraction., L’étude de l’état solide d’un principe actif (PA) est une étape cruciale dans le développement d’un médicament. En fonction des conditions de cristallisation, une molécule peut apparaitre sous différentes formes ou phases (polymorphes, solvates, sels, co-cristaux, amorphes) ayant chacune des propriétés physico-chimiques qui leurs sont propres comme la solubilité, la morphologie, l’hygroscopicité et la stabilité. Le but est donc d’identifier la forme solide permettant d’obtenir les propriétés les plus adaptées et de déterminer ses conditions de cristallisation. Pour cela, un criblage complet est nécessaire. De plus, la nucléation étant un phénomène stochastique, un grand nombre d’expériences doivent être réalisées pour obtenir des données fiables, ce qui entraine une consommation importante de matière première. Dans ce contexte, des techniques de criblage à haut débit ont été développées. Parmi celles-ci, la microfluidique à base de goutte permet la génération de centaine de gouttes monodispersées (isolées les unes des autres par de l’huile) pouvant être considérées comme des nano-cristallisoirs indépendants (volume de l’ordre de la centaine de nanolitres).Cette thèse présente le développement d’une plateforme microfluidique pour l’étude de l’état solide de PA. Dans un premier temps, cette plateforme permet de partir directement de la poudre, de la dissoudre dans différents solvants, d’estimer ses solubilités en fonction de la température et de préparer des solutions sursaturées. Puis dans un deuxième temps, le PA est cristallisé dans des gouttes avec différentes conditions expérimentales : compositions chimiques, concentration et température. Enfin, les cristaux obtenus sont caractérisés in situ à l’aide d’un microscope Raman ou par Diffraction des Rayons X. Si nécessaire les cristaux peuvent être récupérés pour des analyses plus précises en ex-situ.

The aim of this work is the development of a versatile microfluidique platform for protein crystallization, combining statistical studies, material saving, speed of execution and ease of use. The aim is to develop a unique tool to address the different issues of proteins crystallization: identification of a robust crystallization condition via screening and optimization, and co-crystallization of a protein with ligands for structure-based drug design. For this purpose, a versatile droplet-based microfluidic method without adding any surfactant is used. It allows the generation of hundreds or even thousands of droplets of a few nanoliters in which the crystallization takes place independently. Droplets composition is controlled by the flow rates of the different solutions using programmable syringe pumps and checked on-line by UV-visible spectroscopy. Many different crystallization conditions can thus be tested quickly: nature and concentration of crystallization agent(s), addition of a ligand, etc. In addition, the crystals obtained using this microfluidic platform are characterized in situ and ex situ by X-ray diffraction.The platform is applied to the crystallization of two proteins, first a model protein, lysozyme, and then a protein of pharmaceutical interest, quinone reductase 2. Thus we have developed a tool suitable to constraints of pharmaceutical industry, in order to be transferred to research laboratories for routine use by non-specialists of microfluidics. This platform permits a high throughput screening approach, or HTS, and tends to the automation of both screening and X-ray diffraction., Cette thèse porte sur le développement d’une plateforme microfluidique polyvalente pour la cristallisation des protéines alliant études statistiques, économie de matière, rapidité d’exécution et facilité d’utilisation. L’objectif est de développer un outil unique pour répondre aux différentes problématiques de la cristallisation des protéines : l’identification d’une condition de cristallisation robuste via le criblage et l’optimisation, ainsi que la co-cristallisation d’une protéine et de ligands pour le structure-based drug design. Pour cela, une méthode microfluidique versatile à base de gouttes et sans tensioactif est utilisée. Elle permet la génération de centaines voire milliers de gouttes de quelques nanolitres dans lesquelles la cristallisation peut avoir lieu indépendamment. La composition des gouttes est contrôlée par les débits des différentes solutions à l’aide de pousse-seringues programmable et vérifiée en ligne par spectrométrie UV-visible. De nombreuses conditions de cristallisation différentes peuvent ainsi être testées rapidement : nature et concentration de(s) agent(s) de cristallisation, ajout d’un ligand, etc. De plus, les cristaux obtenus à l’aide de cette plateforme microfluidique sont caractérisés in situ et ex situ par diffraction des rayons X. Cette plateforme est appliquée à la cristallisation de deux protéines, tout d’abord une protéine modèle, le lysozyme, puis une protéine d’intérêt pharmaceutique, la quinone réductase de type 2. Ainsi, nous avons développé un outil adapté aux contraintes de l’industrie pharmaceutique qui peut être transféré dans un laboratoire de recherche pour une utilisation de routine par des non-spécialistes de la microfluidique. La plateforme permet d’avoir une approche de criblage à haut débit, ou HTS, et de tendre vers l’automatisation à la fois du criblage et de la diffraction des rayons X.