This chapter presents an overview of microfluidic devices reported in the literature, used to develop methodologies for nucleation of biomolecules, with crystal size control, and for collecting thermodynamic and kinetic data. Section 2 is dedicated to the properties of microfluidic devices through materials used for their fabrication and for crystal analysis. Section 3 describes the variety of microfluidic devices available and how to handle them to produce flows, droplets, and/or wells of micrometer size. These devices use crystallization methods inspired by batch processes, and they are mainly used for protein crystallization. Section 4 focuses on fundamental properties of biomolecule crystallization determined using droplet-based microfluidics: nucleation kinetics, nucleation rate, and effective interfacial energy crystal/solution. Section 5 explains how the kinetic effect of confinement due to micrometer size, and so nanovolumes, leads to isolation of different phases. These latter are characterized by X-ray diffraction (XRD), and methods to minimize manual handling of crystals for XRD are also presented, with appropriate equipment to store the crystals.

A new crystallization platform coupling droplet-based microfluidics with two photon and second harmonic generation microscopy operating with a femtosecond pulsed wave laser of tunable wavelength is presented. The present paper demonstrates the performance of this set-up by conducting a large number of independent crystallization experiments in small (nL) unstirred reactors. We used ortho-aminobenzoic acid (o-ABA) as a model substance: it is a polymorphic substance known to exhibit concomitant polymorphism. It was possible to rapidly image and discriminate crystals with a centrosymmetric structure from crystals with a non-centrosymmetric structure by using this new coupling system. By conducting thorough crystallization studies, our set up allowed the impact of small volume on the nucleation behavior of o-ABA polymorphs to be investigated and the occurrence of both solvent mediated transformation and solid-solid transition to be evidenced. In particular, we show that small volume favors the nucleation of the most stable crystal form of o-ABA.

In this work, insights into the structural rearrangements occurring in aqueous solution, prior to the nucleation of different 4'-hydroxyacetophenone (HAP) forms from water, were obtained, through a combination of thermomicroscopy, microdifferential scanning calorimetry, density and speed of sound measurements, and molecular dynamics simulations. The results confirmed our previous observation that cooling crystallization of HAP is intermediated by liquid-liquid phase separation (LLPS) and, depending on the initially selected concentration range, selectively leads to the formation of different crystal forms. Analysis of the solution properties before the onset of LLPS revealed that, in the HAP mole fraction range x(HAP) < 0.004 (Zone I), where hydrate H2 ultimately crystallizes, small, solvated clusters are initially present in solution, which remain approximately invariant in size, shape, and HAP/H2O proportion as the temperature decreases. For the x(HAP) > 0.005 range (Zone III), where anhydrous form I crystallizes, large HAP/water aggregates (that can even percolate the whole system as x(HAP) approaches the 0.005 limit) are already initially present in solution. As cooling progresses, they become more compact, a process accompanied by a reduction in water content, which is more significant as the solution concentration increases. The 0.004 < x(HAP) < 0.005 (Zone II) range corresponds to a transition region where, as x(HAP) increases, the physical properties of the solution initially evolve from those typical of Zone I and, at a certain point, abruptly change and start converging to those typical of Zone III. In all zones, the colloidal particles formed upon LLPS (from which crystallization results) can also reduce their water content on cooling, but the extent of this process increases as x(HAP) moves from Zones I and II, where hydrates are formed, to Zone III, where anhydrous form I is produced.

We developed a rapid polymorphic screening approach based on contracting sessile microdroplets, which offers several advantages: (1) achieves very high supersaturation to facilitate formation of metastable forms; (2) allows systematic labeling of samples; (3) gives access to the statistical distribution of polymorphic selectivity as a function of experimental conditions; (4) ensures the formation of crystal for each droplet, addressing the problem of uncrystallized droplets in traditional microfluidics. We studied the competitive nucleation of d-mannitol polymorphs and investigated the effect of droplet volume on polymorphic selectivity. We showed that our observed polymorph distributions at different volumes are qualitatively consistent with the predictions of classical nucleation theory except for very small volumes where thermodynamic confinement or surface effects could play a substantial role. Overall, our microfluidic approach can be a promising tool not only for routine screening of pharmaceutical polymorphs in the industrial context but also in the fundamental understanding of the mechanisms underlying the competitive nucleation of polymorphs. Using microdroplets, we developed an approach to rapidly measure the nucleation statistics of polymorphs. It can be useful in the routine screening of pharmaceutical solid forms, and in the study of the competitive nature of polymorphic nucleation.

Solubility and interfacial energy are two fundamental parameters underlying the competitive nucleation of polymorphs. However, solubility measurement of metastable phases comes with a risk of solventmediated transformations which can render the results unreliable. In this work, we present a rapid microfluidic technique for measuring aqueous solubility of the metastable form using KDP Phase IV as a model system. This bracketing approach involves analyzing the dissolution behavior of crystals in contact with supersaturated microdroplets generated via evaporation. Then, with the help of our recently developed nucleation time measurement technique, together with Mersmann calculation of interfacial energies from solubilities, we were able to access the interfacial energies of both metastable and stable phases. To gain further insights into the observed nucleation behavior, we employed the Classical Nucleation Theory (CNT) to model the competition of polymorphs using our measured solubility and calculated interfacial energies. The results show that the stable form is favored at lower supersaturation while the metastable form is favored at higher supersaturation, in good agreement with our observations and experimental reports in the literature. Overall, our microfluidic approach allows access to unprecedentedly deep levels of supersaturation and reveals an interesting interplay between thermodynamics and kinetics in polymorphic nucleation. The experimental methods and insights presented herein can be of great interest, notably in the mineral processing and pharmaceutical industry.

This study describes the preparation, characterization, and influence of the enantiopure vs. racemic coformer on the physico-chemical properties of a pharmaceutical cocrystal. For that purpose, two new 1:1 cocrystals, namely lidocaine:dl-menthol and lidocaine:d-menthol, were prepared. The menthol racemate-based cocrystal was evaluated by means of X-ray diffraction, infrared spectroscopy, Raman, thermal analysis, and solubility experiments. The results were exhaustively compared with the first menthol-based pharmaceutical cocrystal, i.e., lidocaine:l-menthol, discovered in our group 12 years ago. Furthermore, the stable lidocaine/dl-menthol phase diagram has been screened, thoroughly evaluated, and compared to the enantiopure phase diagram. Thus, it has been proven that the racemic vs. enantiopure coformer leads to increased solubility and improved dissolution of lidocaine due to the low stable form induced by menthol molecular disorder in the lidocaine:dl-menthol cocrystal. To date, the 1:1 lidocaine:dl-menthol cocrystal is the third menthol-based pharmaceutical cocrystal, after the 1:1 lidocaine:l-menthol and the 1:2 lopinavir:l-menthol cocrystals reported in 2010 and 2022, respectively. Overall, this study shows promising potential for designing new materials with both improved characteristics and functional properties in the fields of pharmaceutical sciences and crystal engineering.

Nucleation, the birth of a stable cluster from a 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, 1.5, and 5.5 pl microdroplets reveals an interesting interplay between confinement effects and shifting of nucleation mechanisms. Overall, our findings highlight the need to treat nucleation stochastically rather than deterministically to bridge the gap between theory and experiment.

We investigated the pressure dependence of gas flow and field ion intensity of a coaxial ion source operating at room temperature over a wide pressure range, testing various gases and ionisation voltages. Flow conductance measurements taking into account the different gases' viscosity and molecular mass consistently exhibit a generic pattern. Three different flow regimes appear with increasing upstream pressure. Since the coaxial ion source supplies the gas locally, very near the apex of the tip where ionisation occurs, large ionisation currents can be obtained without degrading the propagation conditions of the beam. Compared with field ionisation in a partial pressure chamber, using the coaxial ion source increases the ion current a hundredfold for the same residual low pressure. We also show that the gas flow regime does not impact ionisation yield. Although a fuller characterisation remains to be performed, brightness reaches 3 x 10 11 A/m 2 /sr at 12kV extracting voltage. a) https://www.cinam.univ-mrs.fr/

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 repeated 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 set-up 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.

In eukaryotic cells, membrane proteins play a crucial role. They fall into three categories: intrinsic proteins, extrinsic proteins, and proteins that are essential to the human genome (30% of which is devoted to encoding them). Hydrophobic interactions inside the membrane serve to stabilize integral proteins, which span the lipid bilayer. This review investigates a number of computational and experimental methods used to study membrane proteins. It encompasses a variety of technologies, including electrophoresis, X-ray crystallography, cryogenic electron microscopy (cryo-EM), nuclear magnetic resonance spectroscopy (NMR), biophysical methods, computational methods, and artificial intelligence. The link between structure and function of membrane proteins has been better understood thanks to these approaches, which also hold great promise for future study in the field. The significance of fusing artificial intelligence with experimental data to improve our comprehension of membrane protein biology is also covered in this paper. This effort aims to shed light on the complexity of membrane protein biology by investigating a variety of experimental and computational methods. Overall, the goal of this review is to emphasize how crucial it is to understand the functions of membrane proteins in eukaryotic cells. It gives a general review of the numerous methods used to look into these crucial elements and highlights the demand for multidisciplinary approaches to advance our understanding.

Kristen Fichthorn opened the discussion of the paper by Jutta Rogal: I find the shapes of the nuclei that form in your studies interesting, as there is evidence that fivefold twinned nuclei and nuclei with stacking faults can occur1,2 in nucleation of fcc systems. 1 J. C. E, L. Wang, Y. C

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 numbers 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 60 pL and 4 pL microdroplets 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.

Kristen Fichthorn opened the discussion of the introductory lecture by James De Yoreo: I noted that in your potential of mean force (PMF) plots for various particle orientations, fluctuations occur such that different orientations could be preferred at a fixed distance. Could this affect the ag

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 variation of the solution density and water activity due to the evolving solute concentration, and the diffusive interaction between neighboring droplets. 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.

This review examines the preparation of alginate hydrogel microparticles by using droplet-based microfluidics, a technique widely employed for its ease of use and excellent control of physicochemical properties, with narrow size distribution. The gelation of alginate is realized "on-chip" and/or "off-chip", depending on where cross-linkers are introduced. Various strategies are described and compared. Microparticle properties such as size, shape, concentration, stability and mechanical properties are discussed. Finally, we consider future perspectives for the preparation of hydrogel microparticles and their potential applications.

The use of a multi-well system allowed us to measure the solubility of two anhydrous forms of olanzapine in pure solvents with different polarities in a temperature range of 20 to 60° Celsius (°C) with a small quantity of material. After that, we carried out cooling crystallization experiments in a stagnant medium with a small volume of 0.8 mL and a small quantity of material. We found that the solubility increased with the solvent polarity in the order: methanol > ethanol > acetonitrile > ethyl acetate > tetrahydrofuran > toluene. We demonstrated the effect of the solvents and the cooling rates on the crystal habit, crystal phase, and crystal size distribution.

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{\textdegree}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.

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.