CNT effective interfacial energy and pre-exponential kinetic factor from measured NaCl crystal nucleation time distributions in contracting microdroplets.

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.

Exploring the World of Membrane Proteins: Techniques and Methods for Understanding Structure, Function, and Dynamics.

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.

Evaporation Dynamics of Sessile Saline Microdroplets in Oil.

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.

Nucleation in sessile saline microdroplets: induction time measurement via deliquescence-recrystallization cycling.

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.

Localizing and inducing primary nucleation.

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.

Highly efficient chiral resolution of DL-arginine by cocrystal formation followed by recrystallization under preferential-enrichment conditions.

An excellent chiral symmetry-breaking spontaneous enantiomeric resolution phenomenon, denoted preferential enrichment, was observed on recrystallization of the 1:1 cocrystal of dl-arginine and fumaric acid, which is classified as a racemic compound crystal with a high eutectic ee value (>95 %), under non-equilibrium crystallization conditions. On the basis of temperature-controlled video microscopy and in situ time-resolved solid-state (13) C NMR spectroscopic studies on the crystallization process, a new mechanism of phase transition that can induce preferential enrichment is proposed.

Monitoring picoliter sessile microdroplet dynamics shows that size does not matter.

We monitor the dissolution of arrayed picoliter-size sessile microdroplets of the aqueous phase in oil, generated using a recently developed fluidic device. Initial pinning of the microdroplet perimeter leads to a nearly constant contact diameter, thus contraction proceeds via microdroplet (micrometer-diameter) height and contact angle reductions. This confirms that picoliter microdroplets contraction or dissolution due to the selective diffusion of water in oil has comparable dynamics with microliter droplet evaporation in air. We observe a constant microdroplet dissolution rate in different aqueous solutions. The application of this simple model to solvent-diffusion-driven crystallization experiments in confined volumes, for instance, would allow us to determine precisely the concentration in the microdroplet during an experiment and particularly at nucleation.

Microcrystals with enhanced emission prepared from hydrophobic analogues of the green fluorescent protein chromophore via reprecipitation.

Certain synthetic analogues of the green fluorescent protein (GFP) chromophore are almost nonfluorescent in dilute solutions but are strongly light-emissive in the solid state, thus exhibiting aggregation-induced emission (AIE) behavior. In the present work, two such hydrophobic derivatives of the GFP chromophore known to be fluorescent in the crystalline state (p-hexyloxy- and p-dodecyloxybenzylideneimidazolinone) were used to prepare aqueous suspensions of particles via a mild solvent-exchange reprecipitation (RP) method. This evolution was monitored at various experimental conditions by UV-vis absorption and fluorescence spectroscopy, fluorescence microscopy, as well as electron transmission and scanning microscopy. Both compounds spontaneously produced platelet-like microcrystals, the size and shape of which were influenced by the experimental conditions. The dodecyl derivative also led to the concomitant formation of nanofibers, a tendency reinforced by addition of poly(acrylic acid) to the RP medium. The photoluminescence properties of the solids produced by RP were compared to pristine microcrystalline powders obtained by crystallization in an organic solvent. Significant differences in the emission properties were found and are discussed. This study illustrates the fact that AIE fluorescence is strongly dependent on the nature of the particles and hence on the preparation methods. Being aware of these variations is important for the preparation and subsequent use of AIE-active compounds as fluorescent materials.

New Lidocaine-Based Pharmaceutical Cocrystals: Preparation, Characterization, and Influence of the Racemic vs. Enantiopure Coformer on the Physico-Chemical Properties.

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.

Predictive nucleation of crystals in small volumes and its consequences.

We propose another way of getting to the bottom of nucleation by using finite volume systems. Here we show, using a sharp tip, that a single nucleation event is launched as soon as the tip touches the supersaturated confined metastable solution. We thus control spatial and temporal location and demonstrate that confinement allows us to carry out predictive nucleation experiments. This control is a major step forward in understanding the factors influencing the nucleation process and its underlying physics.

Preparation of alginate hydrogel microparticles by gelation introducing cross-linkers using droplet-based microfluidics: a review of methods.

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 2C putative helicase of echovirus 30 adopts a hexameric ring-shaped structure.

The 2C protein, which is an essential ATPase and one of the most conserved proteins across the Picornaviridae family, is an emerging antiviral target for which structural and functional characterization remain elusive. Based on a distant relationship to helicases of small DNA viruses, piconavirus 2C proteins have been predicted to unwind double-stranded RNAs. Here, a terminally extended variant of the 2C protein from echovirus 30 has been studied by means of enzymatic activity assays, transmission electron microscopy, atomic force microscopy and dynamic light scattering. The transmission electron-microscopy technique showed the existence of ring-shaped particles with ∼12 nm external diameter. Image analysis revealed that these particles were hexameric and resembled those formed by superfamily 3 DNA virus helicases.

Advances in the Use of Microfluidics to Study Crystallization Fundamentals.

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.

Crystallization via tubing microfluidics permits both in situ and ex situ X-ray diffraction.

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.

Exploring bovine pancreatic trypsin inhibitor phase transitions.

This paper presents an investigation of the phase diagram of BPTI (bovine pancreatic trypsin inhibitor)/350 mM KSCN at pH 4.9 by direct observation and numerical simulations. We report optical microscopy and light and X-ray scattering experiments coupled with theoretical data analysis using numerical tools. The phase diagram is thoroughly determined, as a function of temperature. Two polymorphs are observed by video microscopy and their solubility measured. In this phase diagram, the liquid-liquid phase separation (LLPS) is metastable with respect to the solid-liquid phase separation. Above the T(L-L) boundary curve, solutions are composed of a mixture of BPTI monomers and decamers. Attractive interactions are stronger between decamers than between monomers. Below the T(L-L) boundary curve, the dense phase is highly concentrated in protein and composed of BPTI decamers alone. Thus, the driving force for liquid-liquid or liquid-solid phase separation is the attraction between decamers at low pH. The structure factors of the dense phases are characteristic of repulsive dense phases because of a hard sphere repulsion core, meaning that in the dense phase proteins are actually in contact (interparticle distance of 53 A). In agreement with the Oswald rule of stages, LLPS occurs prior to and impedes the solid nucleation.