Two-step Nucleation Mechanism Research Articles

Real-Time Observation of Protein Dense Liquid Cluster Evolution during Nucleation in Protein Crystallization

Controlled navigation in the phase diagram of protein crystallization and probing by advanced Dynamic Light Scattering (DLS) technology provided new information and more insight on the early processes during the nucleation process. The observed hydrodynamic radius distribution pattern clearly reveals a two-step mechanism of nucleation and the occurrence of liquid dense protein clusters, which were verified by transmission electron microscopy. The growth kinetics of these protein clusters, forming distinct radii fractions, is analyzed in real time. Further, the data confirmed that critical nuclei show a distinctly different radius distribution than the liquid dense clusters. The data and results provide experimental evidence that during nucleation, a formation of distinct liquid clusters with high protein concentration occur prior to a transition to crystal nuclei by increasing the internal structural order of these clusters, subsequently.

Two-Step Amyloid Aggregation: Sequential Lag Phase Intermediates

The self-assembly of proteins into fibrillar structures called amyloid fibrils underlies the onset and symptoms of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. However, the molecular basis and mechanism of amyloid aggregation are not completely understood. For many amyloidogenic proteins, certain oligomeric intermediates that form in the early aggregation phase appear to be the principal cause of cellular toxicity. Recent computational studies have suggested the importance of nonspecific interactions for the initiation of the oligomerization process prior to the structural conversion steps and template seeding, particularly at low protein concentrations. Here, using advanced single-molecule fluorescence spectroscopy and imaging of a model SH3 domain, we obtained direct evidence that nonspecific aggregates are required in a two-step nucleation mechanism of amyloid aggregation. We identified three different oligomeric types according to their sizes and compactness and performed a full mechanistic study that revealed a mandatory rate-limiting conformational conversion step. We also identified the most cytotoxic species, which may be possible targets for inhibiting and preventing amyloid aggregation.

Isothermal Crystallization of PC61BM in Thin Layers Far below the Glass Transition Temperature

An isothermal study was performed on 1.25 μm thin layers of PC61BM, a benchmark acceptor for organic photovoltaics. Using fast scanning chip calorimetry allowed for a scanning rate of 30 000 K·s–1, ensuring that all non-isothermal effects were avoided. The effect of isothermal treatments above and far below the glass transition was investigated. It was proven that PC61BM can crystallize below its glass transition, a behavior seen before for several organic glasses. Isothermal treatments as far as ca. 100 K below the glass transition lead to an initial enthalpic relaxation process around the glass transition, which over time gives rise to crystals melting around 300 °C. These observations show a remarkable similarity with the two-step nucleation mechanism documented in the literature. A standard crystallization process was observed for treatments above the glass transition. By combining chip calorimetry pretreatments with atomic force microscopy, different crystal morphologies were visualized for these two...

Do protein crystals nucleate within dense liquid clusters?

Protein-dense liquid clusters are regions of high protein concentration that have been observed in solutions of several proteins. The typical cluster size varies from several tens to several hundreds of nanometres and their volume fraction remains below 10(-3) of the solution. According to the two-step mechanism of nucleation, the protein-rich clusters serve as locations for and precursors to the nucleation of protein crystals. While the two-step mechanism explained several unusual features of protein crystal nucleation kinetics, a direct observation of its validity for protein crystals has been lacking. Here, two independent observations of crystal nucleation with the proteins lysozyme and glucose isomerase are discussed. Firstly, the evolutions of the protein-rich clusters and nucleating crystals were characterized simultaneously by dynamic light scattering (DLS) and confocal depolarized dynamic light scattering (cDDLS), respectively. It is demonstrated that protein crystals appear following a significant delay after cluster formation. The cDDLS correlation functions follow a Gaussian decay, indicative of nondiffusive motion. A possible explanation is that the crystals are contained inside large clusters and are driven by the elasticity of the cluster surface. Secondly, depolarized oblique illumination dark-field microscopy reveals the evolution from liquid clusters without crystals to newly nucleated crystals contained in the clusters to grown crystals freely diffusing in the solution. Collectively, the observations indicate that the protein-rich clusters in lysozyme and glucose isomerase solutions are locations for crystal nucleation.

Advances in Modeling of Solidification Microstructures

In different manufacturing processes, such as casting, welding, and additive manufacturing, metallic materials go through solidification to produce parts with different sizes and shapes. Microstructures that develop during solidification control the property and performance of the manufactured parts, thereby predicting the solidification microstructures as functions of alloying composition and processing parameters is essential to control the quality of products. The multiscale nature of solidification microstructures, which depend on temperature distribution, solute concentration, capillary forces, and kinetic length, make the understanding and prediction of these microstructures exceptionally difficult. In recent years, and because of emergence of new and powerful supercomputers, it has become more feasible to computationally simulate the materials nanoand microstructures in large scales and with finest details. The computational models for solidification of metallic materials need to be developed based on the actual multiphysics of solidification, while considering the need for efficient numerical algorithms to solve the governing equations of the models. To quantitatively predict solidification microstructures, computational models at mesoscale need information from theory, experiments, lower scale models (e.g., density functional theory calculations, molecular dynamics simulations, etc.), and phase diagram calculations. The current computational models for simulating dendritic growth at the microscopic scale are based on different methods such as phase field (diffusive interface), level set, direct interface tracking, and cellular automaton methods. Each of these methods has its advantages and disadvantages: some can simulate the finest details of solidification microstructures with high accuracy, while others can simulate dendritic growth in large-scale domains with high computational efficiency. In this Journal of Metals topic, we present recent contributions in modeling of solidification microstructures. Damien Tourret et al. present a three-dimensional (3D) version of the dendritic needle network (DNN) model for directional solidification. They apply the DNN model to predict the stable range of primary dendritic spacings for an Al-9.8 wt%Si alloy over a range of growth velocities. They compare their predictions to spacings measured from in situ x-ray imaging of directional solidification experiments. Mohsen Eshraghi et al. present a parallel 3D lattice Boltzmann-cellular automaton model to simulate dendritic growth during solidification of metallic binary alloys. Their large-scale simulations show a great scale-up performance up to 40,000 computing cores and an excellent speed-up performance on up to 1000 computing cores. Yasushi Shibuta and co-workers performed simulations of solidification from atomic to microstructural levels using a graphics processing unit (GPU) architecture. They use million-atom molecular dynamics simulations to study nucleation of solid phase in undercooled melt and to capture evolution of anisotropy for solid seeds. Using a quantitative phasefield model, they simulate dendrite growth in directional solidification at millimeter scale in two– dimensional (2D) and 3D by multi-GPU computation on a supercomputer. These two articles present promising techniques to predict solidification microstructures in large macro-scale domains with good computational efficiency. Alexander Monas et al. present 3D phase-field simulations of solidification microstructures of MgAl alloy. They use the Calphad method to obtain the phase diagram and necessary parameters for their simulations. Michael Rappaz and Guven Kurtuldu discuss a new nucleation mechanism in liquid metallic alloys based on thermodynamics arguments. They explain that the two-step nucleation mechanism starts with Mohsen Asle Zaeem is the guest editor for the Solidification Committee of the TMS Materials Processing & Manufacturing Division, and coordinator of the topic Advances in Modeling of Solidification Microstructures in this issue. JOM, Vol. 67, No. 8, 2015

A Multi-step Thermodynamic Model for Alumina Formation during Aluminum Deoxidation in Fe–O–Al Melt

Based on the two-step nucleation mechanism, a multi-step thermodynamic model for alumina inclusion formation during aluminum deoxidation process was proposed in Fe–O–Al melt. Thermodynamic properties of metastable intermediates including (Al2O3) n clusters for prenucleation and α-Al2O3 nanoparticle for growth process were calculated using density functional theory. Furthermore, Gibbs free energy change of forming the intermediate by reaction between the dissolved aluminum (Al) and oxygen (O) in the melt was calculated. The results indicated that the thermodynamics of (Al2O3) n at steelmaking temperature are dependent on their structures, while that of α-Al2O3 nanoparticle are dependent on their size. The nuclei of α-Al2O3 which was originated from (Al2O3) n aggregated under a high supersaturation ratio of Al and O(R s) in the melt. There existing excess oxygen because of the low R s, but the secondary inclusions will be formed during the cooling process due to the excess oxygen. The nuclei lager than 20 nm can grow up spontaneously and instantaneously into primary inclusions because of thermodynamic drive. It is difficult to control the size of α-Al2O3 to be less than 20 nm, in the aluminum deoxidation process of the current conditions of steelmaking.

Learning about the nucleation pathway of MgO·Al2O3spinel from an Fe–O–Al–Mg melt using a two-step nucleation mechanism

MgO·Al2O3spinel nucleation is attributed to the organization of clusters into ordered lattice structures by a multi-step nucleation pathway.

Recent advances in the understanding of two-step nucleation of protein crystals.

The two-step mechanism of nucleation of crystals in solutions posits that the formation of crystal nuclei occurs within structures of extended lifetimes, in which the nucleating solute is at high concentration. The validity of this mechanism has been demonstrated for proteins, small-molecule organic and inorganic materials, colloids, and polymers. Due to large molecule sizes, proteins are an ideal system to study the details of this nucleation pathway, in particular the formation mechanisms of the nucleation precursors and the associated physico-chemical rules. The precursors of protein crystal nuclei are protein-rich clusters of sizes ∼100 nm that contain 10,000-100,000 molecules and occupy less than 10(-3) of the total solution volume. Here we demonstrate, using oblique illumination microscopy, the liquid nature of the clusters of the protein lysozyme and reveal their inhomogeneous structure. We test a hypothesis put forth by theory that clusters primarily consist of transient protein oligomers. For this, we explore how varying the strength of the Coulomb interaction affects the cluster characteristics. We find that the cluster's size is insensitive to variations of pH and ionic strength. In contrast, the addition of urea, a chaotropic agent that leads to protein unfolding, strongly decreases the cluster size. Shear stress, a known protein denaturant, induced by bubbling of the solutions with an inert gas, elicits a similar response. These observations support partial protein unfolding, followed by dimerization, as the mechanism of cluster formation. The amide hydrogen-deuterium exchange, monitored by nuclear magnetic resonance, highlights that lysozyme conformational flexibility is a condition for the formation of the protein-rich clusters and facilitates the nucleation of protein crystals.

On the question of two-step nucleation in protein crystallization.

We report a real-time study on protein crystallization in the presence of multivalent salts using small angle X-ray scattering (SAXS) and optical microscopy, focusing particularly on the nucleation mechanism as well as on the role of the metastable intermediate phase (MIP). Using bovine beta-lactoglobulin as a model system in the presence of the divalent salt CdCl2, we have monitored the early stage of crystallization kinetics which demonstrates a two-step nucleation mechanism: protein aggregates form a MIP, which is followed by the nucleation of crystals within the MIP. Here we focus on characterizing and tuning the structure of the MIP using salt and the related effects on the two-step nucleation kinetics. The results suggest that increasing the salt concentration near the transition zone pseudo-c** enhances the energy barrier for both MIPs and crystal nucleation, leading to slow growth. The structural evolution of the MIP and its effect on subsequent nucleation is discussed based on the growth kinetics. The observed kinetics can be well described, using a rate-equation model based on a clear physical two-step picture. This real-time study not only provides evidence for a two-step nucleation process for protein crystallization, but also elucidates the role and the structural signature of the MIPs in the nonclassical process of protein crystallization.

Nucleation is second order: an apparent kinetically effective nucleus of two for Ir(0)n nanoparticle formation from [(1,5-COD)Ir(I)·P2W15Nb3O62]8- plus hydrogen.

Nucleation initiates phase changes across nature. A fundamentally important, presently unanswered question is if nucleation begins as classical nucleation theory (CNT) postulates, with n equivalents of monomer A forming a "critical nucleus", A(n), in a thermodynamic (equilibrium) process. Alternatively, is a smaller nucleus formed at a kinetically limited rate? Herein, nucleation kinetics are studied starting with the nanoparticle catalyst precursor, [A] = [(Bu4N)5Na3(1,5-COD)Ir(I)·P2W15Nb3O62], forming soluble/dispersible, B = Ir(0)(∼300) nanoparticles stabilized by the P2W15Nb3O62(9-) polyoxoanion. The resulting sigmoidal kinetic curves are analyzed using the 1997 Finke-Watzky (hereafter FW) two-step mechanism of (i) slow continuous nucleation (A → B, rate constant k(1obs)), then (ii) fast autocatalytic surface growth (A + B → 2B, rate constant k(2obs)). Relatively precise homogeneous nucleation rate constants, k(1obs), examined as a function of the amount of precatalyst, A, reveal that k(1obs) has an added dependence on the concentration of the precursor, k(1obs) = k(1obs(bimolecular))[A]. This in turn implies that the nucleation step of the FW two-step mechanism actually consists of a second-order homogeneous nucleation step, A + A → 2B (rate constant, k(1obs(bimol))). The results are significant and of broad interest as an experimental disproof of the applicability of the "critical nucleus" of CNT to nanocluster formation systems such as the Ir(0)n one studied herein. The results suggest, instead, the experimentally-based concepts of (i) a kinetically effective nucleus and (ii) the concept of a first-observable cluster, that is, the first particle size detectable by whatever physical methods one is currently employing. The 17 most important findings, associated concepts, and conclusions from this work are provided as a summary.

Two-step nucleation mechanism in solid–solid phase transitions

The microscopic kinetics of ubiquitous solid-solid phase transitions remain poorly understood. Here, by using single-particle-resolution video microscopy of colloidal films of diameter-tunable microspheres, we show that transitions between square and triangular lattices occur via a two-step diffusive nucleation pathway involving liquid nuclei. The nucleation pathway is favoured over the direct one-step nucleation because the energy of the solid/liquid interface is lower than that between solid phases. We also observed that nucleation precursors are particle-swapping loops rather than newly generated structural defects, and that coherent and incoherent facets of the evolving nuclei exhibit different energies and growth rates that can markedly alter the nucleation kinetics. Our findings suggest that an intermediate liquid should exist in the nucleation processes of solid-solid transitions of most metals and alloys, and provide guidance for better control of the kinetics of the transition and for future refinements of solid-solid transition theory.

Probing methane hydrate nucleation through the forward flux sampling method.

Understanding the nucleation of hydrate is the key to developing effective strategies for controlling methane hydrate formation. Here we present a computational study of methane hydrate nucleation, by combining the forward flux sampling (FFS) method and the coarse-grained water model mW. To facilitate the application of FFS in studying the formation of methane hydrate, we developed an effective order parameter λ on the basis of the topological analysis of the tetrahedral network. The order parameter capitalizes the signature of hydrate structure, i.e., polyhedral cages, and is capable of efficiently distinguishing hydrate from ice and liquid water while allowing the formation of different hydrate phases, i.e., sI, sII, and amorphous. Integration of the order parameter λ with FFS allows explicitly computing hydrate nucleation rates and obtaining an ensemble of nucleation trajectories under conditions where spontaneous hydrate nucleation becomes too slow to occur in direct simulation. The convergence of the obtained hydrate nucleation rate was found to depend crucially on the convergence of the spatial distribution for the spontaneously formed hydrate seeds obtained from the initial sampling of FFS. The validity of the approach is also verified by the agreement between the calculated nucleation rate and that inferred from the direct simulation. Analyzing the obtained large ensemble of hydrate nucleation trajectories, we show hydrate formation at 220 K and 500 bar is initiated by the nucleation events occurring in the vicinity of water-methane interface, and facilitated by a gradual transition from amorphous to crystalline structure. The latter provides the direct support to the proposed two-step nucleation mechanism of methane hydrate.

Mechanism of particle formation in radical emulsion copolymerization of styrene with α-tert-butoxy-ω-vinylbenzyl-polyglycidol macromonomer

In the batch emulsion copolymerization of styrene and α-tert-butoxy-ω-vinylbenzyl-polyglycidol macromonomer, carried out at macromonomer concentrations exceeding the critical micelle concentration (cmc), particles are formed by a two-step coagulative nucleation mechanism. This mechanism leaves its mark on morphology of particle interface, rate of polymerization and on molecular weight distribution of the obtained polymer. AFM studies revealed that the interface of particles is composed of objects with dimensions close to dimensions of the primary particles. Compartmentalization of styrene in the macromonomer micelles leads to the higher initial rate of styrene conversion than in the similar macromonomer free homopolymerization of styrene. The initial polymerization in the monomer-swollen macromonomer micelles, similar to the microemulsion polymerization, is responsible for the formation of the highest molecular weight component. In the mature particles there are two different polymerization loci: the interfacial layer and the core. This leads to bimodal molecular weight distribution of the formed polymer.

Electrogeneration of Single Nanobubbles at Sub-50-nm-Radius Platinum Nanodisk Electrodes

The electrochemical generation of individual H(2) nanobubbles at Pt nanodisk electrodes immersed in a 0.5 M H(2)SO(4) solution is reported. A sudden drop in current associated with the transport-limited reduction of protons is observed in the i–V response at Pt nanodisk electrodes with radii of less than 50 nm. This decrease in current (~95% blockage) corresponds to the formation of a single H(2) nanobubble attached to the nanoelectrode that blocks proton transport to the surface. The current at which nanobubble formation occurs, i(nb)(p), is independent of scan rate and H(2)SO(4) concentration (for [H(2)SO(4)] > 0.1 M), indicating a critical concentration profile of electrogenerated H(2) required to nucleate a nanobubble. Finite element simulation based on Fick’s first law, combined with the Young–Laplace equation and Henry’s law, indicates that the concentration of H(2) near the nanoelectrode surface at i(nb)(p) exceeds the saturation concentration necessary to generate a nanobubble with a size comparable to the electrode size. The rapid dissolution of the nanobubble due to the high inner Laplace pressure is precisely balanced by the electrogeneration of H(2) at the partially exposed Pt surface, resulting in a dynamically stabilized nanobubble. Preliminary measurements of the i–t response during nanobubble formation indicate a two-step nucleation and growth mechanism with time scales on the order of 100 μs (or less) and ~1 ms, respectively.

Characterization of Preferred Crystal Nucleation Sites on Mica Surfaces

Rates of heterogeneous nucleation can be greatly increased not only through control of the chemistry of a surface, but also of its topography. Following previous work in which we showed that scratching a mica surface significantly enhances crystal nucleation from vapor, we here use a new experimental approach to understand better the effect of topography on crystal nucleation. The compounds carbon tetrabromide, camphor, norbornane, and hexachloroethane were deposited from vapor onto mica sheets containing various surface defects, and their nucleation was studied using optical microscopy. Following subsequent evaporation of the crystals, examination of the sites where these had nucleated with a scanning electron microscope enabled the nature of each material’s preferred nucleation sites to be determined, and all four compounds appeared to exhibit a strong preference for sites characterized by delamination of the layered mica structure. Indeed, comparison of the four compounds on the same mica substrates showed that they all favored the same nucleation sites. These observations are attributed to the presence of an acute wedge geometry at the delamination lines, which can provide a thermodynamic reduction in the free energy barrier to nucleation directly from vapor; alternatively, the results are also consistent with a two-step nucleation mechanism via a liquid capillary condensate.

Nucleation of Gas Hydrates within Constant Energy Systems

The early stage of formation of gas hydrates has recently attracted attention as amorphous intermediate gas hydrate structures have been observed, apparently contrary to a classical model of nucleation and some experimental observations. To date, essentially all reported molecular simulations of the nucleation of gas hydrates have been under constant temperature conditions, which does not consider the possible impacts of heat transfer on the nucleation processes. Here we show, using constant energy molecular simulations, that the nuclei at an early stage of the hydrate formation have relatively more crystalline order in comparison with those observed in previous isothermal (NPT or NVT) work. The current work suggests a more transient role for intermediate amorphous structures during hydrate nucleation, thereby providing a stronger link between molecular simulation and experimental observations. Our NVE results nevertheless support the two-step nucleation mechanism proposed in previous simulation studies under constant temperature conditions which features the initial formation of amorphous hydrate-like structures.

Insights into the polymorphism of glycine: membrane crystallization in an electric field

In this work we studied glycine crystallization with two main objectives: (i) to get improved control of crystal growth and polymorphic selectivity of organic molecules; (ii) to achieve additional insights into the nucleation mechanisms of glycine polymorphs. To reach these goals, membrane crystallization technology, a tool which allows improved control of supersaturation in solution crystallization, was used under different operating conditions: the variable solvent removal rate, acidic and almost neutral pH, the presence of a pulsed electric field. The traditional explanation for the crystallization of α and γ glycine polymorphs from aqueous solution is based on the general cyclic dimer hypothesis and the self-poisoning mechanism. In contrast with both the conventional theories, experimental results suggest that the relative nucleation rates with respect to the relative growth kinetics of the two forms under the different conditions play a dominant role in determining the polymorphic outcome. Our results instead support a molecular nucleation route where open chain dimers can behave as building units for both γ- and α-glycines in the rate determining structuring step of the two-step nucleation mechanism.

Nucleation of protein condensed phases

Proteins in solution form a number of condensed phases. Even omitting the amyloid structures formed after partial protein unfolding, these phases include crystals, polymers, and other solid aggregates, as well as dense liquids and gels. Some of these condensed phases underlie pathological conditions, while others play a crucial role in the biological function of the respective protein or are an essential part of its laboratory or industrial processing. In this review, we summarize the fundamentals and recent findings on the kinetics of nucleation of dense liquid droplets and crystals. We define the transition from nucleation to spinodal decomposition for these two phase transitions. We review the two-step mechanism of protein crystal nucleation, in which mesoscopic metastable protein clusters serve as precursors to the ordered crystal nuclei. The concepts and mechanisms reviewed here provide powerful tools for control of the nucleation process, by varying the solution thermodynamic parameters.

Nucleation.

Crystallization starts with nucleation and control of nucleation is crucial for the control of the number, size, perfection, polymorphism and other characteristics of crystalline materials. This is particularly true for crystallization in solution, which is an essential part of processes in the chemical and pharmaceutical industries and a major step in physiological and pathological phenomena. There have been significant recent advances in the understanding of the mechanism of nucleation of crystals in solution. The foremost of these are the two-step mechanism of nucleation and the notion of the solution-crystal spinodal. According to the two-step mechanism, the crystalline nucleus appears inside pre-existing metastable clusters of size several hundred nanometers, which consist of dense liquid and are suspended in the solution. While initially proposed for protein crystals, the applicability of this mechanism has been demonstrated for small molecule organic materials, colloids, polymers, and biominerals. This mechanism helps to explain several long-standing puzzles of crystal nucleation in solution: nucleation rates which are many orders of magnitude lower than theoretical predictions, the significance of the dense protein liquid, and others. At high supersaturations typical of most crystallizing systems, the generation of crystal embryos occurs in the spinodal regime, where the nucleation barrier is negligible. The solution-crystal spinodal helps to understand the role of heterogeneous substrates in nucleation and the selection of crystalline polymorphs. Importantly, these ideas provide powerful tools for control of the nucleation process by varying the solution thermodynamic parameters.

Highly Surface-Textured ZnO:Al Films Fabricated by Controlling the Nucleation and Growth Separately for Solar Cell Applications

Highly surface-textured ZnO:Al (AZO) thin films have been fabricated at room temperature by a two-step magnetron sputtering process and using an oxygen-deficient ZnO target with small grain sizes. The as-deposited AZO films are composed of a highly oriented seed layer and a closely packed columnar overlayer with pyramidal growth fronts, supporting a two-step mechanism of crystallite nucleation and grain growth. The structural, optical, and electrical properties of the AZO films can be tuned by the deposition conditions. The optimal two-step AZO film with a maximum root-mean-square roughness of 40.2 nm reaches a very low square resistance of 0.66 Ω/sq (ρ = 1.32 × 10−4 Ω·cm) with an average transparency of 87.9% in the range of 400−1100 nm. The maximum haze factor of the as-deposited film is 60.7% at 360 nm, and the average haze factor is 14.8%. These properties are comparable to or exceed the reported values of surface-textured SnO2- and ZnO-based transparent conducting oxide films, making our films suitable for transparent electrode applications, especially in thin-film solar cells.