Magnet-in-ferroelectric crystals exhibiting photomultiferroicity.

Growing crystallographically incommensurate and dissimilar organic materials is fundamentally intriguing but challenging for the prominent cross-correlation phenomenon enabling unique magnetic, electronic, and optical functionalities. Here, we report the growth of molecular layered magnet-in-ferroelectric crystals, demonstrating photomanipulation of interfacial ferroic coupling. The heterocrystals exhibit striking photomagnetization and magnetoelectricity, resulting in photomultiferroic coupling and complete change of their color while inheriting ferroelectricity and magnetism from the parent phases. Under a light illumination, ferromagnetic resonance shifts of 910 Oe are observed in heterocrystals while showing a magnetization change of 0.015 emu/g. In addition, a noticeable magnetization change (8% of magnetization at a 1,000 Oe external field) in the vicinity of ferro-to-paraelectric transition is observed. The mechanistic electric-field-dependent studies suggest the photoinduced ferroelectric field effect responsible for the tailoring of photo-piezo-magnetism. The crystallographic analyses further evidence the lattice coupling of a magnet-in-ferroelectric heterocrystal system.

Chemically Fueled Dissipative Cross-Linking of Protein Hydrogels Mediated by Protein Unfolding.

Cells assemble dynamic protein-based nanostructures far from equilibrium, such as microtubules, in a process referred to as dissipative assembly. Synthetic analogues have utilized chemical fuels and reaction networks to form transient hydrogels and molecular assemblies from small molecule or synthetic polymer building blocks. Here, we demonstrate dissipative cross-linking of transient protein hydrogels using a redox cycle, which exhibit protein unfolding-dependent lifetimes and mechanical properties. Fast oxidation of cysteine groups on bovine serum albumin by hydrogen peroxide, the chemical fuel, formed transient hydrogels with disulfide bond cross-links that degraded over hours by a slow reductive back reaction. Interestingly, despite increased cross-linking, the hydrogel lifetime decreased as a function of increasing denaturant concentration. Experiments showed that the solvent-accessible cysteine concentration increased with increasing denaturant concentration due to unfolding of secondary structures. The increased cysteine concentration consumed more fuel, which led to less direction oxidation of the reducing agent and affected a shorter hydrogel lifetime. Increased hydrogel stiffness, disulfide cross-linking density, and decreased oxidation of redox-sensitive fluorescent probes at a high denaturant concentration provided evidence supporting the unveiling of additional cysteine cross-linking sites and more rapid consumption of hydrogen peroxide at higher denaturant concentrations. Taken together, the results indicate that the protein secondary structure mediated the transient hydrogel lifetime and mechanical properties by mediating the redox reactions, a feature unique to biomacromolecules that exhibit a higher order structure. While prior works have focused on the effects of the fuel concentration on dissipative assembly of non-biological molecules, this work demonstrates that the protein structure, even in nearly fully denatured proteins, can exert similar control over reaction kinetics, lifetime, and resulting mechanical properties of transient hydrogels.

Electric Field-Induced Water Condensation Visualized by Vapor-Phase Transmission Electron Microscopy.

Understanding the nanoscale water condensation dynamics in strong electric fields is important for improving the atmospheric modeling of cloud dynamics and emerging technologies utilizing electric fields for direct air moisture capture. Here, we use vapor-phase transmission electron microscopy (VPTEM) to directly image nanoscale condensation dynamics of sessile water droplets in electric fields. VPTEM imaging of saturated water vapor stimulated condensation of sessile water nanodroplets that grew to a size of ∼500 nm before evaporating over a time scale of a minute. Simulations showed that electron beam charging of the silicon nitride microfluidic channel windows generated electric fields of ∼108 V/m, which depressed the water vapor pressure and effected rapid nucleation of nanosized liquid water droplets. A mass balance model showed that droplet growth was consistent with electric field-induced condensation, while droplet evaporation was consistent with radiolysis-induced evaporation via conversion of water to hydrogen gas. The model quantified several electron beam-sample interactions and vapor transport properties, showed that electron beam heating was insignificant, and demonstrated that literature values significantly underestimated radiolytic hydrogen production and overestimated water vapor diffusivity. This work demonstrates a method for investigating water condensation in strong electric fields and under supersaturated conditions, which is relevant to vapor-liquid equilibrium in the troposphere. While this work identifies several electron beam-sample interactions that impact condensation dynamics, quantification of these phenomena here is expected to enable delineating these artifacts from the physics of interest and accounting for them when imaging more complex vapor-liquid equilibrium phenomena with VPTEM.

Nanoscale Mapping of Nonuniform Heterogeneous Nucleation Kinetics Mediated by Surface Chemistry.

Nucleation underlies the formation of many liquid-phase synthetic and natural materials with applications in materials chemistry, geochemistry, biophysics, and structural biology. Most liquid-phase nucleation processes are heterogeneous, occurring at specific nucleation sites at a solid-liquid interface; however, the chemical and topographical identity of these nucleation sites and how nucleation kinetics vary from site-to-site remain mysterious. Here we utilize in situ liquid cell electron microscopy to unveil counterintuitive nanoscale nonuniformities in heterogeneous nucleation kinetics on a macroscopically uniform solid-liquid interface. Time-resolved in situ electron microscopy imaging of silver nanoparticle nucleation at a water-silicon nitride interface showed apparently randomly located nucleation events at the interface. However, nanometric maps of local nucleation kinetics uncovered nanoscale interfacial domains with either slow or rapid nucleation. Interestingly, the interfacial domains vanished at high supersaturation ratio, giving way to rapid spatially uniform nucleation kinetics. Atomic force microscopy and nanoparticle labeling experiments revealed a topographically flat, chemically heterogeneous interface with nanoscale interfacial domains of functional groups similar in size to those observed in the nanometric nucleation maps. These results, along with a semiquantitative nucleation model, indicate that a chemically nonuniform interface presenting different free energy barriers to heterogeneous nucleation underlies our observations of nonuniform nucleation kinetics. Overall, our results introduce a new imaging modality, nanometric nucleation mapping, and provide important new insights into the impact of surface chemistry on microscopic spatial variations in heterogeneous nucleation kinetics that have not been previously observed.

Direct Visualization of Planar Assembly of Plasmonic Nanoparticles Adjacent to Electrodes in Oscillatory Electric Fields.

Electric field-directed assembly of colloidal nanoparticles (NPs) has been widely adopted for fabricating functional thin films and nanostructured surfaces. While first-order electrokinetic effects on NPs are well-understood in terms of classical models, effects of second-order electrokinetics that involve induced surface charge are still poorly understood. Induced charge electroosmotic phenomena, such as electrohydrodynamic (EHD) flow, have long been implicated in electric field-directed NP assembly with little experimental basis. Here, we use in situ dark-field optical microscopy and plasmonic NPs to directly observe the dynamics of planar assembly of colloidal NPs adjacent to a planar electrode in low-frequency (<1 kHz) oscillatory electric fields. We exploit the change in plasmonic NP color resulting from interparticle plasmonic coupling to visualize the assembly dynamics and assembly structure of silver NPs. Planar assembly of NPs is unexpected because of strong electrostatic repulsion between NPs and indicates that there are strong attractive interparticle forces oriented perpendicular to the electric field direction. A parametric investigation of the voltage- and frequency-dependent phase behavior reveals that planar NP assembly occurs over a narrow frequency range below which irreversible ballistic deposition occurs. Two key experimental observations are consistent with EHD flow-induced NP assembly: (1) NPs remain mobile during assembly and (2) electron microscopy observations reveal randomly close-packed planar assemblies, consistent with strong interparticle attraction. We interpret planar assembly in terms of EHD fluid flow and develop a scaling model that qualitatively agrees with the measured phase regions. Our results are the first direct in situ observations of EHD flow-induced NP assembly and shed light on long-standing unresolved questions concerning the formation of NP superlattices during electric field-induced NP deposition.

Toward a modular multi-material nanoparticle synthesis and assembly strategy via bionanocombinatorics: bifunctional peptides for linking Au and Ag nanomaterials.

Materials-binding peptides represent a unique avenue towards controlling the shape and size of nanoparticles (NPs) grown under aqueous conditions. Here, employing a bionanocombinatorics approach, two such materials-binding peptides were linked at either end of a photoswitchable spacer, forming a multi-domain materials-binding molecule to control the in situ synthesis and organization of Ag and Au NPs under ambient conditions. These multi-domain molecules retained the peptides' ability to nucleate, grow, and stabilize Ag and Au NPs in aqueous media. Disordered co-assemblies of the two nanomaterials were observed by TEM imaging of dried samples after sequential growth of the two metals, and showed a clustering behavior that was not typically observed without both metals and the linker molecules. While TEM evidence suggested the formation of AuNP/AgNP assemblies upon drying, SAXS analysis indicated that no extended assemblies existed in solution, suggesting that sample drying plays an important role in facilitating NP clustering. Molecular simulations and experimental data revealed tunable materials-binding based upon the isomerization state of the photoswitchable unit and metal employed. This work is a first step in generating externally actuated biomolecules with specific material-binding properties that could be used as the building blocks to achieve multi-material switchable NP assemblies.

Dark-Field Scanning Transmission Ion Microscopy via Detection of Forward-Scattered Helium Ions with a Microchannel Plate.

A microchannel plate was used as an ion sensitive detector in a commercial helium ion microscope (HIM) for dark-field transmission imaging of nanomaterials, i.e. scanning transmission ion microscopy (STIM). In contrast to previous transmission HIM approaches that used secondary electron conversion holders, our new approach detects forward-scattered helium ions on a dedicated annular shaped ion sensitive detector. Minimum collection angles between 125 mrad and 325 mrad were obtained by varying the distance of the sample from the microchannel plate detector during imaging. Monte Carlo simulations were used to predict detector angular ranges at which dark-field images with atomic number contrast could be obtained. We demonstrate atomic number contrast imaging via scanning transmission ion imaging of silica-coated gold nanoparticles and magnetite nanoparticles. Although the resolution of STIM is known to be degraded by beam broadening in the substrate, we imaged magnetite nanoparticles with high contrast on a relatively thick silicon nitride substrate. We expect this new approach to annular dark-field STIM will open avenues for more quantitative ion imaging techniques and advance fundamental understanding of underlying ion scattering mechanisms leading to image formation.

Direct observation of aggregative nanoparticle growth: kinetic modeling of the size distribution and growth rate.

Direct observations of solution-phase nanoparticle growth using in situ liquid transmission electron microscopy (TEM) have demonstrated the importance of "non-classical" growth mechanisms, such as aggregation and coalescence, on the growth and final morphology of nanocrystals at the atomic and single nanoparticle scales. To date, groups have quantitatively interpreted the mean growth rate of nanoparticles in terms of the Lifshitz-Slyozov-Wagner (LSW) model for Ostwald ripening, but less attention has been paid to modeling the corresponding particle size distribution. Here we use in situ fluid stage scanning TEM to demonstrate that silver nanoparticles grow by a length-scale dependent mechanism, where individual nanoparticles grow by monomer attachment but ensemble-scale growth is dominated by aggregation. Although our observed mean nanoparticle growth rate is consistent with the LSW model, we show that the corresponding particle size distribution is broader and more symmetric than predicted by LSW. Following direct observations of aggregation, we interpret the ensemble-scale growth using Smoluchowski kinetics and demonstrate that the Smoluchowski model quantitatively captures the mean growth rate and particle size distribution.

Electrolyte-Dependent Aggregation of Colloidal Particles near Electrodes in Oscillatory Electric Fields.

Colloidal particles adjacent to electrodes have been observed to exhibit drastically different aggregation behavior depending on the identity of the suspending electrolyte. For example, particles suspended in potassium chloride aggregate laterally near the electrode upon application of a low-frequency (∼100 Hz) oscillatory electric field, but the same particles suspended in potassium hydroxide are instead observed to separate. Previous work has interpreted the particle aggregation or separation in terms of various types of electrically induced fluid flow around the particle, but the details remain poorly understood. Here we present experimental evidence that the aggregation rate is highly correlated to both the particle zeta potential and the electric field amplitude, both of which depend on the electrolyte type. Measurement of the aggregation rate in 26 unique electrolyte-particle combinations demonstrates that the aggregation rate decreases with increasing zeta potential magnitude (i.e., particles with a large zeta potential tended to separate regardless of sign). Likewise, direct measurements of the oscillatory electric field in different electrolytes revealed that the aggregation rate was negatively correlated with solution conductivity and thus positively correlated with the field strength. We tested the experimentally measured aggregation rates against a previously developed point dipole model and found that the model fails to capture the observed electrolyte dependence. The results point to the need for more detailed modeling to capture the effect of electrolyte on the zeta potential and solution conductivity to predict fluid flow around colloids near electrodes.

Machine intelligence-accelerated discovery of all-natural plastic substitutes.

One possible solution against the accumulation of petrochemical plastics in natural environments is to develop biodegradable plastic substitutes using natural components. However, discovering all-natural alternatives that meet specific properties, such as optical transparency, fire retardancy and mechanical resilience, which have made petrochemical plastics successful, remains challenging. Current approaches still rely on iterative optimization experiments. Here we show an integrated workflow that combines robotics and machine learning to accelerate the discovery of all-natural plastic substitutes with programmable optical, thermal and mechanical properties. First, an automated pipetting robot is commanded to prepare 286 nanocomposite films with various properties to train a support-vector machine classifier. Next, through 14 active learning loops with data augmentation, 135 all-natural nanocomposites are fabricated stagewise, establishing an artificial neural network prediction model. We demonstrate that the prediction model can conduct a two-way design task: (1) predicting the physicochemical properties of an all-natural nanocomposite from its composition and (2) automating the inverse design of biodegradable plastic substitutes that fulfils various user-specific requirements. By harnessing the model's prediction capabilities, we prepare several all-natural substitutes, that could replace non-biodegradable counterparts as exhibiting analogous properties. Our methodology integrates robot-assisted experiments, machine intelligence and simulation tools to accelerate the discovery and design of eco-friendly plastic substitutes starting from building blocks taken from the generally-recognized-as-safe database.

Dynamic surface chemistry and interparticle interactions mediating chemically fueled dissipative assembly of colloids.

HYPOTHESIS: Dissipative assembly of colloids involves using a chemical fuel to temporarily activate organic colloid surface ligands to an assembly prone state. Colloids assemble into transient aggregates that disintegrate after the fuel is consumed. The underlying colloidal interactions controlling dissipative assembly have not been rigorously identified or quantified. We expect that fuel concentration dependent dissipative assembly behavior can be reconciled with measurements of dynamic colloid surface chemistry and colloidal interactions. EXPERIMENTS: Carbodiimide chemistry was utilized to induce dissipative assembly of carboxylic acid functionalized polystyrene colloids. We measured aggregation kinetics, colloid surface hydrophobicity, and zeta potential as a function of time, which established that colloids underwent dissipative assembly for fuel concentrations between 5 and 12.5 mM and irreversible aggregation at higher fuel concentrations due to transient changes in surface chemistry. FINDINGS: We formulated a pairwise colloidal interaction potential model including hydrophobic interactions quantified by fluorescence binding experiments. Fuel concentrations causing dissipative assembly displayed a transient increase in secondary minimum depth and a primary maximum much larger than the thermal potential. Fuel concentrations leading to irreversible aggregation displayed a primary maximum smaller than the thermal potential. This is the first study to quantify surface chemistry and interparticle interactions during dissipative colloid assembly and represents a foundational step in rationally designing more complex dissipative assembly systems.

Visualizing formation of high entropy alloy nanoparticles with liquid phase transmission electron microscopy.

High entropy alloy (HEA) nanoparticles hold promise as active and durable (electro)catalysts. Understanding their formation mechanism will enable rational control over composition and atomic arrangement of multimetallic catalytic surface sites to maximize their activity. While prior reports have attributed HEA nanoparticle formation to nucleation and growth, there is a dearth of detailed mechanistic investigations. Here we utilize liquid phase transmission electron microscopy (LPTEM), systematic synthesis, and mass spectrometry (MS) to demonstrate that HEA nanoparticles form by aggregation of metal cluster intermediates. AuAgCuPtPd HEA nanoparticles are synthesized by aqueous co-reduction of metal salts with sodium borohydride in the presence of thiolated polymer ligands. Varying the metal : ligand ratio during synthesis showed that alloyed HEA nanoparticles formed only above a threshold ligand concentration. Interestingly, stable single metal atoms and sub-nanometer clusters are observed by TEM and MS in the final HEA nanoparticle solution, suggesting nucleation and growth is not the dominant mechanism. Increasing supersaturation ratio increased particle size, which together with observations of stable single metal atoms and clusters, supported an aggregative growth mechanism. Direct real-time observation with LPTEM imaging showed aggregation of HEA nanoparticles during synthesis. Quantitative analyses of the nanoparticle growth kinetics and particle size distribution from LPTEM movies were consistent with a theoretical model for aggregative growth. Taken together, these results are consistent with a reaction mechanism involving rapid reduction of metal ions into sub-nanometer clusters followed by cluster aggregation driven by borohydride ion induced thiol ligand desorption. This work demonstrates the importance of cluster species as potential synthetic handles for rational control over HEA nanoparticle atomic structure.

Unraveling chemical processes during nanoparticle synthesis with liquid phase electron microscopy and correlative techniques.

Liquid phase transmission electron microscopy (LPTEM) has enabled unprecedented direct real time imaging of physicochemical processes during solution phase synthesis of metallic nanoparticles. LPTEM primarily provides images of nanometer scale, and sometimes atomic scale, metal nanoparticle crystallization processes, but provides little chemical information about organic surface ligands, metal-ligand complexes and reaction intermediates, and redox reactions. Likewise, complex electron beam-solvent interactions during LPTEM make it challenging to pinpoint the chemical processes, some involving exotic highly reactive radicals, impacting nanoparticle formation. Pairing LPTEM with correlative solution synthesis, ex situ chemical analysis, and theoretical modeling represents a powerful approach to gain a holistic understanding of the chemical processes involved in nanoparticle synthesis. In this feature article, we review recent work by our lab and others that has focused on elucidating chemical processes during nanoparticle synthesis using LPTEM and correlative chemical characterization and modeling, including mass and optical spectrometry, fluorescence microscopy, solution chemistry, and reaction kinetic modeling. In particular, we show how these approaches enable investigating redox chemistry during LPTEM, polymeric and organic capping ligands, metal deposition mechanisms on plasmonic nanoparticles, metal clusters and complexes, and multimetallic nanoparticle formation. Future avenues of research are discussed, including moving beyond electron beam induced nanoparticle formation by using light and thermal stimuli during LPTEM. We discuss prospects for real time LPTEM imaging and online chemical analysis of reaction intermediates using microfluidic flow reactors.

Examining Silver Deposition Pathways onto Gold Nanorods with Liquid-Phase Transmission Electron Microscopy.

Liquid-phase transmission electron microscopy (LP-TEM) enables one to directly visualize the formation of plasmonic nanoparticles and their postsynthetic modification, but the relative contributions of plasmonic hot electrons and radiolysis to metal precursor reduction remain unclear. Here we show silver deposition onto plasmonic gold nanorods (AuNRs) during LP-TEM is dominated by water radiolysis-induced chemical reduction. Silver was observed with LP-TEM to form bipyramidal shells at higher surfactant coverage and tip-preferential lobes at lower surfactant coverage. Ex situ silver photodeposition formed nanometer-thick shells on AuNRs with preferential deposition in inter-rod gaps, while chemical reduction deposited silver at AuNR tips at low surfactant coverage and formed pyramidal shells at higher surfactant coverage, consistent with LP-TEM. Silver deposition locations during LP-TEM were inconsistent with simulated near-field enhancement and hot electron generation hot spots. Collectively, the results indicate chemical reduction dominated during LP-TEM, indicating observation of plasmonic hot electron-induced photoreduction will necessitate suppression of radiolysis.

Real-time imaging of metallic supraparticle assembly during nanoparticle synthesis.

Observations of nanoparticle superlattice formation over minutes during colloidal nanoparticle synthesis elude description by conventional understanding of self-assembly, which theorizes superlattices require extended formation times to allow for diffusively driven annealing of packing defects. It remains unclear how nanoparticle position annealing occurs on such short time scales despite the rapid superlattice growth kinetics. Here we utilize liquid phase transmission electron microscopy to directly image the self-assembly of platinum nanoparticles into close packed supraparticles over tens of seconds during nanoparticle synthesis. Electron-beam induced reduction of an aqueous platinum precursor formed monodisperse 2-3 nm platinum nanoparticles that simultaneously self-assembled over tens of seconds into 3D supraparticles, some of which showed crystalline ordered domains. Experimentally varying the interparticle interactions (e.g., electrostatic, steric interactions) by changing precursor chemistry revealed that supraparticle formation was driven by weak attractive van der Waals forces balanced by short ranged repulsive steric interactions. Growth kinetic measurements and an interparticle interaction model demonstrated that nanoparticle surface diffusion rates on the supraparticles were orders of magnitude faster than nanoparticle attachment, enabling nanoparticles to find high coordination binding sites unimpeded by incoming particles. These results reconcile rapid self-assembly of supraparticles with the conventional self-assembly paradigm in which nanocrystal position annealing by surface diffusion occurs on a significantly shorter time scale than nanocrystal attachment.

Revealing Reactions between the Electron Beam and Nanoparticle Capping Ligands with Correlative Fluorescence and Liquid-Phase Electron Microscopy.

Liquid-phase transmission electron microscopy (LP-TEM) enables real-time imaging of nanoparticle self-assembly, formation, and etching with single nanometer resolution. Despite the importance of organic nanoparticle capping ligands in these processes, the effect of electron beam irradiation on surface-bound and soluble capping ligands during LP-TEM imaging has not been investigated. Here, we use correlative LP-TEM and fluorescence microscopy (FM) to demonstrate that polymeric nanoparticle ligands undergo competing crosslinking and chain scission reactions that nonmonotonically modify ligand coverage over time. Branched polyethylenimine (BPEI)-coated silver nanoparticles were imaged with dose-controlled LP-TEM followed by labeling their primary amine groups with fluorophores to visualize the local thickness of adsorbed capping ligands. FM images showed that free ligands crosslinked in the LP-TEM image area over imaging times of tens of seconds, enhancing local capping ligand coverage on nanoparticles and silicon nitride membranes. Nanoparticle surface ligands underwent chain scission over irradiation times of minutes to tens of minutes, which depleted surface ligands from the nanoparticle and silicon nitride surface. Conversely, solutions of only soluble capping ligand underwent successive crosslinking reactions with no chain scission, suggesting that nanoparticles enhanced the chain scission reactions by acting as radiolysis hotspots. The addition of a hydroxyl radical scavenger, tert-butanol, eliminated chain scission reactions and slowed the progression of crosslinking reactions. These experiments have important implications for performing controlled and reproducible LP-TEM nanoparticle imaging as they demonstrate that the electron beam can significantly alter ligand coverage on nanoparticles in a nonintuitive manner. They emphasize the need to understand and control the electron beam radiation chemistry of a given sample to avoid significant perturbations to the nanoparticle capping ligand chemistry, which are invisible in electron micrographs.

Visualizing Ligand-Mediated Bimetallic Nanocrystal Formation Pathways with in Situ Liquid-Phase Transmission Electron Microscopy Synthesis.

Colloidal synthesis of alloyed multimetallic nanocrystals with precise composition control remains a challenge and a critical missing link in theory-driven rational design of functional nanomaterials. Liquid-phase transmission electron microscopy (LP-TEM) enables direct visualization of nanocrystal formation mechanisms that can inform discovery of design rules for nanocrystal synthesis, but it remains unclear whether the salient flask synthesis chemistry is preserved under electron beam irradiation during LP-TEM. Here, we demonstrate controlled in situ LP-TEM synthesis of alloyed AuCu nanocrystals while maintaining the molecular structure of electron beam sensitive metal thiolate precursor complexes. Ex situ flask synthesis experiments formed alloyed nanocrystals containing on average 70 atomic% Au using heteronuclear metal thiolate complexes as a precursor, while gold-rich alloys with nearly no copper formed in their absence. Systematic dose rate-controlled in situ LP-TEM synthesis experiments established a range of electron beam synthesis conditions that formed alloyed AuCu nanocrystals that had statistically indistinguishable alloy composition, aggregation state, and particle size distribution shape compared to ex situ flask synthesis, indicating the flask synthesis chemistry was preserved under these conditions. Reaction kinetic simulations of radical-ligand reactions revealed that polymer capping ligands acted as effective hydroxyl radical scavengers during LP-TEM synthesis and prevented oxidation of metal thiolate complexes at low dose rates. Our results revealed a key role of the capping ligands aside from their well-known functions, which was to prevent copper oxidation and facilitate formation of prenucleation cluster intermediates via formation of metal thiolate complexes. This work demonstrates that complex ion precursor chemistry can be maintained during LP-TEM imaging, enabling probing nonclassical nanocrystal formation mechanisms with LP-TEM under reaction conditions representative of ex situ flask synthesis.

Effects of Protein Unfolding on Aggregation and Gelation in Lysozyme Solutions.

In this work, we investigate the role of folding/unfolding equilibrium in protein aggregation and formation of a gel network. Near the neutral pH and at a low buffer ionic strength, the formation of the gel network around unfolding conditions prevents investigations of protein aggregation. In this study, by deploying the fact that in lysozyme solutions the time of folding/unfolding is much shorter than the characteristic time of gelation, we have prevented gelation by rapidly heating the solution up to the unfolding temperature (~80 °C) for a short time (~30 min.) followed by fast cooling to the room temperature. Dynamic light scattering measurements show that if the gelation is prevented, nanosized irreversible aggregates (about 10-15 nm radius) form over a time scale of 10 days. These small aggregates persist and aggregate further into larger aggregates over several weeks. If gelation is not prevented, the nanosized aggregates become the building blocks for the gel network and define its mesh length scale. These results support our previously published conclusion on the nature of mesoscopic aggregates commonly observed in solutions of lysozyme, namely that aggregates do not form from lysozyme monomers in their native folded state. Only with the emergence of a small fraction of unfolded proteins molecules will the aggregates start to appear and grow.

Detection and Sizing of Submicron Particles in Biologics With Interferometric Scattering Microscopy.

We demonstrate the application of interferometric scattering microscopy (IFS) for characterizing submicron particles in stir-stressed monoclonal antibody. IFS uses a layered silicon sensor and modified optical microscope to rapidly visualize and determine the particle size distribution (PSD) of submicron particles based on their scattering intensity, which is directly proportional to particle mass. Limits for particle size and optimal solution concentration were established for IFS characterization of submicron particles. We critically compare IFS data with dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) and find IFS is superior to NTA and DLS for determining the realistic shape of the number-based PSD, whereas NTA and DLS provide superior information about absolute particle size. Together, IFS, NTA, and DLS provide complementary information on submicron particles and enable quantitative characterization of the PSD of submicron aggregates. Finally, we explore quantifying particle size with IFS by developing a calibration curve for particle scattering intensity based on correlative scanning electron microscopy imaging. We found that only a subset of isotropic-shaped particles followed the expected proportionality between IFS intensity and particle mass. Overall, this study demonstrates IFS is a simple approach for detecting and quantifying submicron aggregate PSD in protein-based therapeutics.

Discovery of Anion Insertion Electrochemistry in Layered Hydroxide Nanomaterials.

Electrode materials which undergo anion insertion are a void in the materials innovation landscape and a missing link to energy efficient electrochemical desalination. In recent years layered hydroxides (LHs) have been studied for a range of electrochemical applications, but to date have not been considered as electrode materials for anion insertion electrochemistry. Here, we show reversible anion insertion in a LH for the first time using Co and Co-V layer hydroxides. By pairing in situ synchrotron and quartz crystal microbalance measurements with a computational unified electrochemical band-diagram description, we reveal a previously undescribed anion-insertion mechanism occurring in Co and Co-V LHs. This proof of concept study demonstrates reversible electrochemical anion insertion in LHs without significant material optimization. These results coupled with our foundational understanding of anion insertion electrochemistry establishes LHs as a materials platform for anion insertion electrochemistry with the potential for future application to electrochemical desalination.