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.

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.

Cold atmospheric plasma-induced protein modification: Novel nonthermal processing technology to improve protein quality, functionality, and allergenicity reduction.

With the constant increase in protein demand globally, it is expedient to develop a strategy to effectively utilize protein, particularly those extracted from plant origin, which has been associated with low digestibility, poor techno-functional properties, and inherent allergenicity. Several thermal modification approaches have been developed to overcome these limitations and showed excellent results. Nevertheless, the excessive unfolding of the protein, aggregation of unfolded proteins, and irregular protein crosslinking have limited its application. Additionally, the increased consumer demand for natural products with no chemical additives has created a bottleneck for chemical-induced protein modification. Therefore, researchers are now directed toward other nonthermal technologies, including high-voltage cold plasma, ultrasound, high-pressure protein, etc., for protein modification. The techno-functional properties, allergenicity, and protein digestibility are greatly influenced by the applied treatment and its process parameters. Nevertheless, the application of these technologies, particularly high-voltage cold plasma, is still in its primary stage. Furthermore, the protein modification mechanism induced by high-voltage cold plasma has not been fully explained. Thus, this review meets the necessity to assemble the recent information on the process parameters and conditions for modifying proteins by high-voltage cold plasma and its impact on protein techno-functional properties, digestibility, and allergenicity.

Reinforcing canola protein matrix with chemically tailored nanocrystalline cellulose improves the functionality of canola protein-based packaging materials.

Canola protein derived from the canola industry byproduct is a potent biopolymer source to develop sustainable food packaging materials, but it has limitations due to its poor mechanical and barrier properties. Nanomaterials such as nanocrystalline cellulose (NCC) have shown promising potential in improving material properties. The current study aimed to enhance the functionality of canola protein-based films using TEMPO ((2,2,6,6-Tetramethylpiperidin-1-yl)oxyl) modified nanocrystalline cellulose (TM-NCC). TEMPO modification was performed using TEMPO/NaClO/NaBr based oxidation. Modified and unmodified nanocrystalline cellulose (U-NCC) were used at different weight ratios to prepare the films. TEMPO-mediated oxidation converted 19.61 ± 3.53 % of primary -OH groups into -COOH groups. The addition of U-NCC and TM-NCC significantly increased the tensile strength reporting the highest value of 8.36 ± 0.85 MPa for 5% TM-NCC, which was only 3.43 ± 0.66 MPa for control films. Interestingly, both U-NCC and TM-NCC enhanced the films' water barrier and thermal properties compared to control.

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.

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.

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.