A mechanistic examination of salting out in protein-polymer membrane interactions.

Developing a mechanistic understanding of protein dynamics and conformational changes at polymer interfaces is critical for a range of processes including industrial protein separations. Salting out is one example of a procedure that is ubiquitous in protein separations yet is optimized empirically because there is no mechanistic description of the underlying interactions that would allow predictive modeling. Here, we investigate peak narrowing in a model transferrin-nylon system under salting out conditions using a combination of single-molecule tracking and ensemble separations. Distinct surface transport modes and protein conformational changes at the negatively charged nylon interface are quantified as a function of salt concentration. Single-molecule kinetics relate macroscale improvements in chromatographic peak broadening with microscale distributions of surface interaction mechanisms such as continuous-time random walks and simple adsorption-desorption. Monte Carlo simulations underpinned by the stochastic theory of chromatography are performed using kinetic data extracted from single-molecule observations. Simulations agree with experiment, revealing a decrease in peak broadening as the salt concentration increases. The results suggest that chemical modifications to membranes that decrease the probability of surface random walks could reduce peak broadening in full-scale protein separations. More broadly, this work represents a proof of concept for combining single-molecule experiments and a mechanistic theory to improve costly and time-consuming empirical methods of optimization.

Quantitative Analysis of Nanorod Aggregation and Morphology from Scanning Electron Micrographs Using SEMseg.

General methods to achieve better physical insight about nanoparticle aggregation and assembly are needed because of the potential role of aggregation in a wide range of materials, environmental, and biological outcomes. Scanning electron microscopy (SEM) is fast and affordable compared to transmission electron microscopy, but SEM micrographs lack contrast and resolution due to lower beam energy, topographic contrast, edge effects, and charging. We present a new segmentation algorithm called SEMseg that is robust to the challenges inherent in SEM micrograph analysis and demonstrate its utility for analyzing gold (Au) nanorod aggregates. SEMseg not only supports nanoparticle size analysis for dispersed nanoparticles, but also discriminates between nanoparticles within an aggregate. We compare our algorithm to those incorporated into the commonly used software ImageJ and demonstrate improved segmentation of aggregate structures. New physical insight about aggregation is demonstrated by the introduction of an order parameter describing side-by-side structure in nanoparticle aggregates. We also present the segmentation and fitting algorithms included in SEMseg within a user-friendly graphical user interface. The resulting code is provided with an open-source interface to provide quantitative image processing tools for researchers to characterize both dispersed nanoparticles and nanoparticle assemblies in SEM micrographs with high throughput.

Single Particle Tracking: From Theory to Biophysical Applications.

After three decades of developments, single particle tracking (SPT) has become a powerful tool to interrogate dynamics in a range of materials including live cells and novel catalytic supports because of its ability to reveal dynamics in the structure-function relationships underlying the heterogeneous nature of such systems. In this review, we summarize the algorithms behind, and practical applications of, SPT. We first cover the theoretical background including particle identification, localization, and trajectory reconstruction. General instrumentation and recent developments to achieve two- and three-dimensional subdiffraction localization and SPT are discussed. We then highlight some applications of SPT to study various biological and synthetic materials systems. Finally, we provide our perspective regarding several directions for future advancements in the theory and application of SPT.

Nanoelectrode-emitter spectral overlap amplifies surface enhanced electrogenerated chemiluminescence.

Electrogenerated chemiluminescence (ECL) is a promising technique for low concentration molecular detection. To improve the detection limit, plasmonic nanoparticles have been proposed as signal boosting antennas to amplify ECL. Previous ensemble studies have hinted that spectral overlap between the nanoparticle antenna and the ECL emitter may play a role in signal enhancement. Ensemble spectroscopy, however, cannot resolve heterogeneities arising from colloidal nanoparticle size and shape distributions, leading to an incomplete picture of the impact of spectral overlap. Here, we isolate the effect of nanoparticle-emitter spectral overlap for a model ECL system, coreaction of tris(2,2'-bipyridyl)dichlororuthenium(ii) hexahydrate and tripropylamine, at the single-particle level while minimizing other factors influencing ECL intensities. We found a 10-fold enhancement of ECL among 952 gold nanoparticles. This signal enhancement is attributed exclusively to spectral overlap between the nanoparticle and the emitter. Our study provides new mechanistic insight into plasmonic enhancement of ECL, creating opportunities for low concentration ECL sensing.

Plasmon Energy Transfer in Hybrid Nanoantennas.

Plasmonic metal nanoparticles exhibit large dipole moments upon photoexcitation and have the potential to induce electronic transitions in nearby materials, but fast internal relaxation has to date limited the spatial range and efficiency of plasmonic mediated processes. In this work, we use photo-electrochemistry to synthesize hybrid nanoantennas comprised of plasmonic nanoparticles with photoconductive polymer coatings. We demonstrate that the formation of the conductive polymer is selective to the nanoparticles and that polymerization is enhanced by photoexcitation. In situ spectroscopy and simulations support a mechanism in which up to 50% efficiency of nonradiative energy transfer is achieved. These hybrid nanoantennas combine the unmatched light-harvesting properties of a plasmonic antenna with the similarly unmatched device processability of a polymer shell.

Nanoscale Surface-Induced Unfolding of Single Fibronectin Is Restricted by Serum Albumin Crowding.

Understanding nanoscale protein conformational changes at solid-liquid interfaces is critical for predicting how proteins will impact the performance of biomaterials in vivo. Crowding is an important contributor to conformational stability. Here we apply single-molecule high resolution imaging with photobleaching to directly measure dye-conjugated fibronectin's unfolding in varying conditions of crowding with human serum albumin on aminosilanized glass. Using this approach, we identify serum albumin's crowding mechanism. We find that fibronectin achieves larger degrees of unfolding when not crowded by coadsorbed serum albumin. Serum albumin does not as effectively constrict fibronectin's conformation if it is sequentially, rather than simultaneously, introduced, suggesting that serum albumin's crowding mechanism is dependent on its ability to sterically block fibronectin's unfolding during the process of adsorption. Because fibronectin's conformation is dependent on interfacial macromolecular crowding under in vitro conditions, it is important to consider the role of in vivo crowding on protein activity.

Unraveling the origin of chirality from plasmonic nanoparticle-protein complexes.

Plasmon-coupled circular dichroism has emerged as a promising approach for ultrasensitive detection of biomolecular conformations through coupling between molecular chirality and surface plasmons. Chiral nanoparticle assemblies without chiral molecules present also have large optical activities. We apply single-particle circular differential scattering spectroscopy coupled with electron imaging and simulations to identify both structural chirality of plasmonic aggregates and plasmon-coupled circular dichroism induced by chiral proteins. We establish that both chiral aggregates and just a few proteins in interparticle gaps of achiral assemblies are responsible for the ensemble signal, but single nanoparticles do not contribute. We furthermore find that the protein plays two roles: It transfers chirality to both chiral and achiral plasmonic substrates, and it is also responsible for the chiral three-dimensional assembly of nanorods. Understanding these underlying factors paves the way toward sensing the chirality of single biomolecules.

Spectral Response of Plasmonic Gold Nanoparticles to Capacitive Charging: Morphology Effects.

We report a study of the shape-dependent spectral response of the gold nanoparticle surface plasmon resonance at various electron densities to provide mechanistic insight into the role of capacitive charging, a topic of some debate. We demonstrate a morphology-dependent spectral response for gold nanoparticles due to capacitive charging using single-particle spectroscopy in an inert electrochemical environment. A decrease in plasmon energy and increase in spectral width for gold nanospheres and nanorods was observed as the electron density was tuned through a potential window of -0.3 to 0.1 V. The combined observations could not be explained by existing theories. A new quantum theory for charging based on the random phase approximation was developed. Additionally, the redox reaction of gold oxide formation was probed using single-particle plasmon voltammetry to reproduce the reduction peak from the bulk cyclic voltammetry. These results deepen our understanding of the relationship between optical and electronic properties in plasmonic nanoparticles and provide insight toward their potential applications in directed electrocatalysis.