Bo Shuang

Adsorption and Unfolding of a Single Protein Triggers Nanoparticle Aggregation

The response of living systems to nanoparticles is thought to depend on the protein corona, which forms shortly after exposure to physiological fluids and which is linked to a wide array of pathophysiologies. A mechanistic understanding of the dynamic interaction between proteins and nanoparticles and thus the biological fate of nanoparticles and associated proteins is, however, often missing mainly due to the inadequacies in current ensemble experimental approaches. Through the application of a variety of single molecule and single particle spectroscopic techniques in combination with ensemble level characterization tools, we identified different interaction pathways between gold nanorods and bovine serum albumin depending on the protein concentration. Overall, we found that local changes in protein concentration influence everything from cancer cell uptake to nanoparticle stability and even protein secondary structure. We envision that our findings and methods will lead to strategies to control the associated pathophysiology of nanoparticle exposure in vivo.

Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations

Significance Adsorption of proteins underlies the purification of biopharmaceuticals, as well as therapeutic apheresis, immunoassays, and biosensors. In particular, separation of proteins by interactions with charged ligands on surfaces (ion-exchange chromatography) is an essential tool of the modern pharmaceutical industry. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create functional adsorption sites and that even chemically identical ligands create sites of varying kinetic properties that depend on steric availability at the interface. Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally.

Tuning the acoustic frequency of a gold nanodisk through its adhesion layer

To fabricate robust metallic nanostructures with top-down patterning methods such as electron-beam lithography, an initial nanometer-scale layer of a second metal is deposited to promote adhesion of the metal of interest. However, how this nanoscale layer affects the mechanical properties of the nanostructure and how adhesion layer thickness controls the binding strength to the substrate are still open questions. Here we use ultrafast laser pulses to impulsively launch acoustic phonons in single gold nanodisks with variable titanium layer thicknesses, and observe an increase in phonon frequencies as a thicker adhesion layer facilitates stronger binding to the glass substrate. In addition to an all-optical interrogation of nanoscale mechanical properties, our results show that the adhesion layer can be used to controllably modify the acoustic phonon modes of a gold nanodisk. This direct coupling between optically excited plasmon modes and phonon modes can be exploited for a variety of emerging optomechanical applications.

High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study

The retention and elution of proteins in ion-exchange chromatography is routinely controlled by adjusting the mobile phase salt concentration. It has repeatedly been observed, as judged from adsorption isotherms, that the apparent heterogeneity of adsorption is lower at more-eluting, higher ionic strength. Here, we present an investigation into the mechanism of this phenomenon using a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT). We observed that the number of functional adsorption sites was smaller at high ionic strength and that these sites had reduced desorption kinetic heterogeneity, and thus narrower predicted elution profiles, for the anion-exchange adsorption of α-lactalbumin on an agarose-supported, clustered-charge ligand stationary phase. Explanations for the narrowing of the functional population such as inter-protein interactions and protein or support structural changes were investigated through kinetic analysis, circular dichroism spectroscopy, and microscopy of agarose microbeads, respectively. The results suggest the reduction of heterogeneity is due to both electrostatic screening between the protein and ligand and tuning the steric availability within the agarose support. Overall, we have shown that single molecule spectroscopy can aid in understanding the influence of ionic strength on the population of functional adsorbent sites participating in the ion-exchange chromatographic separation of proteins.

Fast Step Transition and State Identification (STaSI) for Discrete Single-Molecule Data Analysis

We introduce a step transition and state identification (STaSI) method for piecewise constant single-molecule data with a newly derived minimum description length equation as the objective function. We detect the step transitions using the Student’s t test and group the segments into states by hierarchical clustering. The optimum number of states is determined based on the minimum description length equation. This method provides comprehensive, objective analysis of multiple traces requiring few user inputs about the underlying physical models and is faster and more precise in determining the number of states than established and cutting-edge methods for single-molecule data analysis. Perhaps most importantly, the method does not require either time-tagged photon counting or photon counting in general and thus can be applied to a broad range of experimental setups and analytes.

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.

Improved analysis for determining diffusion coefficients from short, single-molecule trajectories with photoblinking.

Two maximum likelihood estimation (MLE) methods were developed for optimizing the analysis of single-molecule trajectories that include phenomena such as experimental noise, photoblinking, photobleaching, and translation or rotation out of the collection plane. In particular, short, single-molecule trajectories with photoblinking were studied, and our method was compared to existing analytical techniques applied to simulated data. The optimal method for various experimental cases was established, and the optimized MLE method was applied to a real experimental system: single-molecule diffusion of fluorescent molecular machines known as nanocars.

Charge-Dependent Transport Switching of Single Molecular Ions in a Weak Polyelectrolyte Multilayer

The tunable nature of weak polyelectrolyte multilayers makes them ideal candidates for drug loading and delivery, water filtration, and separations, yet the lateral transport of charged molecules in these systems remains largely unexplored at the single molecule level. We report the direct measurement of the charge-dependent, pH-tunable, multimodal interaction of single charged molecules with a weak polyelectrolyte multilayer thin film, a 10 bilayer film of poly(acrylic acid) and poly(allylamine hydrochloride) PAA/PAH. Using fluorescence microscopy and single-molecule tracking, two modes of interaction were detected: (1) adsorption, characterized by the molecule remaining immobilized in a subresolution region and (2) diffusion trajectories characteristic of hopping (D ∼ 10–9 cm2/s). Radius of gyration evolution analysis and comparison with simulated trajectories confirmed the coexistence of the two transport modes in the same single molecule trajectories. A mechanistic explanation for the probe and condition mediated dynamics is proposed based on a combination of electrostatics and a reversible, pH-induced alteration of the nanoscopic structure of the film. Our results are in good agreement with ensemble studies conducted on similar films, confirm a previously-unobserved hopping mechanism for charged molecules in polyelectrolyte multilayers, and demonstrate that single molecule spectroscopy can offer mechanistic insight into the role of electrostatics and nanoscale tunability of transport in weak polyelectrolyte multilayers.

Generalized recovery algorithm for 3D super-resolution microscopy using rotating point spread functions

Super-resolution microscopy with phase masks is a promising technique for 3D imaging and tracking. Due to the complexity of the resultant point spread functions, generalized recovery algorithms are still missing. We introduce a 3D super-resolution recovery algorithm that works for a variety of phase masks generating 3D point spread functions. A fast deconvolution process generates initial guesses, which are further refined by least squares fitting. Overfitting is suppressed using a machine learning determined threshold. Preliminary results on experimental data show that our algorithm can be used to super-localize 3D adsorption events within a porous polymer film and is useful for evaluating potential phase masks. Finally, we demonstrate that parallel computation on graphics processing units can reduce the processing time required for 3D recovery. Simulations reveal that, through desktop parallelization, the ultimate limit of real-time processing is possible. Our program is the first open source recovery program for generalized 3D recovery using rotating point spread functions.

Optimization of Spectral and Spatial Conditions to Improve Super-Resolution Imaging of Plasmonic Nanoparticles

Interactions between fluorophores and plasmonic nanoparticles modify the fluorescence intensity, shape, and position of the observed emission pattern, thus inhibiting efforts to optically super-resolve plasmonic nanoparticles. Herein, we investigate the accuracy of localizing dye fluorescence as a function of the spectral and spatial separations between fluorophores (Alexa 647) and gold nanorods (NRs). The distance at which Alexa 647 interacts with NRs is varied by layer-by-layer polyelectrolyte deposition while the spectral separation is tuned by using NRs with varying localized surface plasmon resonance (LSPR) maxima. For resonantly coupled Alexa 647 and NRs, emission to the far field through the NR plasmon is highly prominent, resulting in underestimation of NR sizes. However, we demonstrate that it is possible to improve the accuracy of the emission localization when both the spectral and spatial separations between Alexa 647 and the LSPR are optimized.