Untying the Gordian KNOT: Unbiased Single Particle Tracking Using Point Clouds and Adaptive Motion Analysis.

Achieving mechanistic understanding of transport in complex environments such as inside cells or at polymer interfaces is challenging. We need better ways to image transport in 3-D and better single particle tracking algorithms to determine transport that are not systemically biased toward any classical motion model. Here we present an unbiased single particle tracking algorithm: Knowing Nothing Outside Tracking (KNOT).

Single-Molecule Dynamics Reflect IgG Conformational Changes Associated with Ion-Exchange Chromatography.

Conformational changes of antibodies and other biologics can decrease the effectiveness of pharmaceutical separations. Hence, a detailed mechanistic picture of antibody-stationary phase interactions that occur during ion-exchange chromatography (IEX) can provide critical insights. This work examines antibody conformational changes and how they perturb antibody motion and affect ensemble elution profiles.

A new metric for relating macroscopic chromatograms to microscopic surface dynamics: the distribution function ratio (DFR).

Heterogeneous stationary phase chemistry causes chromatographic tailing that lowers separation efficiency and complicates optimizing mobile phase conditions. Model-free metrics are attractive for assessing optimal separation conditions due to the low quantity of information required, but often do not reveal underlying mechanisms that cause tailing, for example, heterogeneous retention modes. We report a new metric, which we call the Distribution Function Ratio (DFR), based on a graphical comparison between the chromatogram and Gaussian cumulative distribution functions, achieving correspondence to ground truth surface dynamics with a single chromatogram.

Transforming Separation Science with Single-Molecule Methods.

Empirical optimization of the multiscale parameters underlying chromatographic and membrane separations leads to enormous resource waste and production costs. A bottom-up approach to understand the physical phenomena underlying challenges in separations is possible with single-molecule observations of solute-stationary phase interactions. We outline single-molecule fluorescence techniques that can identify key interactions under ambient conditions.

Unraveling peak asymmetry in chromatography through stochastic theory powered Monte Carlo simulations.

An overarching theory of chromatography capable of modeling all analyte-stationary phase interactions would enable predictive design of pharmaceutically relevant separations. The stochastic theory of chromatography has been postulated as a suitable basis to achieve this goal. Here, we implement Dondi and Cavazzini's Monte Carlo framework that utilizes experimentally accessible single molecule kinetics and use it to correlate heterogenous adsorption statistics at the stationary phase to shifts in asymmetry.

Imaging Switchable Protein Interactions with an Active Porous Polymer Support.

Mechanistic details about how local physicochemistry of porous interfaces drives protein transport mechanisms are necessary to optimize biomaterial applications. Cross-linked hydrogels made of stimuli-responsive polymers have potential for active protein capture and release through tunable steric and chemical transformations. Simultaneous monitoring of dynamic changes in both protein transport and interfacial polymer structure is an experimental challenge.

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.

From a Protein's Perspective: Elution at the Single-Molecule Level.

Column chromatography is a widely used analytical technique capable of identifying and isolating a desired chemical species from a more complicated mixture. Despite the method's prevalence, theoretical descriptions have not advanced to accommodate today's common analyte, proteins. Proteins are increasingly used as biologics, a term that refers to biological pharmaceuticals, and present new complexities for chromatographic separation.

Enhancing Analytical Separations Using Super-Resolution Microscopy.

Super-resolution microscopy is becoming an invaluable tool to investigate structure and dynamics driving protein interactions at interfaces. In this review, we highlight the applications of super-resolution microscopy for quantifying the physics and chemistry that occur between target proteins and stationary-phase supports during chromatographic separations. Our discussion concentrates on the newfound ability of super-resolved single-protein spectroscopy to inform theoretical parameters via quantification of adsorption-desorption dynamics, protein unfolding, and nanoconfined transport.

The structure-energy landscape of NMDA receptor gating.

N-Methyl-D-aspartate (NMDA) receptors are the main calcium-permeable excitatory receptors in the mammalian central nervous system. The NMDA receptor gating is complex, exhibiting multiple closed, open, and desensitized states; however, central questions regarding the conformations and energetics of the transmembrane domains as they relate to the gating states are still unanswered. Here, using single-molecule Förster resonance energy transfer (smFRET), we map the energy landscape of the first transmembrane segment of the Rattus norvegicus NMDA receptor under resting and various liganded conditions.

Variable Lysozyme Transport Dynamics on Oxidatively Functionalized Polystyrene Films.

Tuning protein adsorption dynamics at polymeric interfaces is of great interest to many biomedical and material applications. Functionalization of polymer surfaces is a common method to introduce application-specific surface chemistries to a polymer interface. In this work, single-molecule fluorescence microscopy is utilized to determine the adsorption dynamics of lysozyme, a well-studied antibacterial protein, at the interface of polystyrene oxidized via UV exposure and oxygen plasma and functionalized by ligand grafting to produce varying degrees of surface hydrophilicity, surface roughness, and induced oxygen content.

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.

Effects of Solute-Solvent Hydrogen Bonding on Nonaqueous Electrolyte Structure.

We investigate the source of Raman background signal commonly misidentified as fluorescence in nonaqueous electrolytes via a variety of spectroscopies (Raman, fluorescence, NMR) and find evidence of hydrogen-bonding interactions. This hydrogen bonding gives rise to broadband anharmonic vibrational modes and suggests that anions play an important and underappreciated role in the structure of nonaqueous electrolytes. Controlling electrolyte structure has important applications in advancing in operando spectroscopy measurements as well as understanding the stability of high concentration electrolytes for next-generation electrochemical energy storage devices.