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.ion-exchange chromatography | single-molecule kinetics | bioseparations | optical nanoscopy T he hundred-billion-dollar global pharmaceutical industry relies increasingly on the painstaking purification of therapeutic biomolecules such as proteins and nucleic acids (1). Separation of biologics is often performed using ion-exchange chromatography on stationary phases supporting singly charged ligands (2, 3) and constitutes an expensive, bottlenecking step in production. Improving bioseparations is thus highly desirable (4, 5); yet, a molecular-scale, mechanistic understanding is lacking, for ionexchange chromatography in particular (6). Mechanistic detail is lost in ensemble analyses, reflecting the inherent heterogeneity of both the adsorbed biomolecules and the porous stationary phase supports (7). Ensemble adsorption isotherms, however, suggest the likelihood that protein and nucleic acid separations in ion-exchange columns may involve random ligand clustering (8-10). Additional support for such an assertion lies in the implementation of stationary phases of very high charge density by polymerization of charged monomers or layer-by-layer deposition (11-13), and in the demonstration that patches of high charge density on proteins often play a disproportionate role in their adsorption (4,6,(14)(15)(16)(17). In this work, we provide direct evidence of the importance of charge clustering in ion-exchange systems by direct observation of individual adsorption sites.Although the role of multivalency is broadly accepted and exploited in a wide range of associative and adsorption ...

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.

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.

We present a hardware-based method that can improve single molecule fluorophore observation time by up to 1500% and super-localization by 47% for the experimental conditions used. The excitation was modulated using an acousto-optic modulator (AOM) synchronized to the data acquisition and inherent data conversion time of the detector. The observation time and precision in super-localization of four commonly used fluorophores were compared under modulated and traditional continuous excitation, including direct total internal reflectance excitation of Alexa 555 and Cy3, non-radiative Förster resonance energy transfer (FRET) excited Cy5, and direct epi-fluorescence wide field excitation of Rhodamine 6G. The proposed amplitude-modulated excitation does not perturb the chemical makeup of the system or sacrifice signal and is compatible with multiple types of fluorophores. Amplitude-modulated excitation has practical applications for any fluorescent study utilizing an instrumental setup with time-delayed detectors.