Browsing by Author "Landes, Christy F."
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Item Fluorescence correlation spectroscopy study of protein transport and dynamic interactions with clustered?charge peptide adsorbents(Journal of Molecular Recognition, 2013-08) Daniels, Charlisa R.; Kisley, Lydia; Kim, Hannah; Chen, Wen-Hsiang; Poongavanam, Mohan-Vivekanandan; Reznik, Carmen; Kourentzi, Katerina D.; Willson, Richard C.; Landes, Christy F.Ion?exchange chromatography relies on electrostatic interactions between the adsorbent and the adsorbate and is used extensively in protein purification. Conventional ion?exchange chromatography uses ligands that are singly charged and randomly dispersed over the adsorbent, creating a heterogeneous distribution of potential adsorption sites. Clustered?charge ion exchangers exhibit higher affinity, capacity, and selectivity than their dispersed?charge counterparts of the same total charge density. In the present work, we monitored the transport behavior of an anionic protein near clustered?charge adsorbent surfaces using fluorescence correlation spectroscopy. We can resolve protein?free diffusion, hindered diffusion, and association with bare glass, agarose?coated, and agarose?clustered peptide surfaces, demonstrating that this method can be used to understand and ultimately optimize clustered?charge adsorbent and other surface interactions at the molecular scale.Item Permeability of anti-fouling PEGylated surfaces probed by fluorescence correlation spectroscopy(Colloids Surf B Biointerfaces, 2017-10) Daniels, Charlisa R.; Reznik, Carmen; Kilmer, Rachel; Felipe, Mary J.; Tria, Maria C. R.; Kourentzi, Katerina D.; Chen, Wen-Hsiang; Advincula, Rigoberto C.; Willson, Richard C.; Landes, Christy F.The present work reports on in situ observations of the interaction of organic dye probe molecules and dye-labeled protein with different poly(ethylene glycol) (PEG) architectures (linear, dendron, and bottle brush). Fluorescence correlation spectroscopy (FCS) and single molecule event analysis were used to examine the nature and extent of probe朠EG interactions. The data support a sieve-like model in which size-exclusion principles determine the extent of probe朠EG interactions. Small probes are trapped by more dense PEG architectures and large probes interact more with less dense PEG surfaces. These results, and the tunable pore structure of the PEG dendrons employed in this work, suggest the viability of electrochemically-active materials for tunable surfaces.Item Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations(Proceedings of the National Academy of Sciences, 2014-02) Kisley, Lydia; Chen, Jixin; Mansur, Andrea P.; Shuang, Bo; Kourentzi, Katerina D.; Poongavanam, Mohan-Vivekanandan; Chen, Wen-Hsiang; Dhamane, Sagar; Willson, Richard C.; Landes, Christy F.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.