Dye Diffusion at Surfaces: Charge Matters

Daniels, Charlisa R.; Reznik, Carmen; Landes, Christy F.

doi:10.1021/la904749z

Cited by 25 publications

(43 citation statements)

References 60 publications

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“…This control demonstrates that there are no long-lived (>3 ms) (SI Appendix, Fig. S2) interactions between the glass substrate and the α-lactalbumin, consistent with previous observations (58,60).…”

Section: Resultssupporting

confidence: 90%

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

Kisley¹,

Chen²,

Mansur³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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124

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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 ...

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Section: Resultssupporting

confidence: 90%

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

Kisley¹,

Chen²,

Mansur³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

124

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“…2(a and b) for R6G and Alexa, respectively. As it has been described previously [38], the cationic R6G dye exhibited coulombic interactions with the glass while the anionic Alexa dye did not measurably interact with the surface. This can be seen in comparisons of the measurement close to and including the surface (0.5 μm, see in Section 4 describing geometry of focal volume) versus within the bulk solution (2.0 μm).…”

Section: Resultssupporting

confidence: 65%

“…We used confocal FCS and single event analysis to quantify the presence and extent of interactions between PEG and the cationic Rhodamine 6G (R6G) and anionic AlexaFluor 555 ® (Alexa) dyes as a function of distance from the surface. We have successfully used these techniques to study transport at charged and crowded interfaces and with heterogeneous mixtures [38–41]. Others have recently reported the successful use of FCS to understand probe–polymer brush interactions [42].…”

Section: Introductionmentioning

confidence: 99%

“…Others have recently reported the successful use of FCS to understand probe–polymer brush interactions [42]. When measuring close to the substrate interface, it is important to consider dye–surface interactions [38]. In order to offer insight into these questions we report evidence of the interaction of small molecules and proteins with linear and other forms of PEG brushes (Fig.…”

Section: Introductionmentioning

confidence: 99%

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Permeability of anti-fouling PEGylated surfaces probed by fluorescence correlation spectroscopy

Daniels¹,

Reznik²,

Kilmer³

et al. 2011

Colloids and Surfaces B: Biointerfaces

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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–PEG interactions. The data support a sieve-like model in which size-exclusion principles determine the extent of probe–PEG 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.

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“…34 It will be also not further discussed how FCS can assist in analysing the photokinetics in conjugated polymers, 35 or detect molecular mobility at interfaces, 36,37 nor will this review cover FCS studies performed with nanoparticles. [38][39][40][41][42][43][44] With respect to the latter topic, I refer to the review on FCS studies in the journal "Current Opinion in Colloid & Interface Science" by Koynov et al 45 Fluorescence correlation spectroscopy…”

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confidence: 99%