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 binding of a DNA aptamer (5'-CCGTCTTCCAGACAAGAGTGCAGGG-3') to recombinant human vascular endothelial growth factor (VEGF(165)) was characterized using surface plasmon resonance (SPR), fluorescence anisotropy and isothermal titration calorimetry (ITC). Results from both fluorescence anisotropy and ITC indicated that a single aptamer molecule binds to each VEGF homodimer, unlike other VEGF inhibitors that exhibit 2(ligand):1(VEGF homodimer) stoichiometry. In addition, ITC revealed that the association of the aptamer to VEGF at 20 degrees C is enthalpically driven, with an unfavorable entropy contribution. SPR kinetic studies, with careful control of possible mass transfer effects, demonstrated that the aptamer binds to VEGF with an association rate constant k(on) = 4.79 +/- 0.03 x 10(4) M(-1) s(-1) and a dissociation rate constant k(off) = 5.21 +/- 0.02 x 10(-4) s(-1) at 25 degrees C. Key recognition hot-spots were determined by a combination of aptamer sequence substitutions, truncations, and extensions. Most single-nucleotide substitutions, particularly within an mfold-predicted stem, suppress binding, whereas those within a predicted loop have a minimal effect. The 5'-end of the aptamer plays a key role in VEGF recognition, as a single-nucleotide truncation abolished VEGF binding. Conversely, an 11-fold increase in the association rate (and affinity) is observed with a single cytosine nucleotide extension, due to pairing of the 3'-GGG with 5'-CCC in the extended aptamer. Our approach effectively maps the secondary structural elements in the free aptamer, which present the unpaired interface for high affinity VEGF recognition. These data demonstrate that a directed binding analysis can be used in concert with library screening to characterize and improve aptamer/ligand recognition.

Demand for highly sensitive, robust diagnostics and environmental monitoring methods has led to extensive research in improving reporter technologies. Inorganic phosphorescent materials exhibiting persistent luminescence are commonly found in electroluminescent displays and glowing paints but are not widely used as reporters in diagnostic assays. Persistent luminescence nanoparticles (PLNPs) offer advantages over conventional photoluminescent probes, including the potential for enhanced sensitivity by collecting time-resolved measurements or images with decreased background autofluorescence while eliminating the need for expensive optical hardware, superior resistance to photobleaching, amenability to quantitation, and facile bioconjugation schemes. We isolated rare-earth doped strontium aluminate PLNPs from larger-particle commercial materials by wet milling and differential sedimentation and water-stabilized the particles by silica encapsulation using a modified Stöber process. Surface treatment with aldehyde silane followed by reductive amination with heterobifunctional amine-poly(ethylene glycol)-carboxyl allowed covalent attachment of proteins to the particles using standard carbodiimide chemistry. NeutrAvidin PLNPs were used in lateral flow assays (LFAs) with biotinylated lysozyme as a model analyte in buffer and monoclonal anti-lysozyme HyHEL-5 antibodies at the test line. Preliminary experiments revealed a limit of detection below 100 pg/mL using the NeutrAvidin PLNPs, which was approximately an order of magnitude more sensitive than colloidal gold.

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.

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In previous work, Vibrio proteolyticus 5S rRNA was shown to stabilize 13-50 nucleotide "guest" RNA sequences for expression in Escherichia coli. The expressed chimeric RNAs accumulated to high levels in E. coli without being incorporated into ribosomes and without obvious effects on the host cells. In this work, we inserted sequences encoding known aptamers recognizing a protein and an organic dye into the 5S rRNA carrier and showed that aptamer function is preserved in the chimeras. A surface plasmon resonance competitive binding assay demonstrated that a vascular endothelial growth factor (VEGF) aptamer/5S rRNA chimera produced in vitro by transcriptional runoff could compete with a DNA aptamer for VEGF, implying binding of the growth factor by the VEGF "ribosomal RNA aptamer." Separately, a 5S rRNA chimera displaying an aptamer known to increase the fluorescence of malachite green (MG) also enhanced MG fluorescence. Closely related control rRNA molecules showed neither activity. The MG aptamer/5S rRNA chimera, like the original MG aptamer, also increased the fluorescence of other triphenyl methane (TPM) dyes such as crystal violet, methyl violet, and brilliant green, although less effectively than with MG. These results indicate that the molecular recognition properties of aptamers are not lost when they are expressed in the context of a stable 5S rRNA carrier. Inclusion of the aptamer in a carrier may facilitate production of large quantities of RNA aptamers, and may open an approach to screening aptamer libraries in vivo.

Two types of viral nanoparticles were functionalized with target-specific antibodies and multiple copies of an enzymatic reporter (horseradish peroxidase). The particles were successfully integrated into an immunochromatographic assay detecting MS2 bacteriophage, a model for viral pathogens. The sensitivity of the assay was greatly superior to conventional gold nanoparticle lateral flow assays, and results could easily be evaluated, even without advanced lab instruments.