We report the characterization of gold nanoparticles modified with thiol-terminated single stranded DNA (ssDNA) using analytical ultracentrifugation. Dynamic light scattering was used to measure the diameter of bare and ssDNA modified gold nanoparticles to corroborate the predictions of our models. Sedimentation coefficients of nominally 10 and 20 nm diameter gold nanoparticles modified with thiol-terminated thymidine homo-oligonucleotides, 5-30 bases in length, were determined with analytical ultracentrifugation. The sedimentation coefficients of gold nanoparticles modified with ssDNA were found to decrease with increasing coverage of ssDNA and increasing length of ssDNA. The sedimentation coefficients of ssDNA modified gold particles were most closely predicted when the strands were modeled as fully extended chains (FEC). Apparent particle densities of bare gold nanoparticles calculated from measured sedimentation coefficients decreased significantly below that of bulk gold with decreasing size of nanoparticles. This finding suggests that hydration layer effects are an important factor in the sedimentation behavior for both bare and short ssDNA chain modified gold particles.
We report a glycoanalysis method in which lectins are used to probe the glycans of therapeutic glycoproteins that are adsorbed on gold nanoparticles. A model mannose-presenting glycoprotein, ribonuclease B (RNase B), and the therapeutic monoclonal antibody (mAb) rituximab, were found to adsorb spontaneously and non-specifically to bare gold nanoparticles such that glycans were accessible for lectin binding. Addition of a multivalent binding lectin, such as concanavalin A (Con A), to a solution of the modified gold nanoparticles resulted in cross-linking of the nanoparticles. This phenomenon was evidenced within 1 min by a change in the hydrodynamic diameter, D(H), measured by dynamic light scattering (DLS) and a shift and increase in absorbance of the plasmon resonance band of the gold nanoparticles. By combining the sugar-binding specificity and the cross-linking capabilities of lectins, the non-specific adsorption of glycoproteins to gold surfaces, and the unique optical reporting properties of gold nanoparticles, a glycosylation pattern of rituximab could be generated. This assay provides advantages over currently used glycoanalysis methods in terms of short analysis time, simplicity of the conjugation method, convenience of simple spectroscopic detection, and feasibility of providing glycan characterization of the protein drug product by using a variety of binding lectins.
Henry’s constants of 2-propanone, 2-butanone, 2-pentanone, 2-hexanone, and 2-heptanone in aqueous solutions containing inorganic salts or quaternary ammonium salts were determined at temperatures between 313 and 343 K, and salt ionic strengths below 5.0. Our data show that inorganic salts generally salt-out the 2-ketones, whereas quaternary ammonium salts generally salt-in the 2-ketones. However, 2-propanone is salted-out by both types of salts. We have also extended a published model based on dilute solution theory to solutions containing two inorganic salts, as well as to solutions containing quaternary ammonium salts. We show that it is possible to correlate all data for salting-out behavior using a single salt-effect parameter for each system. Salting-in behavior can also be correlated with a single parameter, although agreement with experiment is not as good as in the case of salting-out behavior. This is because the model does not explicitly account for the role of dispersion forces in these systems, which may be inferred from the trends exhibited by the salt-effect parameter as the size of the 2-ketone increases in solutions containing quaternary ammonium salts.
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