High-throughput single-molecule total internal reflection fluorescence microscopy was used to investigate the effects of pH and ionic strength on bovine serum albumin (BSA) adsorption, desorption, and interfacial diffusion at the aqueous-fused silica interface. At high pH and low ionic strength, negatively charged BSA adsorbed slowly to the negatively charged fused silica surface. At low pH and low ionic strength, where BSA was positively charged, or in solutions at higher ionic strength, adsorption was approximately 1000 times faster. Interestingly, neither surface residence times nor the interfacial diffusion coefficients of BSA were influenced by pH or ionic strength. These findings suggested that adsorption kinetics were dominated by energy barriers associated with electrostatic interactions, but once adsorbed, protein-surface interactions were dominated by short-range nonelectrostatic interactions. These results highlight the ability of single-molecule techniques to isolate elementary processes (e.g., adsorption and desorption) under steady-state conditions, which would be impossible to measure using ensemble-averaging methods.

The synthesis of end-functionalized hydrogen-bonding polymers and the investigation of their melt phase blend behavior are reported. Monofunctional and telechelic ureidopyrimidinone (UPy)-functionalized poly(styrene)s and poly(methyl methacrylate)s were synthesized using atom transfer radical polymerization (ATRP) followed by atom transfer radical coupling (ATRC). Aggregation of UPy end-groups in solution was observed using size exclusion chromatography. The effect of UPy end-groups on blend miscibility in the melt state was studied using optical microscopy and differential scanning calorimetry. The incorporation of associating groups onto one end of either blend component decreases miscibility relative to unfunctionalized parent blends. Lower miscibility was also observed for blends in which both components were monofunctionalized with associating end-groups. The largest decrease in miscibility was observed for blends containing telechelic UPy-functionalized polymers, which were immiscible across the entire composition range.

The presence of single-stranded (ssDNA) vs. double-stranded (dsDNA) DNA at a surfactant-laden aqueous-nematic liquid crystal (LC) interface results in distinctly different orientations of the LC molecular axis; this is of practical interest as a method to detect DNA hybridization. Results presented here provide new insights into the molecular-level mechanisms of these phenomena. The adsorption of ssDNA to a cationic surfactant-laden aqueous-LC interface caused LC reorientation, leading to coexistence between homeotropic and planar (birefringent) oriented regions. Fluorescence microscopy revealed that ssDNA preferentially partitioned into the birefingent regions, presumably causing a decreased surface coverage of surfactant and the resultant planar LC orientation. Both electrostatic and hydrophobic effects were found to be critical to inducing LC reorientation. In particular, insufficient ssDNA adsorption occurred in the absence of a cationic surfactant (e.g. with no surfactant or with a non-ionic surfactant), demonstrating the importance of electrostatic interactions with the polyanionic ssDNA. Even in the presence of a cationic surfactant, however, polyanions without hydrophobic side-group moieties (poly[acrylic acid] and dsDNA) caused no LC reorientation, while polyanions with hydrophobic side groups (polystyrene sulfonate and ssDNA) initiated the desired LC reorientation. These observations are consistent with the fact that interfacial hybridization of adsorbed probe ssDNA to complementary target ssDNA caused a reorientation from planar back to homeotropic. We propose that ssDNA forms an electrostatic interfacial complex with cationic surfactant where the hydrophobic nucleobases associate directly with the LC phase, effectively competing with surfactant molecules for interfacial sites. Upon hybridization, the hydrophobic character of the ssDNA is lost and the nucleobases no longer associate directly with the LC phase, allowing the surfactant molecules to pack more closely at the interface.

No abstract

Using high throughput single-molecule total internal reflection fluorescence microscopy (TIRFM), we have acquired molecular trajectories of bovine serum albumin (BSA) and hen egg white lysozyme during protein layer formation at the silicone oil-water interface. These trajectories were analyzed to determine the distribution of molecular diffusion coefficients, and for signatures of molecular crowding/caging, including subdiffusive motion and temporal anticorrelation of the instantaneous velocity vector. The evolution of these properties with aging time of the interface was compared with dynamic interfacial tension measurements. For both lysozyme and BSA, we observed an overall slowing of protein objects, the onset of both subdiffusive and anticorrelated motion (associated with crowding), and a decrease in the interfacial tension with aging time. For lysozyme, all of these phenomena occurred virtually simultaneously, consistent with a homogeneous model of layer formation that involves gradual crowding of weakly interacting proteins. For BSA, however, the slowing occurred first, followed by the signatures of crowding/caging, followed by a decrease in interfacial tension, consistent with a heterogeneous model of layer formation involving the formation of protein clusters. The application of microrheological methods to single molecule trajectories described here provides an unprecedented level of mechanistic interpretation of interfacial events that occurred over a wide range of interfacial protein coverage.