Single component and selective competitive protein adsorption in a patchy polymer brush: opposition between steric repulsions and electrostatic attractions

This work explores the use of "patchy" polymer brushes to control protein adsorption rates on engineered surfaces and to bind targeted species from protein mixtures with high selectivity but without invoking molecular recognition. The brushes of interest contain embedded cationic "patches" composed of isolated adsorbed poly(l-lysine) coils (PLL) that are about 10 nm in diameter and are randomly arranged on a silica substrate. Around these patches is a protein-resistant poly(ethylene glycol) (PEG) brush that is formed from the adsorption of a PLL-g-PEG graft copolymer on the remaining silica surface. Electrostatic attractions between individual cationic patches and the negative regions of approaching proteins may be energetically insufficient to bind proteins. Furthermore, protein-patch attractions are reduced by steric repulsions between proteins and the PEG brush. We show that protein adsorption, gauged by ultimate short-term coverages and by the initial protein adsorption rate, exhibits an adhesion threshold: pure PEG brushes of the architectures employed here and brushes containing sparse loadings of PLL patches do not adsorb protein. Above a critical PLL patch loading or threshold, protein adsorption proceeds, often dramatically. The PLL patch thresholds are specific to the protein of interest, allowing surfaces to be engineered to adhesively discriminate different proteins within a mixture. The separation achieved is remarkably sharp: one protein adsorbs, but the second is completely rejected from the interface. The surfaces in this study, by virtue of their well-controlled and well-characterized patchy nature, distinguish themselves from multicomponent brushes or brushes used to end-tether peptide sequences and nucleotides.     

Particle capture via discrete binding elements: systematic variations in binding energy for randomly distributed nanoscale surface features

This work examines how the binding strength of surface-immobilized "stickers" (representative of receptors or, in nonbiological systems, chemical heterogeneities) influences the adhesion between surfaces that are otherwise repulsive. The study focuses on a series of surfaces designed with fixed average adhesive energy per unit area and demonstrates quantitatively how a redistribution of the adhesive functionality into progressively larger clusters (stronger stickers) increases the probability of adhesive events. The work employs an electrostatic model system: relatively uniform, negative 1 μm silica spheres flow gently over negative silica flats. The flats contain small amounts of randomly positioned nanoscale cationic patches. The silica-silica interaction is repulsive; however, the cationic patches (present at sufficiently low levels that the overall surface charge remains substantially negative) produce local attractions. In this study, the attractions are relatively weak so that multiple patches engage to capture flowing particles. Experiments reveal an adhesion signature characteristic of a renormalized random distribution when the sticker strength is increased at an overall fixed binding strength per unit area of surface. The form of the particle capture curves are in good quantitative agreement with a simple model that assumes only a fixed adhesion energy needed for particle capture. Aside from the quantitative details that provide a simple formalism for anticipating particle adhesion, this work demonstrates how increasing the heterogeneities in the surface functionality can cause a system to go from being nonadhesive to becoming strongly adhesive. Indeed, systems containing small amounts of discretized adhesive functionality are always more adhesive than systems in which the same functionality is distributed uniformly over the surface (the mean field scenario).     

Synthèse de l'oxo-7 7H cyclohepta[b] pyridine et de quelques dérivés

This communication describes the synthesis of 7-one (7H) cyclohepta[b]pyridine and some derivatives (6,8-dimethyl; 8-methyl; 8-ethyl….). These compounds have been obtained by condensation of ketones with 2,3-diformylpyridine.     

Micrometer scale adhesion on nanometer-scale patchy surfaces: adhesion rates, adhesion thresholds, and curvature-based selectivity

Using a model system based on electrostatics, we probe interactions between spherical particles (negative silica) and planar surfaces that present randomly placed discrete attractive regions, 10 nm in size, in a repulsive background (silica flats carrying cationic surface constructs). Experiments measure the adhesion rates of particles onto the patchy collecting surfaces from flowing dispersions, as a function of the surface loading of the attractive patches, for different particle sizes (0.5 and 1 mum diameter spheres) and different ionic strengths. Surfaces densely populated with patches, such that they present net electrostatic attractions to approaching particles, capture particles at the transport-limited (maximum) rate. Surfaces sparsely loaded with attractive patches (which present a repulsive mean field to approaching particles) are usually still adhesive, but the particle adhesion rate depends on particle size, ionic strength, and patch loading. Most significant is an adhesion threshold, a critical density of patches needed to capture particles. This threshold, which occurs at average patch spacings of 30 nm and larger and which can be tuned through ionic strength, comprises the ability of the patchy surfaces to selectively distinguish particles of different sizes or objects of different local curvature or roughness. The observation of such an adhesion threshold implicates spatial fluctuations in patch arrangement. In addition to experiments, this paper develops arguments for lengthscales that govern adhesion rate behavior, comparing particle geometry and fluctuation lengthscales, and then demonstrating qualitative consistency with the localized colloidal potentials involved.     

Adhesion plaque formation dynamics between polymer vesicles in the limit of highly concentrated binding sites

This work examines the process of adhesion plaque formation between pairs of copolymer vesicles presenting dense surface concentrations of avidin (NeutrAvidin) and biotin. Micropipet aspiration maintains constant membrane tension, as the low-tension vesicle membrane spreads over a second, more tensed vesicle. Spreading rates near 1 microm/s but as high as 7 microm/s (the adhesion plaque diameter) and contact angle growth rates of 2-14 deg/s are observed. The ultimate contact angles, in the range of 120-140 degrees, are independent of membrane tension and also exceed those previously reported. Adhesion plaque formation occurs in three phases: an initial step in which contact is established, typically lasting from a few seconds to a minute, an abrupt jump into contact in which both vesicles undergo substantial deformation, and a slower continued growth of the contact angle and area. Vesicle pairs are irreversibly bound at the plaque such that attempts to peel them apart cause membrane rupture at critical tensions as high as 4 mN/m, setting a lower bound on the interfacial strength. When the quantity tau(1 - cos theta) (with tau the membrane tension and theta the contact angle) is plotted as a function of time during plaque formation for different values of tau, the curves fail to collapse, indicating the chemical driving force for adhesion greatly exceeds the mechanical resisting tension.     

The adhesion kinetics of sticky vesicles in tension: the distinction between spreading and receptor binding

We investigate the kinetics of spreading and adhesion between polymer vesicles decorated with avidin and biotin, held in micropipettes to maintain fixed tension and suppress membrane bending fluctuations. In this study, the density of avidin (actually Neutravidin) and biotin was varied, but was always sufficiently high so that lateral diffusion in the membrane was unimportant to the adhesive mechanism or rate. For a stunning result, we report a concentration-dependent distinction between adhesion and spreading: At low surface densities of avidin and biotin, irreversible vesicle adhesion is strong enough to break the membrane when vesicle separation is attempted, yet there is no spreading or "wetting". By this we mean that there is no development of an adhesion plaque beyond the initial radius of contact and there is no development of a meaningful contact angle. Conversely, at 30% functionalization and greater, membrane adhesion is manifest through a spreading process in which the vesicle held at lower tension partially engulfs the second vesicle, and the adhesion plaque grows, as does the contact angle. Generally, when spreading occurs, it starts abruptly, following a latent contact period whose duration decreases with increasing membrane functionality. A nucleation-type rate law describes the latency period, determined by competition between bending and sticking energy. The significance of this result is that, not only are membrane mechanics important to the development of adhesion in membranes of nanometer-scale thickness, mechanics can dominate and even mask adhesive features such as contact angle. This renders contact angle analyses inappropriate for some systems. The results also suggest that there exist large regions of parameter space where adhesive polymeric vesicles will behave qualitatively differently from their phospholipid counterparts. This motivates different strategies to design polymeric vesicles for applications such as targeted drug delivery and biomimetic scavengers.     

Sensitivity of protein adsorption to architectural variations in a protein-resistant polymer brush containing engineered nanoscale adhesive sites

Patchy polymer brushes contain nanoscale (5-15 nm) adhesive elements, such as polymer coils or nanoparticles, embedded at their base at random positions on the surface. The competition between the brush's steric (protein resistant) repulsions and the attractions from the discrete adhesive elements provides a precise means to control bioadhesion. This differs from the classical approach, where functionality is placed on the brush's periphery. The current study demonstrates the impact of poly(etheylene glycol) (PEG) brush architecture and ionic strength on fibrinogen adsorption on brushes containing embedded poly-l-lysine (PLL, 20K MW) coils or "patches". The consistent appearance of a fibrinogen adsorption threshold, a minimum loading of patches on the surface, below which protein adsorption does not occur, suggests multivalent protein capture: Adsorbing proteins simultaneously engage several patches. The surface composition (patch loading) at the threshold is extremely sensitive to the brush height and ionic strength, varying up to a factor of 5 in the surface loading of the PLL patches (~50% of the range of possible surfaces). Variations in ionic strength have a similar effect, with the smallest thresholds seen for the largest Debye lengths. While trends with brush height were the clearest and most dominant, consideration of the PEG loading within the brush or its persistence length did not reveal a critical brush parameter for the onset of adsorption. The lack of straightforward correlation on brush physics was likely a result of multivalent binding, (producing an additional dependence on patch loading), and might be resolved for univalent adsorption onto more strongly binding patches. While studies with similar brushes placed uniformly on a surface revealed that the PEG loading within the brush is the best indicator of protein resistance, the current results suggest that brush height is more important for patchy brushes. Likely the interactions producing brush extension normal to the interface act similarly to drive lateral tether extension to obstruct patches.     

The effect of topical anesthesia on vocal fold motion

The objective of this study was to determine if topical anesthesia to the larynx and pharynx affects vocal fold motion during dynamic voice evaluation with transnasal flexible endoscopy. Transnasal dynamic laryngeal examinations of 10 patients with no voice complaints were evaluated by five blinded fellowship-trained laryngologists. Each patient was examined before and after application of topical anesthetic. Reviewers rated briskness of right and left vocal fold movement and longitudinal tension on a visual analogue scale. Statistical comparisons were made between individual subject scores before and after anesthetic application. Inter-rater reliability was also assessed. No statistical difference was observed between subject scores before and after anesthetic application. Average intraclass correlation coefficients were 0.643 and 0.591 for pre- and postanesthesia scores, respectively. Application of topical anesthesia to the larynx and pharynx does not affect vocal fold motion.     

Protein spreading kinetics at liquid-solid interfaces via an adsorption probe method

We report the areal growth kinetics of fibrinogen adsorbed on model hydrophobic and hydrophilic surfaces measured via an adsorption probe method. This approach exploits the adsorption of probe molecules to determine the evolution of fibrinogen test molecules under conditions where the fibrinogen test molecules adsorb at relatively dilute surface conditions, minimizing interactions between them. It is found that fibrinogen test molecules spread from an average initial footprint of 100 nm2 to a final footprint near 500 nm2 per molecule on the hydrophobic surface, with a single-exponential decay of 1735 s. On a hydrophilic monolayer, the area increases from 100 to 160 nm2 with a characteristic time of 6740 s. These results demonstrate the power of the adsorption probe approach and comprise the first measurements of the averaged area relaxations of adsorbed proteins. The observation of single-exponential dynamics is remarkable, given the extensive relaxation on the hydrophobic surface, which must involve fibrinogen denaturing.     

Adsorption and Exchange Dynamics in Aging Hydroxyethylcellulose Layers on Silica

The adsorption kinetics of hydroxyethylcellulose (HEC) on silica and relaxations in adsorbed HEC layers were probed using total internal reflectance fluorescence and near-Brewster reflectivity. Like many random-coil polymers, HEC was found to adsorb at the transport-limited rate. Relaxations occurred at nearly constant interfacial mass when HEC layers were exposed to aqueous solvent, causing the subsequent exchange of chains between the layer and the free solution to become increasingly hindered. Eventually, on the time scale of a day, layers became immobilized and unable to accommodate chains from free solution. A continued fluorescence decay, beyond time scales that could be probed with self exchange, suggested further relaxations of the adsorbed HEC. The polydisperse HEC system (with an average molecular weight near 450,000) behaved qualitatively similar to molecular weight standard polyethylene oxide (PEO) layers on silica. For instance, relaxations in PEO layers occurred on a time scale of 10-20 h, like the HEC layers. Young layers of the latter, however, exhibited self-exchange kinetics that were an order of magnitude slower than PEO layers of similar age. This difference in adsorbed layer dynamics was attributed to HEC's stiffer backbone, compared with flexible PEO. Copyright 2000 Academic Press.