Ronald C. Chatelier

Effect of polysaccharide structure on protein adsorption

Abstract Using X-ray photoelectron spectroscopy for quantification, the adsorption has been studied of chicken egg lysozyme, human serum albumin (HSA), bovine colostrum lactoferrin, and γ-globulin (IgG) from single solutions onto surface-immobilised polysaccharide coatings, which were produced by the covalent attachment of a series of carboxymethyldextrans (CMDs) onto aminated fluoropolymer surfaces. CMDs with differing degrees of carboxymethyl substitution were synthesized by the reaction of dextran with bromoacetic acid under different reactant ratios. Substantial amounts of protein adsorption onto these coatings were observed with the majority of the coating/protein combinations. On the most extensively substituted CMD (1 carboxyl group per 2 dextran units), lysozyme and lactoferrin adsorbed to approximately monolayer amounts whereas there was minimal adsorption of HSA, indicating the importance of electrostatic interfacial interactions. CMD 1:14 was similar whereas the least substituted, least dense coating, from CMD 1:30, adsorbed less lysozyme and lactoferrin but more HSA. Adsorption of the large multidomain protein IgG varied little with the coating. Grazing angle XPS data indicated that for the CMD 1:30 coating there occurred significant in-diffusion of the lower molecular weight proteins. The data suggest that elimination of adsorption of a broad spectrum of proteins is not straightforward with negatively charged polysaccharide coatings; elimination of protein accumulation onto/into such coatings may not be achievable solely with a balance of electrostatic and steric–entropic interfacial forces.

Characterization of the Ageing of Plasma-deposited Polymer Films: Global Analysis of X-ray Photoelectron Spectroscopy Data

A protocol for global analysis of x-ray photoelectron spectra of plasma-deposited polymer films is presented. These materials are difficult to analyse because of the multitude of different chemical groups present. The combination of different primary and secondary binding energy shifts results in relatively broad, featureless photoelectron peaks. The protocol is based on fitting a series of spectra obtained by monitoring the surface composition of a plasma polymer film over extended periods of time after deposition. Information obtained from this first round of curve-fitting is used in the form of additional constraints for a second round of fitting. This leads to a self-consistent procedure which is akin to a global approach to curve-fitting. To illustrate application of this method, results of a study of ann-heptylamine plasma polymer are described. The spectral changes on ageing provided clear evidence for radical-initiated oxidation reactions. These reactions generated additional radicals, which sustained the oxidation process for many months and not only led to substantial incorporation of oxygen into the material (forming a variety of carbon–oxygen functionalities) but also to conversion of most of the existing carbon–nitrogen functionalities to an oxidized form.

Evolution of the surface composition and topography of perfluorinated polymers following ammonia-plasma treatment

Treatment of fluorinated ethylene propylene (FEP) and polytetrafluoroethylene (PTFE) in ammonia plasmas produced surfaces with very high wettability by water, but on storage in air at ambient temperature, the air/water contact angles increased markedly. The evolution of the surface composition and topography was studied by angle-dependent X-ray photoelectron spectroscopy (XPS), derivatization of amine groups with fluorescein isothiocyanate, scanning tunelling microscopy (STM), and atomic force microscopy (AFM). XPS demonstrated a continuous increase in the oxygen content over periods of weeks; this was assigned to oxidation of trapped radicals and subsequent secondary reactions. In addition, the fluorine content also changed markedly on storage; the XPS fluorine signal suggested that there was a substantial amount of fluoride in the freshly treated surfaces, and this component disappeared rapidly on storage. STM and AFM showed no changes in topography with aging but suggested surface hardening on plasma t...

Biomedical coatings by the covalent immobilization of polysaccharides onto gas‐plasma‐activated polymer surfaces

As the surface properties of polymeric biomaterials play an important role in the performance of biomedical devices, highly hydrophilic, ultrathin coatings were applied onto hydrophobic, perfluorinated and organosilicon polymers by the covalent immobilization of polysaccharides using a reductive amination reaction. Gas plasma (r.f. glow discharge) methods were employed to equip the surfaces of these normally unreactive polymeric substrates with chemical groups capable of reacting with polysaccharides in aqueous solution. In one variant, ammonia plasmas were used to introduce into the polymer surfaces a submonolayer of amine groups. Alternatively, an n-heptylamine process vapour was used to deposit a thin plasma polymer film that possessed surface amine groups. The polysaccharides were activated for covalent immobilization by periodate oxidation, which produced hemiacetal structures, as revealed by NMR and XPS. The hemiacetal structures in the polysaccharide chains were reacted with the surface amine groups on the polymers. The resulting Schiff base linkages were stabilized by reduction to secondary amine linkages using sodium cyanoborohydride. Detailed surface analysis is important for verification that the intended chemistries have indeed been achieved in such multilayer coating schemes. X-ray photoelectron spectroscopy provided a thickness estimate of 1 ± 0.3 nm for the polysaccharide coatings in the dehydrated state. Copyright

Correlation of the Nitrogen 1s and Oxygen 1s XPS Binding Energies with Compositional Changes During Oxidation of Ethylene Diamine Plasma Polymers

In the course of plasma deposition of organic-polymeric thin films, radicals are incorporated into the growing film. These radicals initiate spontaneous oxidation reactions that continue over many weeks when the plasma polymers are stored in air. The resultant changes to the composition with time were monitored by XPS for ethylene diamine plasma polymer samples in order to improve understanding of the products of the oxidative reactions. The broadness of all the peaks, and the multitude of functional groups expected to be present, precluded the obtainment of detailed compositional information by curve fitting for components, but shifts in the binding energies (BEs) of the N 1s and O 1s peaks with time provided useful evidence of compositional changes. As the oxygen content of the plasma polymer increased upon oxidation, the binding energy of the N 1s photoelectrons increased from 399.1 to 399.8 eV. Concurrently, the amount of nitrogen in relation to carbon (N/C) decreased from 0.42 to 0.34. As the nitrogen content of the plasma polymer decreased, the binding energy of the O 1s photoelectrons increased from 531.1 to 531.8 eV. The BE values and their shifts with time/compositional changes suggested that the oxidation process predominantly caused oxidation of the carbon atoms that had amine groups attached to them, leading to the formation of amide groups and perhaps also some imides.

Hydrophobic radiofrequency plasma-deposited polymer films : dielectric properties and surface forces'

Abstract Advancing and receding contact angles for various liquids placed on the surface of thin n -hexane plasma polymer films have confirmed that the surface is hydrophobic; comparable to polyethylene. A waveguide technique has been employed to determine the refractive indices of the plasma polymer films in the visible and near infrared regions of the dielectric spectrum. Along with ESCA data that provide polymer group functionality, this information has been used to estimate the n -hexane plasma polymer density and to construct a simple dielectric permeability function (ϵ(iξ), the dielectric constant at imaginary frequency iξ) for the polymer. The dielectric permeability function and Lifshitz theory have been used to calculate the Hamaker function (including non-retarded Hamaker constants) which should characterise the van der Waals interaction in systems that contain n -hexane plasma polymers. The atomic force microscope (AFM) has been employed to measure the force of interaction between n -hexane plasma polymer films deposited on muscovite mica flat plates and either uncoated silicon nitride AFM tips or n -hexane plasma polymer coated glass spheres. The force versus separation curves have been compared with DLVO theory. The homointeraction between n -hexane plasma polymer coatings in water initially displays a long-range attraction additional to van der Waals interaction. There is evidence that after prolonged immersion in water the thin n -hexane plasma polymer films swell.

Contributions of restructuring and oxidation to the aging of the surface of plasma polymers containing heteroatoms

The properties and composition of plasma polymer surfaces stored in air can change considerably over time, especially as a result of oxidative reactions. When plasma polymers contain an element other than O, it is possible to probe for mechanisms in addition to oxidation that contribute to the aging of the surface. Plasma polymers containing N were fabricated from either 1,3-diaminopropane (DAP),n-heptylamine (nHA), or allylamine (AA), and studied by X-ray photo-electron spectroscopy (XPS) and air/water contact angles (CA). For each of the plasma polymers, a multiexponential increase in the O/C ratio was observed over time using XPS. The N/C ratios remained constant (AA) or decreased somewhat (nHA and DAP). In contrast, the trends in CA values differed, declining for the nHA surfaces, rising for the AA, and changing little for the DAP. Surface roughness, assessed by scanning tunnelling or atomic force microscopy, did not change over time. The diverse adjustments in the polarity of each surface and the similar compositional changes between them are reconcilable if the aging of the plasma polymer surface is a manifestation of the superposition of concurrent oxidative reactions and partial surface reorientation; the former introduce polar groups and the latter transports then from the surface to deeper regions beyond the CA probe depth but within the XPS analysis depth. These processes vary between different plasma polymers. Data for the alkylamine plasma polymers is also compared with that for two plasma polymers fabricated from methanol. The change in composition, but not polarity, of the DAP surface after 4 days of storage demonstrates the importance of using multiple techniques to characterize the aging of plasma polymer surfaces.

Hybrid biomaterials: Surface-MALDI mass spectrometry analysis of covalent binding versus physisorption of proteins

Abstract Lysozyme, lactoferrin, trypsin and myoglobin were bound onto thin carboxymethyl-dextran coatings in two ways: by physisorption, and by an intended carbodiimide interfacial linking reaction. XPS analyses recorded substantial protein loadings on all the samples. In some cases, the protein coverages achieved by physisorption were larger than on covalent binding, indicating substantial affinity of the proteins for spontaneous adsorption onto the coatings. The observation of significant physisorption raised the question of to what extent physisorption competed with covalent binding when the intended covalent linking reaction was performed. The technique of Surface-MALDI mass spectrometry was found to be rapid, convenient, sensitive, and eminently suitable for probing for the presence of a physisorbed population among the covalently linked proteins. Control experiments with physisorbed samples showed that all the four proteins investigated gave rise to strong signals. On samples produced using carbodiimide linking, however, no protein signals were detected while photomatrix signals attested to successful evaporation of the matrix. Covalently linked proteins evidently were immune to desorption and detection by Surface-MALDI-MS. Using signal-to-noise ratios of spectra recorded on samples with approximate monolayer coverage of physisorbed proteins, it was estimated that a signal should have been detectable if, among the covalently coupled protein molecules, >1–2% of the total protein coverage present after intended covalent immobilization were physisorbed. Therefore, Surface-MALDI analyses suggest that within experimental accuracy, the carbodiimide chemistry used led to complete covalent interfacial immobilization of all four protein layers.

Concurrent restructuring and oxidation of the surface of n-hexane plasma polymers during aging in air

Angle-dependent XPS and air/water contact angle (CA) measurements were performed on specimens of n-hexane plasma polymers at various times after fabrication in order to monitor the aging of the surfaces in contact with air. XPS revealed incorporation of oxygen over extended periods of time. The depth distribution of O changed in the course of aging. CAs decreased over the first 3 weeks, then increased again, and finally stabilized at 5 weeks. These results were interpreted in terms of two concurrent processes: spontaneous oxidation (initiated by trapped radicals), which increased the surface polarity, and surface restructuring, which caused the partial removal of polar groups from the interface with air. The former process made a larger contribution to the overall aging of the surfaces but the latter process also contributed measurably. Over the first 3 weeks, oxidation was rapid and surface restructuring was not competitive. Subsequently, the oxygen uptake slowed down markedly, and the outermost surface layers became oxygen depleted relative to the deeper layers, due to partial surface reorientation. The surface topography, assessed by STM, did not change on aging. The aging of n-hexane plasma polymer surfaces thus is due to superimposed effects arising from post-deposition oxidative processes and partial surface reorientation.

Universal Correlation of Nitrogen 1s and Oxygen 1s Photoelectron Binding Energies with Chemical Composition in Nitrogen-Containing Plasma Polymers

The incorporation of oxygen into nitrogen-containing plasma deposited polymers was studied by X-ray Photoelectron Spectroscopy (XPS). As the oxygen content of the plasma polymer increased, the binding energy of the N 1s photoelectrons increased. Conversely, the binding energy of the O 1s photoelectrons was inversely proportional to the nitrogen content of the plasma polymer. The data from a large number of samples all obeyed the same “universal” correlations of photoelectron binding energy versus chemical composition. The data were described by the same curve regardless of whether the oxygen was incorporated rapidly into the thin film during plasma deposition or whether the oxygen was added slowly during spontaneous oxidation of the film in air. This implies that the same thermodynamic principles of radical reactions governed the addition of oxygen to the plasma polymer. The shift in the O 1s and N 1s photoelectron binding energies as a function of chemical composition was used to monitor the proximity of nitrogen and oxygen. By contrasting the experimental data with a simple binomial model which described the random addition of oxygen to a lattice containing carbon and nitrogen, we were able to show that oxygen was preferentially added near nitrogen-containing groups in plasma polymers.