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— A fluorescence quenching technique has been used to determine the absolute partition coefficients of a set of n-doxyl stearates which partition between the aqueous and lipid phases of a phospholipid dispersion, and it is shown that the values of the coefficients vary in a systematic way depending on the position of the doxyl group on the acyl chain of the fatty acid. The n-doxyl stearates quench the fluorescence of n-(9-anthroyloxy) fatty acids in lipid bilayers, and consideration of the absolute partition coefficients of the quencher and of the microviscosity of the bilayer is necessary to extract information about the proximity of the fluorophore and quencher. In addition, quenching of the fluorescent fatty acids with a quencher which is small relative to the width of the lipid bilayer allows determination of both the absolute and local partition coefficients, the latter referring to a subvolume of the bilayer within which quenching occurs. The relationship between absolute and local partition coefficients is defined for micelles and bilayers and a method for determining the relative transverse positions of the anthroyloxy rings in these structures is described.

Coatings from carboxymethylated dextrans (CMDs) were fabricated, analyzed by XPS, and investigated for their ability to inhibit corneal epithelial tissue outgrowth and bovine corneal epithelial cell attachment and growth. CMDs with differing degrees of carboxymethyl substitution and various molecular weights were synthesized by the solution reaction of dextrans with bromoacetic acid under different reactant ratios. The CMD compounds thus obtained were attached onto aminated surfaces produced in two ways: by the plasma deposition of a coating from n-heptylamine vapour, and by the plasma deposition of an acetaldehyde coating onto whose surface aldehyde groups the polyamine compounds polylysine, polyethyleneimine and polyallylamine were immobilized to provide platforms for CMD immobilization. XPS spectra showed that the latter route produced thicker coatings than the former approach. CMD molecules attached directly onto the plasma-fabricated amine surface supported some tissue migration; the extent of carboxymethyl substitution and the molecular weight of the CMDs had little influence. For CMDs immobilized via polyamine spacers, on the other hand, tissue outgrowth was completely inhibited, and again there were no discernible effects from the extent of carboxymethyl substitution and the molecular weight of the CMDs. In assays involving cell attachment and growth, analogous observations were found. Thus, the mode of immobilization of these polysaccharide coatings is the dominant factor in their anti-fouling performance, suggesting that optimization of the architecture of polysaccharide coatings may be an important factor for maximizing their cell-repellent abilities.

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.

Identification of the biomolecules that form the first adsorbed monolayer, which thus effect “interface conversion”, in competitive adsorption from multicomponent biological solutions can be challenging because of limitations in mass resolution and sensitivity of established techniques. In this study matrix-assisted laser desorption ionization (MALDI) time of flight mass spectrometry is developed and applied as a novel surface analytical method to enable analysis of adsorbed multicomponent biomolecule layers directly on the biomaterial surfaces. We show that proteins adsorbed in vivo (on human eyes) on contact lenses can be detected rapidly and conveniently by the diagnostic highly resolved mass signals recorded by MALDI mass spectrometry. This new approach allows detection of minor (and major) proteinaceous constituents of biofouled layers at levels substantially below monolayer coverage. Identification is done by comparison with molecular masses of known proteins. Specifically, it is shown that in addition to lysozyme, other low molecular weight proteins adsorb from human tear fluid onto contact lenses; these proteins had not been detected in earlier studies using other techniques. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res, 49, 36–42, 2000.

Fine elastomeric sutures intended for cardiovascular surgery can exhibit “stick-slip” behavior as they are pulled through tissue; the resulting oscillatory force can damage delicate tissue or cause sutures to snap. To eliminate this undesirable effect, sutures were surface-modified using a radiofrequency glow discharge in a vapor of either hexamethyldisiloxane or hexamethyldisilazane, to produce a thin polymeric coating on the suture. The same coatings were also deposited onto aluminized tape to facilitate their characterization by measurement of air/water contact angles and by X-ray photoelectron spectroscopy. Coatings from both monomers were found to be very hydrophobic. The hexamethyldisiloxane glow discharge coatings underwent negligible oxidation when stored in air, and thus remained stable over a shelf-life period akin to what may be required of sutures. The hexamethyldisilazane glow discharge coatings, in contrast, incorporated substantial amounts of oxygen over a 3-month period. The coatings did not measurably alter the tensile properties of the sutures. The frictional properties of coated sutures were assessed by measuring the dynamic friction between the suture and ovine myocardium. Both coatings were effective in removing the inherent stick-slip behavior of polybutester sutures in this model. The coatings remained intact after several passes and proved to be robust and efficacious under various strain regimes. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res (Appl Biomater) 53: 235–243, 2000

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.

A range of carboxymethylated poly(hydroxyethyl methacrylate) (CM-PHEMA) hydrogels with varying degrees of carboxymethylation was synthesized for a systematic study of the effects of ionized groups (‘charge’) on the uptake by hydrogel matrices of the proteins, lysozyme and human serum albumin (HSA). Using a radiolabel-tracer technique, X-ray photoelectron spectroscopy, and laser scanning confocal microscopy, we attempted to differentiate between protein molecules that were irreversibly adsorbed onto the hydrogel surface and those that penetrated into the hydrogel matrix. The effective pore size of the CM-PHEMA hydrogels was modelled and compared with the known molecular dimensions of the two proteins. The effects of the presence of varying amounts of ionized groups in the hydrogel matrix differed for the two proteins. For lysozyme, increased uptake was observed at higher carboxymethylation; this is interpreted as resulting from a combination of electrostatic attraction and increasing ease of penetration of the protein into the more porous hydrogel matrix. For HSA, on the other hand, the uptake was primarily by surface adsorption, with little diffusive penetration into the matrix.

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.

Approaches are described for the analysis of XPS signals from samples comprising thin conformal coatings on substrates with non-ideal surface topography. In particular, attention is focused on arbitrarily shaped rough or curved substrates. Relations are derived for the relative x-ray photoelectron intensities emitted from various elements in such non-ideal samples. The surface topography of arbitrarily rough samples is analysed by atomic force microscopy to provide a frequency histogram of the local slopes, which is incorporated into a global, multi-element analysis method for interpretation of the observed XPS elemental intensities. An analogous approach is used for the analysis of curved samples. Our algorithms also enable simultaneous analysis of successively coated multilayer structures. When the elemental composition of the individual layers is known, the fit procedure enables determination of the thickness values of single-layer and multilayer coatings. The range of validity of the theory is discussed. © 1997 by John Wiley & Sons, Ltd.

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.

Surface restructuring in ambient air of medical grade silicone rubber surfaces modified by repeated RF plasma treatments using various discharge gases including oxygen, argon, carbon dioxide and ammonia, was studied quantitatively. From advancing and receding water contact angle data, the fraction of the surface covered by mobile and immobile polar groups, and a characteristic time constant of the restructuring process were calculated. For argon plasma treated surfaces, the fraction of immobile polar groups increased with repeated plasma treatments, but remained relatively constant for samples repeatedly treated by an ammonia plasma. The use of an oxygen plasma only yielded incorporation of mobile polar groups but not of immobile polar groups. The increase in the restructuring time constants of argon and ammonia plasma treated silicone rubber with the number of plasma treatments suggested enhanced crosslinking of the silicone rubber by these plasmas. In contrast, when an oxygen plasma was repeatedly used, the restructuring time constant decreased suggesting chain cleavage by an oxygen plasma. Tentatively, the carbon dioxide plasma treatment of silicone rubber may initially (up to 3–4 repeated treatments) yield chain cleavage, while the occurrence of crosslinking is indicated after more repetitions.

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.

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.

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.

Optimization of strategies for the covalent attachment of proteins onto polymer surfaces requires the development of analytical methods which can differentiate between proteins that are covalently attached and those that are non-covalently bound (physisorbed). We probed for the surface density of reactive amine, carbonyl, and hydrazide groups using solution phase derivatization reactions to mimic and explore protein immobilization reaction strategies. Labeling compounds investigated were fluorescein derivatives, which were quantified by adsorption spectroscopy, and fluorinated phenyl compounds which were quantified by XPS. Control experiments consisted of performing the same labeling reactions using surfaces without reactive groups, or immersing the polymer surface into the labeling solution after blocking the reactive group of the labeling compound by a covalent reaction in solution. We always found non-negligible contributions arising from physisorption of the derivatization labels. Multiple control surfaces and a novel 'crossover derivatization-XPS' method were studied with the aim of improving compensation for physisorption. Our documentation of surprisingly large physisorption components even for small molecule labels, together with the known propensity of proteins to adsorb onto polymers, suggests caution in quantitative analysis of surface groups by derivatization, and in interpreting covalent protein immobilizations onto polymeric surfaces.

A theoretical description is presented for the macroscopic contact angle (θ) of a solid/vapor/aqueous solution interface when the solid surface is ionizable and relatively hydrophobic. The contact angle is related to the free energy of formation of the ionizable surface in aqueous solution. Therefore, the theoretical approach takes into account the electrostatic free energy of charging the surface and the change in the free energy associated with the acid-base reactions of the surface sites. It is shown that the dependence of θ on pH depends on the number ofionizable surface sites per unit area, the intrinsic acid-base dissociation constant (K a i ) of the surface sites, and the background electrolyte. The negative free energy change which accompanies the acid-base reactions dominates over the positive electrostatic free energy of charging the surface ; therefore, the overall free energy of ionization is negative, and the ionized surfaces are more wettable than the uncharged surfaces. The theoretical description is applied to experimental values of θ as a function of pH, measured on heptylamine plasma polymer surfaces in the presence of either 1 or 10 mM NaCl. The theoretical fit to the advancing and sessile contact angles indicates that the plasma polymer surface contains ca. 4±1x10 17 amines/m 2 and the pK a i of the amine groups is ca. 6. The surface site density is in accord with values obtained by derivatization techniques, and the pK a i is consistent with a low effective dielectric constant for the polymer-water interfacial microenvironment.

Of significance in the routine use of BIAcore is the cost of the sensor chips. This is particularly evident during the phase of method development of an assay where it is not unusual to expend several chips in a day in attempts to optimize immobilization conditions for a novel peptide or protein. In addition, it is accepted practice to discard a chip once its ligand binding capacity has diminished to an unacceptable level. While the high cost of sensor chips has been addressed to some degree through the recent introduction of research-grade sensor chips, we were interested in assessing the possibility of regenerating or reconditioning sensor chips in order to allow them to be reused. In particular, we concerned ourselves with regenerating sensor chips onto which peptide or protein had been immobilized. Our aim was to develop a general procedure that would allow reuse of such chips but would not decrease ligand immobilization capacity or increase nonspecific ligand adsorption properties. We present a method which employs a combination of enzymatic (Pronase E) and chemical (bromoacetic acid) treatments of used sensor chips. Regeneration requires an overnight incubation of the sensor chip ex situ so that one can continue to perform BIAcore experiments. The data demonstrate that this simple two-step procedure substantially removes immobilized proteins such as IgG, Protein G, an HIV-1 envelope glycoprotein (gp120) and a neoglycoprotein based on bovine serum albumin, as determined by reflectance measurements and X-ray photoelectron spectroscopy. In addition, a high density of proteins can be immobilized onto the reconditioned surfaces, since reactive carboxylic acids are regenerated by the procedure.

Polymer surfaces can adapt to surrounding media by reorientation of surface segments ; such motions can produce changes in contact angles (CAs). Building on two existing formalisms for time-independent CAs of heterogeneous surfaces, treatments are derived for the analysis of time-dependent CAs, enabling quantitative evaluation of the restructuring of polymer surfaces. In particular, we treat the case of air/water CAs on polymers which contain some polar surface groups that are immobile and others that are mobile and can migrate away from the surface by rotational and/or translational motions of polymer chains. A first-order process is assumed for the rate-limiting reorientation motion. The resulting equations have three parameters, viz., the fraction of the surface area covered by mobile polar groups, the fraction of the surface area covered by immobile polar groups, and the characteristic time constant (lifetime) of the reorientation process. The theoretical descriptions were applied to sets of contact angles measured on various plasma-modified polymer surfaces over extended periods oftime, enabling quantitative comparison of the effects of identical plasma treatments on the surface restructuring of different polymers.

Fluoropolymers modified by plasma modification were studied for their suitability as surfaces for the adhesion of cells. We compared films made by plasma modification of fluoroethylenepropylene (FEP) using nitrogen-containing gases (ammonia or dimethyl acetamide) with films deposited using oxygen-containing monomers (methanol, methyl methacrylate or sequential treatment with toluene then water). The surfaces were compared for the attachment and spreading of human vein endothelial cells and human dermal fibroblasts. The initial attachment and spreading of cultured fibroblasts and endothelial cells onto films deposited using nitrogen-containing gases were equivalent to that onto films deposited using oxygen-containing monomers, but there were some differences in the mechanism of attachment. With films deposited using oxygen-containing monomers, the initial attachment and spreading of endothelial cells failed when the medium contained 15% (v/v) serum from which both fibronectin (Fn) and vitronectin (Vn) had been removed. Similarly, initial attachment and spreading of endothelial cells onto films deposited using oxygen-containing monomers were reduced by 62-86% when the cells were seeded in medium containing Vn-depleted serum (which contained Fn). Endothelial cells attached and spread onto films made using oxygen-containing monomers, when seeded in medium containing Fn-depleted serum (which contained Vn). On films deposited using nitrogen-containing gases, the adhesion of endothelial cells was only slightly reduced in Vn-depleted medium (as compared to attachment in medium containing unmodified serum). Furthermore, surfaces which had incorporated nitrogen were more effective than were oxygen-containing films in adsorbing sufficient serum Fn as to promote endothelial cell attachment. Similar results were seen for the attachment and spreading of fibroblasts as for the endothelial cells. For fibroblasts, attachment and spreading onto oxygen-containing films and onto nitrogen-containing films were not simply dependent upon either the Vn content or the Fn content of the medium. Maximal attachment and spreading of fibroblasts were, however, dependent upon adsorption of both serum Vn and Fn.

The attachment and growth of human endothelial cells and fibroblasts was studied on polymer surfaces fabricated by the polymerization of volatile amine and amide compounds in a low pressure gas plasma, and by the treatment of various surfaces in ammonia plasmas, which served to increase the nitrogen content of the surface layers. Infrared spectra showed the presence of amide groups, including those cases where the volatile compound ('monomer') did not contain oxygen. The performance of the surfaces in cell attachment correlated with the surface hydrophilicity and the nitrogen content, although for the latter a fair degree of scatter indicated that a more complex relationship applies. All these surfaces supported the attachment and growth of human cells. Generally, amide plasma polymers were best but the individual monomer and the plasma parameters also played a role. From comparisons of the various surfaces, it is suggested that the amide group is the main promoter of cell attachment in nitrogen-containing plasma surfaces.

Treatment of Pellethane 2363-80A--a medical-grade poly(tetramethylene oxide)-based polyurethane elastomer--with 25% (w/w) hydrogen peroxide at 100 degrees C for times ranging from 24 h to 336 h led to significant decreases in ultimate tensile properties and decreases in molecular weight, both at the surface and in the bulk. IR spectral changes were similar to those observed after degradation in vivo. Differential scanning calorimetry showed that hydrogen-peroxide-induced degradation was associated with greater order in the hard domain and greater mobility in the soft domain. Studies conducted with low-molecular-weight model compounds for the hard and soft segments confirmed that methylene groups adjacent to oxygen were susceptible toward oxidation. The extent of degradation of a series of commercial polyurethanes on treatment with hydrogen peroxide (25%, 24 h, 100 degrees C) correlated well with their reported susceptibility to environmental stress cracking in vivo.

A series of novel polyetherurethaneurea elastomers based on m-TMXDI, polyether macrodiols containing 4, 6, 8, or 10 methylene groups between the ether linkages, and aliphatic and aromatic diamines was prepared by two-step solution polymerization. All the materials were thermally processable at 120–160°C except one that was solvent-castable. Mechanical properties and hydrolytic stability (at neutral pH) were found to depend on the macrodiol and chain extender. Poly(tetramethylene oxide)-based polyetherurethaneureas that were chain-extended with primary aliphatic diamines, other than 1,3-propanediamine, exhibited significantly better mechanical properties than those chain-extended with secondary aliphatic or aromatic diamines. The mechanical properties and hydrolytic stability of polyetherurethaneureas containing poly(hexa-, octa-, or decamethylene oxide) and 1,6-hexamethylenediamine were considerably better than those containing 1,2-ethylenediamine. The hydrolytic stability increased with the increasing C/O ratio of the macrodiol. With poly(hexamethylene oxide)-based materials, the phase separation between soft and hard domains was higher than that of the other materials as determined by DSC. © 1993 John Wiley & Sons, Inc.

Novel polyurethane elastomers based on 4,4′-diphenylmethanediisocyanate, 1,4-butanediol, and polyether macrodiols that have 6, 8, or 10 methylene groups between the ether oxygens were prepared by two-step solution polymerization. Their mechanical properties and degradation resistance were compared with poly(tetramethylene oxide)-based polyurethanes. The new polyurethanes were more resistant to hydrolysis and to hydrogen peroxide-induced oxidation than were the poly(tetramethylene oxide) -based materials. The poly(octamethylene oxide) -based polyurethane displayed good resistance to sodium hypochlorite. 1,2-Diaminoethane chain-extended polyurethane–ureas were also prepared using the macrodiols poly(hexamethylene oxide) and poly(octamethylene oxide). They were not thermally processable or solvent-castable. Bulk synthesis of novel polyether polyurethanes yielded, in some cases, materials with improved mechanical properties compared with their solution-polymerized counterparts. The outcome of the bulk syntheses was highly dependent, however, on reaction conditions and stoichiometry.

Hydroxy-terminated poly(alkylene oxide)s with molecular weight M n in the range 700-2400 depending on the reaction time and temperature and hydroxy functionality of 2.0 were obtained by acid-catalysed polycondensation of 1,6-hexanediol, 1,8-octanediol and 1,10-decandiol in the 150-190°C temperature range in the presence of H 2 SO 4 or Nafion-H resin.

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.

A theoretical description is presented for the equilibrium capillary height (heq) of a solution between parallel,flat,solidsurfaceswhichcontainionizablegroups. Theheqisrelatedtothechangeingravitational potential energy, the intrinsic wettability of the un-ionized surface, and the free energy of formation of theionizablesurfaceinaqueoussolution. Thetheoreticalapproachtakesintoaccountboththeelectrostatic free energy of charging the surface and the change in the free energy associated with the acid-base reactions of the surface sites. It is shown that the dependence of heq on pH depends on the number of ionizable surface sites per unit area, the intrinsic acid-base dissociation constant (Ka i ) of the surface sites, andthebackgroundelectrolyte. Thenegativefreeenergychangewhichaccompaniestheacid-basereactions dominates over the positive electrostatic free energy of charging the surface. Consequently, the overall free energy of ionization is negative, and an ionized surface is more wettable than an un-ionized surface. The theoretical description is applied to experimental values ofheqas a function of pH, measured between two heptylamine plasma polymer surfaces in the presence of 1 mM NaCl. The theoretical fit to the data indicates that the plasma polymer surface contains ca. 1017 amines/m2 and the pKa i of the amine groups is ca. 5. The surface site density is in reasonable accord with values obtained by both derivatization techniquesandcontactanglemeasurements. ThepKa i isconsistentwithaloweffectivedielectricconstant for the polymer-water interface.

Dextran was immobilized onto fluorinated ethylene-propylene copolymer surfaces using a three-step procedure. The efficacy of each step was monitored by XPS and by measurement of air/water contact angles (CAs). In the first step, the polymer was treated with a radiofrequency glow discharge struck in a methanol vapour in order to place reactive hydroxyl groups on the surface. The CA results could be related to a single experimental variable, viz, the fractional increase in pressure which accompanied the glow discharge. In the second step, a diepoxy reagent was reacted with the surface hydroxyl groups in the presence of various catalysts. Wettability measurements showed that benzyltrimethylammonium hydroxide was a better catalyst than NaOH. XPS spectra revealed that the epoxy reagents formed a multilayer structure, presumably because the hydroxyl groups generated by the opening of the oxirane ring participated in further ring-opening reactions. In the third step, dextran was immobilized onto the epoxy groups. The resulting surface was hydrophilic and was able to hold a continuous layer of water for more than one minute when the MW of the dextran exceeded 100 kDa.

Dextrans of various molecular weights were immobilized on polymer surfaces using a three-step process. In the first step, dextran was oxidized in aqueous solution with sodium periodate, resulting in the release of formic acid. Titration of the formic acid showed that maximal oxidation of the dextran was achieved in 4–5 hours when two moles of periodate were used per mole of glucose subunits. 1H- and 13C-NMR revealed that the oxidized product contained hemialdal moieties when water was present. The hemialdals were capable of reacting with nucleophilic reagents such as fluorescein thiosemicarbazide. In the second step, a radiofrequency glow discharge through an amine-containing vapour was used to place reactive amine groups on the surface of a polymer. In the third step, the oxidized dextran was bound to the surface amine groups; this reaction produced a hydrolytically unstable Schiff-base linkage. The linkage was reduced by sodium cyanoborohydride to a hydrolytically stable secondary amine. The resulting surfaces were highly wettable, as shown by their ability to hold a visually observable, continuous layer of water for several minutes.

Fluorinated ethylene-propylene (FEP) surfaces modified by ammonia or water vapor plasmas under a range of conditions were stored in contact with air and their surface restructuring on aging was monitored by periodic determination of air/water contact angles (CAs). By use of formalisms for the analysis of time-dependent CAs, the fraction of the surface area initially covered by mobile polar groups, the fraction of the surface area covered by immobile polar groups, and the characteristic time constant (lifetime) of the reorientation process were calculated. For ammonia-plasma-modified FEP surfaces, the fraction of mobile polar groups increased with increasing plasma treatment time up to an optimal treatment time of 45 s, probably due to plasma-induced chain cleavage. This indication of increasing mobility upon extended plasma treatment was, however, not accompanied by a decrease in the lifetime of the reorientation process, implying that the rate-limiting step for surface reorientation may not be associated with the inward motions of plasma-modified surface chains, but instead may depend on the slower outward migration of FEP chains to the surface.

Long-term compositional changes were observed when n-hexane plasma polymers were stored in air at ambient temperature. These post-deposition changes were monitored over periods exceeding one year using XPS, grazing angle FTIR, and SEM. A rapid initial increase in the oxygen content was followed by a slower oxygen uptake, which continued for many months. In contrast to the reported autocatalytic acceleration of the oxidation of polyolefins, the rate of oxidation of n-hexane plasma polymers was found to decrease continuously, and it did not proceed to the same extent toward higher oxidation states. The plasma polymer coatings did not show physical manifestations of oxidative degradation such as cracking, reduction in thickness, or loss of physical integrity. Comparison of the XPS and FTIR data suggested that the top few nanometers of the plasma polymer coatings oxidized at a faster rate than the “bulk.” The experimental data were interpreted using a model comprising reactions known from the oxidative degradation of polyolefins: carbon-centered radicals, trapped in the course of the plasma deposition, combine with atmospheric oxygen to form metastable peroxy radicals and hydroperoxides, which decompose to generate a variety of stable product groups such as hydroxyl, carbonyl, and carboxylic groups. There was broad agreement between the XPS data and the time dependence of particular species in model calculations. © 1994 John Wiley & Sons, Inc.

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 tempe...

Low pressure gas plasma techniques for the fabrication of surfaces designed for compatibility with biological tissue and fluids are investigated. Plasma surface treatment rendered fluorocarbon polymers wettable by polar liquids, but the wettability decreased with time during storage in air. Plasma polymerization, on the other hand, allowed the deposition of thin coatings which were more stable with time. STM of plasma polymers, which were coated with a sputtered film of platinum or gald, showed that these coatings were smooth and continuous.

In in vitro accelerated tests, both novel and commercially produced polyurethanes were subjected to severe hydrolysing and oxidising conditions for 24 hours. Degradation in these tests was compared to degradation of the same materials in an ovine in vivo accelerated test in which polyurethane dumbbells were stretched over poly(methyl methacrylate) holders to a constant strain and implanted for 90 days subcutaneously. Further samples of the same materials were implanted unstressed for 6 months. Degradation in both in vitro and in vivo accelerated tests was correlated with degradation observed in the 6 month in vivo study. The in vitro test is suitable for initial Screening of novel polyurethanes. The accelerated in vivo model generated environmental stress cracking in 90 days and was able to discriminate between materials of differing biostability. Three of the novel materials studied were more biostable than the commercial materials. The accelerated in vivo model provides a technique to identify materials which are more biostable than existing commercial polyurethanes.

Fluorescence quenching techniques have been used extensively in recent years to examine reaction rates and the compartmentalization of components in lipid micelles and membranes. Steady-state fluorescence methods are frequently employed in such studies but the interpretation of the resulting Stern-Volmer plots is often hampered by uncertainties regarding the mode of association of the quencher with the lipid structure and the nature of the quenching mechanism. This paper presents a method for simulating steady-state Stern-Volmer plots in two phase systems, and shows how the forms of such plots are influenced by the type of association of the quencher with the membrane or micelle (partition and/or binding) and by the type of quenching mechanism (dynamic and/or static). Comparisons of simulated plots with experimental data must take into account the possible combinations of quencher association(s) and quenching mechanism(s). The methods presented are applicable to synthetic and natural membranes and provide a basis for comparing the quenching of fluorescent molecules in biological membranes of differing composition.

Dynamic quenching of fluorophores and quenchers in lipid micelles and bilayers can yield information about the bimolecular rate constant for the quenching reaction, and hence information about the microviscosity of the fluorophore-quencher environment. When the fluorophore and quencher have relatively fixed transverse positions in the bilayer, the analysis of Sikaris et al. (Chem. Phys. Lipids. 29 (1981) 23) can be used to separate the microviscosity and proximity contributions to quenching. We now extend this method to show explicitly the effect of static quenching on the analysis. We show by simulation and experiment that a correction factor must be included when static quenching contributes to the observed quenching efficiency.

Relations describing sedimentation equilibrium in solutions containing two macromolecular solute components are derived for the following cases: (1) two nonassociating proteins at arbitrary concentration, (2) one dilute self-associating protein in the presence of a second inert protein at arbitrary concentration, and (3) two proteins at arbitrary concentration that can associate to form a single heterocomplex of arbitrary composition. As in earlier work (R. C. Chatelier and A. P. Minton (1987) Biopolymers, 26, 507–524), the relations are obtained by using scaled particle theory to calculate the thermodynamic activity of each species present at a given radial distance in the centrifuge. The results of numerical simulations of sedimentation equilibrium are presented as the dependence of apparent molecular weights, or apparent weight-average molecular weights, upon solution composition. Semiempirical methods are presented, by means of which the weight-average molecular weights of self- and heteroassociating proteins in highly nonideal solutions may be estimated from experimental data. It is found that the semiempirical methods yield reasonably accurate estimates of the true weight-average molecular weight over a broad range of experimental conditions, providing that the partial specific volumes of two components in a heteroassociating system do not differ by more than about 0.05 mL/g.

The approach of a solution of dilute, monodisperse, globular macromolecules to low-speed sedimentation equilibrium in an ultracentrifuge is simulated by numerical integration of the Lamm equation. Various combinations of overspeed time and angular velocity are used to assess the conditions needed to minimize the time it takes the solution to attain sedimentation equilibrium. The optimal overspeeding time and angular velocity are determined over a wide range of values of the molecular weight (relative molar mass) of the solute and the radial distance between the meniscus and base of the solution. The results may be expressed as simple functions which allow facile calculation of (a) the optimal overspeeding time and velocity, and (b) the time required to reach sedimentation equilibrium. The results are in reasonable agreement with previous analytical solutions which were based on several simplifying assumptions. The parameterized overspeeding procedure is shown to be robust over a wide range of conditions, and typically leads to a greater than 5-fold reduction in centrifugation time.

To extract ligand-cell binding parameters from flow cytometric fluorescence data, one needs a method of converting the measured cellular fluorescence to the actual number of bound molecules producing that fluorescence. Bead standards containing a known number of fluorophores do not necessarily provide the correct conversion factor. We therefore present a method for calibrating flow cytometric bead standards. The technique uses the isoparametric analysis (Chatelier et al: EMBO J 5:1181--1186, 1986) to construct a plot of fluorescence per cell versus the number of bound ligands per cell, thus allowing a direct comparison of the quantum yields of the bead-associated fluorophore with that of the cell-bound fluorophore. The potential of this analysis is demonstrated by contrasting the fluorescent brightness of fluorescein isothiocyanate associated with thymocyte nuclei and synthetic polymer beads with that of fluoresceinated epidermal growth factor bound to A431 cells. When the standards are improperly calibrated, then the number of ligand-binding sites is incorrectly reported, and the derived Scatchard plot may exhibit an apparent positive or negative cooperativity as well as a strong dependence on cell concentration.

Consideration is given to the indefinite self-association of biological molecules assuming that each step-wise addition of monomer is characterized by the same enthalpy change, and that the second virial coefficients adequately describe the thermodynamic nonideality. Statistical thermodynamic models are used to calculate translational and rotational entropy changes accompanying the addition of each monomer to the aggregate. Three polymerization schemes are explored in which the solute species are modeled as right circular cylinders of various length/radius ratios. The analysis is applied to published data on the self-association of bovine liver glutamate dehydrogenase.

Equilibrium statistical-thermodynamic models are presented for the surface adsorption of proteins modeled as regular convex hard particles. The adsorbed phase is treated as a two-dimensional fluid, and the chemical potential of adsorbed protein is obtained from scaled particle theory. Adsorption isotherms are calculated for nonassociating and self-associating adsorbing proteins. Area exclusion broadens adsorption isotherms relative to the Langmuir isotherm (negative cooperativity), whereas self-association steepens them (positive cooperativity). The calculated isotherm for adsorption of hard spheres using scaled particle theory for hard discs agrees well with that calculated from the hard disc virial expansion. As the cross section of the adsorbing protein in the plane of the surface becomes less discoidal, the apparent negative cooperativity manifested in the isotherm becomes more pronounced. The model is extended to the case of simultaneous adsorption of a tracer protein at low saturation and a competitor protein with a different size and/or shape at arbitrary fractional saturation. Area exclusion by competitor for tracer (and vice versa) is shown to substantially enhance the displacement of tracer by competitor and to qualitatively invalidate the standard interpretation of ligand competition experiments, according to which the fractional displacement of tracer by competitor is equal to the fractional saturation by competitor.

A common assumption invoked in the analysis of competition binding assays is that the fractional saturation of sites with the unlabeled ligand is given by 1-(the concentration of bound labeled ligand in the presence of unlabeled ligand)/(the concentration of bound labeled ligand in the absence of unlabeled ligand). This assumption is critically evaluated in the context of several binding models: (a) binding of univalent ligands to multiple classes of equivalent and independent sites, with and without nonspecific binding; (b) cooperative binding of univalent ligands; and (c) binding of multivalent ligands to a single class of univalent acceptors. We show that the conventional assumption is only valid when the labeled ligand is mainly in the free form, occupies a small fraction of the total sites and binds univalently to all sites in an equivalent and independent manner, and when the unlabeled ligand forms 1:1 complexes with the acceptor sites. When these conditions are satisfied, the conventional assumption is valid even if the unlabeled ligand binds to nonequivalent sites or exhibits cooperativity. Finally, we apply the theory derived for case (a) above to the binding of fluoresceinated epidermal growth factor to A431 cells and demonstrate that the analysis of data obtained from both conventional and competition assays provides information which is difficult, if not impossible, to obtain from either assay alone.

Relations describing sedimentation equilibrium in solutions of self-associating macromolecules at arbitrary concentration are presented. These relations are obtained by using scaled-particle theory to calculate the thermodynamic activity of each species present at a given radial distance. The results are expected to be valid for solutions of globular proteins under conditions such that interactions between individual solute molecules may be approximated by a hard-particle potential. Sedimentation equilibria in solutions containing either a nonassociating solute or a solute that self-associates according to several different schemes are simulated using the derived relations. The results of these simulations are presented in terms of the dependence of apparent weight-average molecular weight upon solute concentration. Simple empirical relations are presented for estimating the true weight-average molecular weight from the apparent weight-average molecular weight, without reference to any particular self-association scheme. The weight-average molecular weight estimated in this fashion is within a few percent of the true weight-average molecular weight at all experimentally realizable solute concentrations ( < 400 g/L).