Proteins dominate in the surface layers formed on materials exposed to extracellular polymeric substances from bacterial cultures.

The chemical compositions of the surface conditioning layers formed by different types of solutions (from isolated EPS to whole culture media), involving different bacterial strains relevant for biocorrosion were compared, as they may influence the initial step in biofilm formation. Different substrata (polystyrene, glass, steel) were conditioned and analyzed by X-ray photoelectron spectroscopy. Peak decomposition and assignment were validated by correlations between independent spectral data and the ubiquitous presence of organic contaminants on inorganic substrata was taken into account. Proteins or peptides were found to be a major constituent of all conditioning layers and polysaccharides were not present in appreciable concentrations; the proportion of nitrogen which may be due to DNA was lower than 15%. There was no significant difference between the compositions of the adlayers formed from different conditioning solutions, except for the adlayers produced with tightly bound EPS extracted from D. alaskensis.

Conditioning materials with biomacromolecules: composition of the adlayer and influence on cleanability.

The influence of substrate hydrophobicity and biomacromolecules (dextran, bovine serum albumin - BSA) adsorption on the cleanability of surfaces soiled by spraying aqueous suspensions of quartz particles (10-30µm size), then dried, was investigated using glass and polystyrene as substrates. The cleanability was evaluated using radial flow cell (RFC). The surface composition was determined by X-ray photoelectron spectroscopy (XPS). The interpretation of XPS data allowed the complexity due to the ubiquitous presence of organic contaminants to be coped with, and the surface composition to be expressed in terms of both the amount of adlayer and the mass concentration of adlayer constituents. When soiled with a suspension of particles in water, glass was much less cleanable than polystyrene, which was attributed to its much lower water contact angle, in agreement with previous observations on starch soil. Dextran was easily desorbed and did not affect the cleanability. The presence of BSA at the interface strongly improved the cleanability of glass while the contact angle did not change appreciably. In contrast, soiling polystyrene with quartz particles suspended in a BSA solution instead of water did not change markedly the cleanability, while the contact angle was much lower and the aggregates of soiling particles were more flat. These observations are explained by the major role of capillary forces developed upon drying, which influence the closeness of the contact between the soiling particles and the substrate and, thereby, the adherence of particles. The capillary forces are proportional to the liquid surface tension and depend in a more complex way on contact angles of the particles and of the substrate. The dependence of cleanability on capillary forces, and in particular on the liquid surface tension, is predominant as compared with its dependence on the size and shape of the soiling aggregates, which influence the efficiency of shear forces exerted by the flowing water upon cleaning.

Influence of poly(ethylene oxide)-based copolymer on protein adsorption and bacterial adhesion on stainless steel: modulation by surface hydrophobicity.

The aim of the present work is to study the adhesion of Pseudomonas NCIMB 2021, a typical aerobic marine microorganism, on stainless steel (SS) substrate. More particularly, the potential effect on adhesion of adsorbed poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer is investigated. Bacterial attachment experiments were carried out using a modified parallel plate flow chamber, allowing different surface treatments to be compared in a single experiment. The amount of adhering bacteria was determined via DAPI staining and fluorescence microscopy. X-ray photoelectron spectroscopy (XPS) was used to characterize the surface chemical composition of SS and hydrophobized SS before and after PEO-PPO-PEO adsorption. The adsorption of bovine serum albumin (BSA), a model protein, was investigated to test the resistance of PEO-PPO-PEO layers to protein adsorption. The results show that BSA adsorption and Pseudomonas 2021 adhesion are significantly reduced on hydrophobized SS conditioned with PEO-PPO-PEO. Although PEO-PPO-PEO is also found to adsorb on SS, it does not prevent BSA adsorption nor bacterial adhesion, which is attributed to different PEO-PPO-PEO adlayer structures on hydrophobic and hydrophilic surfaces. The obtained results open the way to a new strategy to reduce biofouling on metal oxide surfaces using PEO-PPO-PEO triblock copolymer.

Physico-chemical mechanisms governing the adherence of starch granules on materials with different hydrophobicities.

The factors influencing the adherence of starch were examined to improve the understanding of the mechanisms affecting soiling and cleanability. Therefore an aqueous suspension of starch granules was sprayed on four model substrates (glass, stainless steel, polystyrene and PTFE) and dried, and the substrates were cleaned using a radial-flow cell. The morphology of the soiled surfaces and the substrate chemical composition were also characterized. By influencing droplet spreading and competition between granule-substrate and granule-granule interfaces regarding the action of capillary forces, substrate wettability affected the shape and compactness of the adhering aggregates, the efficiency of shear forces upon cleaning, and finally the adherence of soiling particles. The rate of drying had an influence explained by the duration left to capillary forces for acting. X-ray photoelectron spectroscopy demonstrated the presence of macromolecules, mainly polysaccharides, which were adsorbed from the liquid phase, or carried by the retracting water film and deposited at the granule-substrate interface. These macromolecules acted as an adhesive joint, the properties of which seemed to be influenced by the detailed history of drying and subsequent exposure to humidity. In summary, the substrate surface energy affects the adherence of starch aggregates by different mechanisms which are all linked together: suspension droplet spreading, action of capillary forces, direct interaction with starch particles and interfacial macromolecules.

Competitive adsorption of fibrinogen and albumin and blood platelet adhesion on surfaces modified with nanoparticles and/or PEO.

In order to evaluate the respective influence of surface nanotopography and chemical composition on blood compatibility, plasma protein adsorption (fibrinogen - Fg and albumin - HSA, quantified simultaneously by dual radioassays) and platelet adhesion were investigated on a range of materials. Reference surfaces were glass, polystyrene and poly(vinyl chloride), as well as pieces of commercial blood bags. Colloidal lithography with 65 and 470 nm polystyrene latex particles was used to prepare nanostructured surfaces with either one layer of colloids or with bimodal roughness. The surfaces were further conditioned by adsorption of poly(ethylene oxide) (PEO)-containing compounds (Pluronic F 68 and PLL-g-PEG). Study of the simultaneous adsorption of Fg and HSA on reference substrates demonstrated that the Fg/HSA adsorbed amount ratio decreases as the substrate hydrophobicity increases, the lower ratio being obtained with commercial blood bag. This is due to the higher resistance of HSA adsorbed on hydrophobic substrates to displacement by proteins from the solution. Such higher resistance was also shown to occur in the case of displacement by constituents of non-diluted blood plasma. Nanostructured substrates gave about the same Fg/HSA ratio as polystyrene and poly(vinyl chloride). Surface conditioning with Pluronic F 68 reduced the adsorption of Fg in competition with HSA on all substrates except glass, while PLL-g-PEG decreased the adsorbed amount of both Fg and HSA on glass but not on the other substrates. Positive correlations between the amount of adhering blood platelets and both the Fg/HSA ratio and the absolute amount of Fg adsorbed in competition with HSA were found for all substrates (reference and nanostructured, as such or after PEO conditioning, except native glass which had to be discarded due to the formation of clots in the liquid phase). These quantities were also related to the state of activation of adhering platelets. This supports the concept that blood compatibility of materials is primarily governed by the presence of Fg in the adsorbed phase, as a result of the competition with other plasma proteins. This is in turn strongly influenced by surface hydrophobicity. Surface nanostructuration as performed here (relief in the range of 50-500 nm) did not affect significantly the relationship between Fg adsorption and platelet adhesion.

Surface chemical composition of diatoms.

Among diatoms, Phaeodactylum tricornutum is a peculiar species that exists in three morphotypes with distinct cell wall structures and low silica content. X-ray photoelectron spectroscopy (XPS) analysis was performed on P. tricornutum and compared with diatom Thalassiosira pseudonana; the results provide new information on the chemical composition (elements, chemical functions, classes of biochemical compounds) of the cell surface. Two types of silicon were found: condensed silica (SiO(2)) and weakly polymerised silicate. Cells of T. pseudonana showed the highest concentration of silicon, with a majority in the form of condensed silica. For the fusiform and triradiate morphotypes of P. tricornutum, the majority of the small concentration of silica found was in the form of weakly polymerised silicate. For all morphotypes of P. tricornutum, higher polysaccharide concentrations replaced silica as a structural part of the cell wall. In both diatoms, a high concentration of lipids was measured, in the form of carboxylic esters. Protonated nitrogen and phosphate were found in correlated amounts and attributed not only to phospholipids but also to phosphoproteins. Chloride ions characterised by a high electron density might be associated to these moieties. Sulfate groups were also detected, principally in P. tricornutum, and attributed to monoesters of polysaccharides.

An AFM, XPS and wettability study of the surface heterogeneity of PS/PMMA-r-PMAA demixed thin films.

A series of homopolymer/random copolymer blends was used to produce heterogeneous surfaces by demixing in thin films. The chosen homopolymer is polystyrene (PS) and the random copolymer is poly(methyl methacrylate)-r-poly(methacrylic acid) (PMMA-r-PMAA), whose acidic functions could be used as reactive sites in view of further surface functionalization. The proportion of each polymer at the interface was deduced from X-ray photoelectron spectroscopy (XPS) data using, on the one hand, the O/C ratio, and on the other hand, decomposition of the carbon peak of the blends in two components corresponding to the carbon peaks of PS and PMMA-r-PMAA. Combining the information from XPS with atomic force microscopy (AFM) images, water contact angle measurements and PS selective dissolution, it appears that the surfaces obtained from blends with a high PS content (90/10 to 70/30) display pits with a bottom made of PMMA-r-PMAA, randomly distributed in a PS matrix. On the other hand, the surfaces obtained from blends with a low PS content (30/70 to 10/90) display randomly distributed PS islands surrounded by a PMMA-r-PMAA matrix. The characteristics of the heterogeneous films are thought to be governed by the higher affinity of PMMA-r-PMAA for the solvent (dioxane), which leads to the elevation of the PS phase compared to the PMMA-r-PMAA phase, and to surface enrichment in PMMA-r-PMAA.

Oxidation of proteins adsorbed on hemodialysis membranes and model materials.

The cleaning of cellulosic hemodialysis membrane Cuprophan and model materials (glass; polystyrene and polypropylene, as such and surface-oxidized), conditioned by adsorption of blood plasma proteins (HSA, fibrinogen, IgG) was investigated in vitro. Sodium hypochlorite (NaClO) and Renalin, a product containing hydrogen peroxide and peroxyacetic acid, were used as cleaning reagents. X-ray photoelectron spectroscopy and the use of radiolabeled fibrinogen demonstrated the presence of varying amounts of a polypeptidic residue, with sulfur brought to a high oxidation stage (sulfonate-like). The trends were the same for the three proteins regarding the effectiveness of the oxidizer and the influence of the surface properties. NaClO was much more effective than Renalin to remove the adsorbed proteins. The proteins adsorbed on Cuprophan were more sensitive to the oxidizers, when compared with proteins adsorbed on other materials. This may be due to both the lower protein-surface affinity, as indicated by radiochemical measurements, and the sensitivity of the material itself to the oxidizer, as revealed by weight loss measurements. These results support the attribution of hemocompatibility improvement after regeneration of Cuprophan with Renalin to the masking of the activating surface by a residue from previously adsorbed proteins.

Direct measurement of hydrophobic forces on cell surfaces using AFM.

Although hydrophobic forces are of great relevance in biological systems, quantifying these forces on complex biosurfaces such as cell surfaces has been difficult owing to the lack of appropriate, ultrasensitive force probes. Here, chemical force microscopy (CFM) with hydrophobic tips was used to measure local hydrophobic forces on organic surfaces and on live bacteria. On organic surfaces, we found an excellent correlation between nanoscale CFM and macroscale wettability measurements, demonstrating the sensitivity of the method toward hydrophobicity and providing novel insight into the nature of hydrophobic forces. Then, we measured hydrophobic forces associated with mycolic acids on the surface of mycobacteria, supporting the notion that these hydrophobic compounds represent an important permeation barrier to drugs.

XPS analysis of chemical functions at the surface of Bacillus subtilis.

The surface chemical composition of nine strains of Bacillus subtilis was determined by X-ray photoelectron spectroscopy. Regressions between elemental concentrations and concentrations associated with different components of C1s, N1s, and O1s peaks provided a more precise validation of the procedure used for peak decomposition and allowed the assignment of the peak components to be completed or strengthened. The component of the O1s peak appearing around 531.2 eV was shown to contain a contribution of oxygen from phosphate groups (PO, PO-), the other contribution being due to oxygen involved in amide functions. The surface negative charge may be fully attributed to phosphate groups, despite the observation of two types of zeta potential vs pH curves. The strains exhibiting a sharp variation of the zeta potential (range of -35 to -55 mV) between pH 2 and 4.7 were characterized by a high phosphate surface concentration and by an excess (about 25%) of phosphate with respect to the sum of potassium, an exchangeable cation, and protonated nitrogen, attributed to protein or to alanine involved in teichoic acids.

Dual radiolabeling to study protein adsorption competition in relation with hemocompatibility.

Human fibrinogen (Fg) and albumin (HSA) were labeled with (3)H and (14)C, respectively. Dual counting allowed the adsorbed amount of the two proteins to be determined simultaneously. Single adsorption, adsorption of the two proteins in competition, but also exchange (substitution by molecules of the same nature) and displacement (desorption under the action of the other protein) experiments were performed on two model surfaces, glass and polystyrene (PS), as well as on pure polyvinylchloride (PVC-s) and on PVC from blood bag (PVC-b). As expected, the adsorbed amount of a single protein is higher on a hydrophobic compared to a hydrophilic surface. When the two proteins are adsorbed in competition, they are found in equal proportion on glass, while HSA is twice more abundant than Fg on PS and PVC-s and about six times more abundant on PVC-b. This trend is related to an increase of the water contact angle of the substrates. For PVC-b, the contact angle is affected by the presence of aliphatic components exposed at the extreme surface, as determined by angle-resolved X-ray photoelectron spectroscopy. In exchange and displacement experiments, the first adsorbed molecules remain dominating on PS while they can be removed from glass. Given the known importance of HSA and Fg adsorption for the fate of materials placed in contact with blood, the method described in this paper may be used as a first approach to orient the design of surfaces with improved hemocompatibility.

Influence of collagen denaturation on the nanoscale organization of adsorbed layers.

Adsorption (at 37 degrees C) of type I collagen, in native and heat-denatured (30 min at 40 and 90 degrees C) forms, on polystyrene was studied using quartz crystal microbalance with energy dissipation monitoring (QCM-D), atomic force microscopy (AFM) in tapping mode and X-ray photoelectron spectroscopy (XPS). The significance of the parameters deduced from QCM-D data was examined by comparing different approaches. The adsorbed layer of native collagen has a complex organization consisting of a thin mat of molecules near the surface, in which fibrils develop depending on concentration and time, and of a thicker overlayer containing protruding molecules or bundles which modify noticeably the local viscosity. As a result of drastic denaturation, the ability of collagen to assemble into fibrils in the adsorbed phase is lost and the protrusion of molecules into the aqueous phase is much less pronounced. The adsorbed layer of denatured collagen appears essentially as a monolayer of flattened coils. At low concentration, this is easily displaced upon drying, leading to particular dewetting figures; at high concentration, aggregates add to the first layer. Moderate denaturation leads to an adsorbed phase which shows properties intermediate between those observed with native and extensively denatured collagen, regarding the ability to form fibrillar structures and the adlayer thickness and viscosity.

Factors and mechanisms determining the formation of fibrillar collagen structures in adsorbed phases.

The adsorption of collagen (type I from calf skin) was studied, comparing different collagen sources and using substrates which differ according to surface hydrophobicity (polystyrene, either native, with OH substitution of each repeat unit, with COOH substitution of a small fraction of repeat units, or surface modified by oxygen plasma discharge). The atomic force microscopy observation of the adsorbed layers showed that aggregation in the solution acts in competition with the formation of fibrils in the adsorbed phase; more aggregated solutions behave like less concentrated solutions regarding adsorption. The fibrils formed in the adsorbed phase are much smaller than the fibrils formed in the suspension, and, in contrast with the latter, do not show regular band pattern. It is confirmed that fibrils formation occurs more readily on more hydrophobic surfaces, which is tentatively attributed to a greater mobility of individual molecules adsorbed on more hydrophobic substrates. This interpretation is supported by previously published radiochemical measurements. However, the comparison of strongly different adsorption procedures (progressive on the one hand; quick and massive on the other hand) did not provide any additional clue.

Resolution of the vertical and horizontal heterogeneity of adsorbed collagen layers by combination of QCM-D and AFM.

Collagen (type I from calf skin) adsorption on polystyrene (PS) and plasma-oxidized polystyrene (PSox) was studied, using a quartz crystal microbalance with energy dissipation measurements (QCM-D) and atomic force microscopy (AFM) in tapping mode. Radio-labeled collagen was used to measure the adsorbed amount and the ability of adsorbed collagen to exchange with molecules in the solution. The results show that the collagen adlayer consists of two parts: a dense and thin sheet in which fibrils are formed (directly observed by AFM) and an overlying thick layer (up to 200 nm) containing protruding molecules or bundles which are in very low concentration but modify noticeably the local viscosity. The thickness and viscosity of the semi-liquid adlayer both increase with adsorption time and collagen concentration. Fibril formation near the surface also increases with time and collagen concentration and occurs more readily on PS compared to PSox. Radiochemical measurements show that this may be related to the larger mobility of molecules adsorbed on PS, presumably owing to a smaller number of binding points.

Influence of the aggregation state in solution on the supramolecular organization of adsorbed type I collagen layers.

In the last years, adsorbed collagen was shown to form layers with a supramolecular organization depending on the substrate surface properties and on the preparation procedure. If the concentration of collagen and the duration of adsorption are sufficient, fibrillar collagen structures are formed, corresponding to assemblies of a few molecules. This occurs more readily on hydrophobic compared to hydrophilic surfaces. This study aims at understanding the origin of such fibrillar structures and in particular at determining whether they result from the deposition of fibrils formed in solution or from the building of assemblies at the interface. Therefore, type I collagen solutions with an increasing degree of aggregation were prepared, using the "neutral-start" approach, by ageing pH 5.8 solutions at 37 degrees C for 15 min, 2 or 7 days. The obtained solutions were used to investigate the influence of collagen aggregation in solution on the supramolecular organization of adsorbed collagen layers, which was characterized by X-ray photoelectron spectroscopy and atomic force microscopy. Polystyrene and plasma-oxidized polystyrene were chosen as substrates for the adsorption. The size and the density of collagen fibrils at the interface decreased upon increasing the degree of aggregation of collagen in solution. This is explained by a competitive adsorption process between monomers and aggregates of the solution, turning at the advantage of the monomers. More aggregated solutions, which are thus depleted in free monomers, behave like less concentrated solutions, i.e. lead to a lower adsorbed amount and less fibril formation at the interface. This study shows that the supramolecular fibrils observed in adsorbed collagen layers, especially on hydrophobic substrates, are not formed in the solution, prior to adsorption, but are built at the interface, through the assembly of free segments of adsorbed molecules.

Aerosolization properties, surface composition and physical state of spray-dried protein powders.

Powder aerosols made of albumin, dipalmitoylphosphatidylcholine (DPPC) and a protein stabilizer (lactose, trehalose or mannitol) were prepared by spray-drying and analyzed for aerodynamic behavior, surface composition and physical state. The powders exited a Spinhaler inhaler as particle aggregates, the size of which depending on composition, spray-drying parameters and airflow rate. However, due to low bulk powder tap density (<0.15 g/cm3), the aerodynamic size of a large fraction of aggregates remained respirable (<5 microm). Fine particle fractions ranged between 21% and 41% in an Andersen cascade impactor operated at 28.3 l/min, with mannitol and lactose providing the most cohesive and free-flowing powders, respectively. Particle surface analysis by X-ray photoelectron spectroscopy (XPS) revealed a surface enrichment with DPPC relative to albumin for powders prepared under certain spray-drying conditions. DPPC self-organized in a gel phase in the particle and no sugar or mannitol crystals were detected by X-ray diffraction. Water sorption isotherms showed that albumin protected lactose from moisture-induced crystallization. In conclusion, a proper combination of composition and spray-drying parameters allowed to obtain dry powders with elevated fine particle fractions (FPFs) and a physical environment favorable to protein stability.

Nanoscale organization of adsorbed collagen: influence of substrate hydrophobicity and adsorption time.

The adsorption of collagen on polystyrene (PS) and polystyrene oxidized by oxygen plasma discharge (PSox) was studied as a function of time using radiolabeling, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). Radiolabeling and XPS indicated that the initial step of adsorption was faster on PS than on PSox. AFM imaging under water revealed very different supramolecular organization of the adsorbed films depending on time and on the nature of the substrate: PS showed patterns of collagen aggregates at all adsorption times (from 1 min to 24 h); PSox was covered with a smooth layer except at long adsorption times (24 h), for which a mesh of collagen structures was observed. After fast drying, the collagen layer remained continuous and showed a morphology which recalled that observed under water. The mechanical stability of the adsorbed films was assessed under water by scraping with the AFM probe at different loading forces: no perturbations were created on PSox; in contrast, the layer adsorbed on PS was sensitive to scraping, the minimum force required to alter the collagen layer morphology increasing with time. These differences in the film properties were correlated with force measurements upon retraction: multiple adhesion forces were observed with collagen adsorbed on PS samples, whereas such an effect was never observed on PSox. The results show that the amount adsorbed and the organization of the adsorbed film respond differently to the adsorption time and that this is influenced by surface hydrophobicity. The quick initial adsorption on PS, compared to PSox, is thought to leave dangling collagen segments that are responsible for the observed morphology, for adhesion forces, and for lower mechanical resistance of the adsorbed layer.

Carbon source-induced modifications in the mycolic acid content and cell wall permeability of Rhodococcus erythropolis E1.

The influence of the carbon source on cell wall properties was analyzed in an efficient alkane-degrading strain of Rhodococcus erythropolis (strain E1), with particular focus on the mycolic acid content. A clear correlation was observed between the carbon source and the mycolic acid profiles as estimated by high-performance liquid chromatography and mass spectrometry. Two types of mycolic acid patterns were observed after growth either on saturated linear alkanes or on short-chain alkanoates. One type of pattern was characterized by the lack of odd-numbered carbon chains and resulted from growth on linear alkanes with even numbers of carbon atoms. The second type of pattern was characterized by mycolic acids with both even- and odd-numbered carbon chains and resulted from growth on compounds with odd-numbered carbon chains, on branched alkanes, or on mixtures of different compounds. Cellular short-chain fatty acids were twice as abundant during growth on a branched alkane (pristane) as during growth on acetate, while equal amounts of mycolic acids were found under both conditions. More hydrocarbon-like compounds and less polysaccharide were exposed at the cell wall surface during growth on alkanes. Whatever the substrate, the cells had the same affinity for aqueous-nonaqueous solvent interfaces. By contrast, bacteria displayed completely opposite susceptibilities to hydrophilic and hydrophobic antibiotics and were found to be strongly stained by hydrophobic dyes after growth on pristane but not after growth on acetate. Taken together, these data show that the cell wall composition of R. erythropolis E1 is influenced by the nutritional regimen and that the most marked effect is a radical change in cell wall permeability.

Direct evidence for the involvement of extracellular proteins in the adhesion of Azospirillum brasilense.

Adhesion of Azospirillum brasilense to glass and polystyrene was investigated by bringing the cells into contact with the support by sedimentation. Adhesion depended on time and temperature: lower adhesion densities were observed when the contact time was only 2 h or 6 h, as compared to 24 h, or when the test was performed at 4 -C, as compared to 30 °. The influence of cell physiology was further demonstrated by the effect of tetracycline, which inhibited adhesion. Scanning electron microscopy showed that cells produced extracellular material when left in contact with a support for 24 h. The surface elemental composition of cells and of polystyrene supports after cell adhesion and subsequent detachment was determined by X-ray photoelectron spectroscopy; this provided information on the relative concentrations of proteins and polysaccharides at the surface. The protein concentration at the surface of a cell sediment increased as a function of time at 30 °, correlating with an increase of adhesion density. A similar correlation between protein concentration and adhesion density was found when comparing exponentialphase cells with stationary-phase cells. The surface composition of polystyrene supports examined after cell detachment was found to be rich in proteins, indicating that proteins are the major constituent at the support surface. Lowering the contact time, or performing adhesion under unfavourable metabolic conditions (4 °) or in the presence of tetracycline, resulted in a decrease in protein concentration at the support surface, which was correlated with a decrease in adhesion density. The correlation between protein concentration at the cell surface or at the support surface and adhesion density, under different experimental conditions, provides a direct demonstration of the involvement of extracellular proteins in the adhesion of A. brasilense to inert surfaces.