Residence time, loading force, pH, and ionic strength affect adhesion forces between colloids and biopolymer-coated surfaces

Exopolymers are thought to influence bacterial adhesion to surfaces, but the time-dependent nature of molecular-scale interactions of biopolymers with a surface are poorly understood. In this study, the adhesion forces between two proteins and a polysaccharide [Bovine serum albumin (BSA), lysozyme, or dextran] and colloids (uncoated or BSA-coated carboxylated latex microspheres) were analyzed using colloid probe atomic force microscopy (AFM). Increasing the residence time of an uncoated or BSA-coated microsphere on a surface consistently increased the adhesion force measured during retraction of the colloid from the surface, demonstrating the important contribution of polymer rearrangement to increased adhesion force. Increasing the force applied on the colloid (loading force) also increased the adhesion force. For example, at a lower loading force of approximately 0.6 nN there was little adhesion (less than -0.47 nN) measured between a microsphere and the BSA surface for an exposure time up to 10 s. Increasing the loading force to 5.4 nN increased the adhesion force to -4.1 nN for an uncoated microsphere to a BSA surface and to as much as -7.5 nN for a BSA-coated microsphere to a BSA-coated glass surface for a residence time of 10 s. Adhesion forces between colloids and biopolymer surfaces decreased inversely with pH over a pH range of 4.5-10.6, suggesting that hydrogen bonding and a reduction of electrostatic repulsion were dominant mechanisms of adhesion in lower pH solutions. Larger adhesion forces were observed at low (1 mM) versus high ionic strength (100 mM), consistent with previous AFM findings. These results show the importance of polymers for colloid adhesion to surfaces by demonstrating that adhesion forces increase with applied force and detention time, and that changes in the adhesion forces reflect changes in solution chemistry.     

Adhesion forces between functionalized latex microspheres and protein-coated surfaces evaluated using colloid probe atomic force microscopy

Proteins are important in bacterial adhesion, but interactions at molecular-scales between proteins and specific functional groups are not well understood. The adhesion forces between four proteins [bovine serum albumin (BSA), protein A, lysozyme, and poly-d-lysine] and COOH, NH2 and OH-functionalized (latex) colloids were examined using colloid probe atomic force microscopy (AFM) as the function of colloid residence time (T) and solution ionic strength (IS). For three of the proteins, OH-functionalized colloids produced higher adhesion forces to proteins (2.6-30.5 nN; IS=1 mM, T=10s) than COOH- and NH2-functionalized colloids (1.6-6.8 nN). However, protein A produced the largest adhesion force (8.1+/-1.0 nN, T=10 s) with the COOH-functionalized colloid, demonstrating the importance of specific and unanticipated protein-functional group interactions. The NH2-functionalized colloid typically produced the lowest adhesion forces with all proteins, likely due to repulsive electrostatic forces and weak bonds for NH2-NH2 interactions. The adhesion force (F) between functionalized colloids and proteins consistently increased with residence time (T), and data was well fitted by F=ATn. The constant value of n=0.21+/-0.07 for all combinations of proteins and functionalized colloids indicated that water exclusion and protein rearrangement were the primary factors affecting adhesion over time. Adhesion forces decreased inversely with IS for all functional groups interacting with surface proteins, consistent with previous findings. These results demonstrate the importance of specific molecular-scale interactions between functional groups and proteins that will help us to better understand factors colloidal adhesion to surfaces.     

Evaluating protein attraction and adhesion to biomaterials with the atomic force microscope

Failure of implanted biomaterials is commonly due to nonspecific protein adsorption, which in turn causes adverse reactions such as the formation of fibrous capsules, blood clots, or bacterial biofilm infections. Current research efforts have focused on modifying the biomaterial interface to control protein reactions. Designing biomaterial interfaces at the molecular level, however, requires an experimental technique that provides detailed, dynamic information on the forces involved in protein adhesion. The goal of this study was to develop an atomic force microscope (AFM)-based technique to evaluate protein adhesion on biomaterial surfaces. In this study, the AFM was used to evaluate (i) protein-protein, (ii) protein-substrate, and (iii) protein-dextran interactions. The AFM was first used to measure the pull-off forces between bovine serum albumin (BSA) tips/BSA surfaces and BSA tips/anti-BSA surfaces. Results from these protein-protein studies were consistent with the literature. More importantly, the successful measurement of antibody-antigen binding interactions demonstrates that both the BSA and anti-BSA proteins retain their folded conformation and remain functional following our immobilization protocol. The AFM was also used to quantify the physiochemical interactions of proteins during adhesion to various self-assembled monolayers (SAMs) and dextran-coated substrates representative of potential biomaterial interface modifications. Dextran, which renders surfaces very hydrophilic, was the only surface coating that BSA protein did not adhere to. Hydrophobic interactions were not found to play a significant role in BSA adhesion. Therefore, the dextran molecules may resist protein adhesion by repulsive steric effects or hydration pressure. Moreover, the AFM-based methodology provides dynamic, quantitative information about protein adhesion at the nanoscale level.     

Atomic force microscopy measurement of heterogeneity in bacterial surface hydrophobicity

The structure and physicochemical properties of microbial surfaces at the molecular level determine their adhesion to surfaces and interfaces. Here, we report the use of atomic force microscopy (AFM) to explore the morphology of soft, living cells in aqueous buffer, to map bacterial surface heterogeneities, and to directly correlate the results in the AFM force-distance curves to the macroscopic properties of the microbial surfaces. The surfaces of two bacterial species, Acinetobacter venetianus RAG-1 and Rhodococcus erythropolis 20S-E1-c, showing different macroscopic surface hydrophobicity were probed with chemically functionalized AFM tips, terminating in hydrophobic and hydrophilic groups. All force measurements were obtained in contact mode and made on a location of the bacterium selected from the alternating current mode image. AFM imaging revealed morphological details of the microbial-surface ultrastructures with about 20 nm resolution. The heterogeneous surface morphology was directly correlated with differences in adhesion forces as revealed by retraction force curves and also with the presence of external structures, either pili or capsules, as confirmed by transmission electron microscopy. The AFM force curves for both bacterial species showed differences in the interactions of extracellular structures with hydrophilic and hydrophobic tips. A. venetianus RAG-1 showed an irregular pattern with multiple adhesion peaks suggesting the presence of biopolymers with different lengths on its surface. R. erythropolis 20S-E1-c exhibited long-range attraction forces and single rupture events suggesting a more hydrophobic and smoother surface. The adhesion force measurements indicated a patchy surface distribution of interaction forces for both bacterial species, with the highest forces grouped at one pole of the cell for R. erythropolis 20S-E1-c and a random distribution of adhesion forces in the case of A. venetianus RAG-1. The magnitude of the adhesion forces was proportional to the three-phase contact angle between hexadecane and water on the bacterial surfaces.     

Effect of surface wettability on the adhesion of proteins

Besides significantly broadening the scope of available data on adhesion of proteins on solid substrates, we demonstrate for the first time that all seven proteins (tested here) behave similarly with respect to adhesion exhibiting a step increase in adhesion as wettability of the solid substrate decreases. Also, quantitative measures of like-protein-protein and like-self-assembled-monolayer (SAM)-SAM adhesive energies are provided. New correlations, not previously reported, suggest that the helix and random content (as measures of secondary structure) normalized by the molecular weight of a protein are significant for predicting protein adhesion and are likely related to protein stability at interfaces. Atomic force microscopy (AFM) was used to directly measure the normalized adhesion or pull-off forces between a set of seven globular proteins and a series of eight well-defined model surfaces (SAMs), between like-SAM-immobilized surfaces and between like-protein-immobilized surfaces in phosphate buffer solution (pH 7.4). Normalized force-distance curves between SAMs (alkanethiolates deposited on gold terminated with functional uncharged groups -CH3, -OPh, -CF3, -CN, -OCH3, -OH, -CONH2, and -EG3OH) covalently attached to an AFM cantilever tip modified with a sphere and covalently immobilized proteins (ribonuclease A, lysozyme, bovine serum albumin, immunoglobulin, gamma-globulins, pyruvate kinase, and fibrinogen) clearly illustrate the differences in adhesion between these surfaces and proteins. The adhesion of proteins with uncharged SAMs showed a general "step" dependence on the wettability of the surface as determined by the water contact angle under cyclooctane (thetaco). Thus, for SAMs with thetaco < approximately 66 degrees, (-OH, -CONH2, and -EG3OH), weak adhesion was observed (>-4 +/- 1 mN/m), while for approximately 66 < thetaco < approximately 104 degrees, (-CH3, -OPh, -CF3, -CN, -OCH3), strong adhesion was observed (< or =8 +/- 3 mN/m) that increases (more negative) with the molecular weight of the protein. Large proteins (170-340 kDa), in contrast to small proteins (14 kDa), exhibit characteristic stepwise decompression curves extending to large separation distances (hundreds of nanometers). With respect to like-SAM surfaces, there exists a very strong adhesive (attractive) interaction between the apolar SAM surfaces and weak interactive energy between the polar SAM surfaces. Because the polar surfaces can form hydrogen bonds with water molecules and the apolar surfaces cannot, these measurements provide a quantitative measure of the so-called mean hydrophobic interaction (approximately -206 +/- 8 mN/m) in phosphate-buffered saline at 296 +/- 1 K. Regarding protein-protein interactions, small globular proteins (lysozyme and ribonuclease A) have the least self-adhesion force, indicating robust conformation of the proteins on the surface. Intermediate to large proteins (BSA and pyruvate kinase-tetramer) show measurable adhesion and suggest unfolding (mechanical denaturation) during retraction of the protein-covered substrate from the protein-covered AFM tip. Fibrinogen shows the greatest adhesion of 20.4 +/- 2 mN/m. Unexpectedly, immunoglobulin G (IgG) and gamma-globulins exhibited very little adhesion for intermediate size proteins. However, using a new composite index, n (the product of the percent helix plus random content times relative molecular weight as a fraction of the largest protein in the set, Fib), to correlate the normalized adhesion force, IgG and gamma-globulins do not behave abnormally as a result of their relatively low helix and random (or high sheet) content.     

Scanning-force techniques to monitor time-dependent changes in topography and adhesion force of proteins on surfaces

Scanning-force microscopy (SFM) investigations were conducted to probe the influences of the interactions of proteins with surfaces relevant in medicine. These interactions are an important feature in the area of biofilm formation. The adsorption of proteins leads to changes in topography, which was monitored for the build up of protein layers of hen egg-white lysozyme and bovine serum albumin (BSA) on mica in real time in phosphate-buffered aqueous solution over a time period of 10 min. Phase imaging was additionally applied to compare material contrasts and to evaluate this method for further application in this field. The adhesion forces that develop on a time scale below 20 s between a protein-modified SFM tip and titanium surfaces (TiO(2), TiAl6V4 and TiAl6Nb7) were investigated. The influences of the parameters loading force and interaction time between the protein and the surface were monitored as well as the influence of protein structure. The interaction time dependency of the adhesion force could be described with a kinetic model of two consecutive first-order reactions. For the maximal adhesion force a correlation to the ratio of the amino acids cysteine, proline and glycine has been proposed.     

Characterisation of membrane surfaces: direct measurement of biological adhesion using an atomic force microscope

An atomic force microscope (AFM) in conjunction with coated colloid probe and cell probe techniques has been used to measure directly the adhesive force between both the protein bovine serum albumin (BSA) and a yeast cell at two different membranes. These were polymeric ultrafiltration membranes of similar MWCO (4000 Da) but of different materials (ES 404 and XP 117, PCI Membrane Systems, UK). The XP 117 membrane is made from a mixture of polymers chosen with the aim of achieving low fouling. The BSA was adsorbed on a 5 μm silica colloid probe formed from a tipless V-shaped AFM cantilever. The cell probe was created by immobilising a single yeast cell on such a tipless cantilever. Measurements were made in 10⁻² M NaCl solution. It was found for both protein and cell systems that the adhesive force at the ES 404 membrane was greater than that at the XP 117 membrane. The paper shows that coated colloid probe and cell probe techniques can provide useful means of directly quantifying the adhesion of biological materials to membrane surfaces.     

Importance of LPS structure on protein interactions with Pseudomonas aeruginosa

Atomic force microscopy (AFM) was used to quantify the adhesion forces between Pseudomonas aeruginosa PAO1 and AK1401, and a representative model protein, bovine serum albumin (BSA). The two bacteria strains differ in terms of the structure of their lipopolysaccharide (LPS) layers. While PAO1 is the wild-type expressing a complete LPS and two types of saccharide units in the O-antigen (A(+) B(+)), the mutant AK1401 expresses only a single unit of the A-band saccharide (A(+) B(-)). The mean adhesion force (F(adh)) between BSA and AK1401 was 1.12 nN, compared to 0.40 nN for F(adh) between BSA and PAO1. In order to better understand the fundamental forces that would control bacterial-protein interactions at equilibrium conditions, we calculated interfacial free energies using the van Oss-Chaudhury-Good (VCG) thermodynamic modeling approach. The hydrogen bond strength was also calculated using a Poisson statistical analysis. AK1401 has a higher ability to participate in hydrogen bonding with BSA than does PAO1, which may be because the short A-band and absence of B-band polymer allowed the core oligosaccharides and lipid A regions to be more exposed and to participate in hydrogen and chemical bonding. Interactions between PAO1 and BSA were weak due to the dominance of neutral and hydrophilic sugars of the A-band polymer. These results show that bacterial interactions with protein-coated surfaces will depend on the types of bonds that can form between bacterial surface macromolecules and the protein. We suggest that strategies to prevent bacterial colonization of biomaterials can focus on inhibiting these bonds.     

Parameters affecting the adhesion strength between a living cell and a colloid probe when measured by the atomic force microscope

In this study, we used the colloid probe atomic force microscopy (AFM) technique to investigate the adhesion force between a living cell and a silica colloid particle in a Leibovitz's L-15 medium (L-15). The L-15 liquid maintained the pharmaceutical conditions necessary to keep the cells alive in the outside environment during the AFM experiment. The force curves in such a system showed a steric repulsion in the compression force curve, due to the compression of the cells by the colloid probe, and an adhesion force in the decompression force curve, due to binding events between the cell and the probe. We also investigated for the first time how the position on the cell surface, the strength of the pushing force, and the residence time of the probe at the cell surface individually affected the adhesion force between a living cell and a 6.84 μm diameter silica colloid particle in L-15. The position of measuring the force on the cell surface was seen not to affect the value of the maximum adhesion force. The loading force was also seen not to notably affect the value of the maximum adhesion force, if it was small enough not to pierce and damage the cell. The residence time of the probe at the cell surface, however, clearly affected the adhesion force, where a longer residence time gave a larger maximum force. From these results, we could conclude that the AFM force measurements should be made using a loading force small enough not to damage the cell and a fixed residence time, when comparing results of different systems.     

Effects of ionic strength and surface charge on protein adsorption at PEGylated surfaces

PEGylated Nb2O5 surfaces were obtained by the adsorption of poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) copolymers, allowing control of the PEG surface density, as well as the surface charge. PEG (MW 2 kDa) surface densities between 0 and 0.5 nm(-2) were obtained by changing the PEG to lysine-mer ratio in the PLL-g-PEG polymer, resulting in net positive, negative and neutral surfaces. Colloid probe atomic force microscopy (AFM) was used to characterize the interfacial forces associated with the different surfaces. The AFM force analysis revealed interplay between electrical double layer and steric interactions, thus providing information on the surface charge and on the PEG layer thickness as a function of copolymer architecture. Adsorption of the model proteins lysozyme, alpha-lactalbumin, and myoglobin onto the various PEGylated surfaces was performed to investigate the effect of protein charge. In addition, adsorption experiments were performed over a range of ionic strengths, to study the role of electrostatic forces between surface charges and proteins acting through the PEG layer. The adsorbed mass of protein, measured by optical waveguide lightmode spectroscopy (OWLS), was shown to depend on a combination of surface charge, protein charge, PEG thickness, and grafting density. At high grafting density and high ionic strength, the steric barrier properties of PEG determine the net interfacial force. At low ionic strength, however, the electrical double layer thickness exceeds the thickness of the PEG layer, and surface charges "shining through" the PEG layer contribute to protein interactions with PLL-g-PEG coated surfaces. The combination of AFM surface force measurements and protein adsorption experiments provides insights into the interfacial forces associated with various PEGylated surfaces and the mechanisms of protein resistance.     

Analysis of bacterial adhesion using a gradient force analysis method and colloid probe atomic force microscopy

The atomic force microscope (AFM) has been used to examine the stickiness of bacteria on the basis of the analysis of approach and retraction force curves between the AFM tip and the bacterial surface. One difficulty in analyzing approach curve data is that the distance between the AFM tip and the surface of the bacterium is difficult to define. The exact distances are difficult to determine because the surface of the bacterium deforms during force imaging, producing a highly nonlinear region in the approach curve. In this study, AFM approach and retraction curves were obtained using a colloid probe AFM for three strains of Escherichia coli (D21, D21f2, and JM109). These strains differed in their relative adhesion to glass surfaces, on the basis of measurements of sticking coefficients in packed bed flow through column tests. A gradient force curve analysis method was developed to model the interactions between the colloid probe and a surface. Gradient analysis of the approach curve revealed four different regions of colloid-surface interactions during the approach and contact of the probe with the bacterial surface: a noninteraction region, a noncontact phase, a contact phase, and a constant compliance region. The noncontact phase, which ranged from 28 to 59 nm for the three bacterial strains, was hypothesized to arise primarily from steric repulsion of the colloid by extracellular polymers on the bacterial surface. The contact phase, spanning 59-113 nm, was believed to arise from the initial pressure of the colloid on the outer membrane of the cell. The constant compliance region likely reflected the response of the colloid probe to the stiff peptidoglycan layer that confers strength and rigidity to gram negative bacteria. It was shown that the sticking coefficients reported for the three E. coli strains were correlated with the length of the noncontact phase but not the properties of the other phases. Sticking coefficients were also not correlated with any parameters determined from retraction force curves such as pull-off distances or separation energies. These results show that gradient analysis is useful for studying the contribution of the length of the exopolymers on the cell surface to bacterial adhesion to glass surfaces.     

The importance of force in microbial cell adhesion

Microbes have evolved sophisticated strategies to colonize biotic and abiotic surfaces. Forces play a central role in microbial cell adhesion processes, yet until recently these were not accessible to study at the molecular scale. Unlike traditional assays, atomic force microscopy (AFM) is capable to study forces in single cell surface molecules and appendages, in their biologically relevant conformation and environment. Recent AFM investigations have demonstrated that bacterial pili exhibit a variety of mechanical responses upon contact with surfaces and that cell surface adhesion proteins behave as force-sensitive switches, two phenomena that play critical roles in cell adhesion and biofilm formation. AFM has also enabled to assess the efficiency of sugars, peptides, and antibodies in blocking cell adhesion, opening up new avenues for the development of antiadhesion therapies against pathogens.     

Bacteria attachment to surfaces--AFM force spectroscopy and physicochemical analyses

Understanding bacterial adhesion to surfaces requires knowledge of the forces that govern bacterial-surface interactions. Biofilm formation on stainless steel 316 (SS316) by three bacterial species was investigated by examining surface force interaction between the cells and metal surface using atomic force microscopy (AFM). Bacterial-metal adhesion force was quantified at different surface delay time from 0 to 60s using AFM tip coated with three different bacterial species: Gram-negative Massilia timonae and Pseudomonas aeruginosa, and Gram-positive Bacillus subtilis. The results revealed that bacterial adhesion forces on SS316 surface by Gram-negative bacteria is higher (8.53±1.40 nN and 7.88±0.94 nN) when compared to Gram-positive bacteria (1.44±0.21 nN). Physicochemical analysis on bacterial surface properties also revealed that M. timonae and P. aeruginosa showed higher hydrophobicity and surface charges than B. subtilis along with the capability of producing extracellular polymeric substances (EPS). The higher hydrophobicity, surface charges, and greater propensity to form EPS by M. timonae and P. aeruginosa led to high adhesive force on the metal surface.     

Importance of molecular details in predicting bacterial adhesion to hydrophobic surfaces

Electrostatic and hydrophobic forces are generally recognized as important in bacterial adhesion. Current continuum models for these forces often wrongly predict measurements of bacterial adhesion forces. The hypothesis tested here is that even qualitative guides to bacterial adhesion often require more than continuum information about hydrophobic forces; they require knowledge about molecular details of the bacteria and substrate surface. In this study, four different strains of bacteria were adsorbed to silica surfaces hydrophobized with alkylsilanes. The thickness of the lipopolysaccharide layers varied on the different bacteria, and the lengths of the alkylsilane molecules were varied from experiment to experiment. Bacterial adhesion was assessed using column experiments and atomic force microscopy (AFM) experiments. Results show that hydrophobized surfaces have higher bacterial sticking coefficients and stronger adhesion forces than bare silica surfaces, as expected. However, adhesion decreased as the solution Debye length became longer than the alkylsilane, perhaps since the silane molecules could not "reach" the bacterial surface. Similarly, those bacteria with a long o-antigen layer had decreased adhesion, perhaps since the silane molecules could not reach surface-bound proteins on the bacteria. This study reveals that macroscopic measurements such as contact angle are not able to fully describe bacterial adhesion; rather, additional details such as the molecular length are required to predict adhesion.     

Force measurements of bacterial adhesion on metals using a cell probe atomic force microscope

The adhesion of microbial cells to metal surfaces in aqueous media is an important phenomenon in both the natural environment and engineering systems. The adhesion of two anaerobic sulfate-reducing bacteria (Desulfovibrio desulfuricans and a local marine isolate) and an aerobe (Pseudomonas sp.) to four polished metal surfaces (i.e., stainless steel 316, mild steel, aluminum, and copper) was examined using a force spectroscopy technique with an atomic force microscope (AFM). Using a modified bacterial tip, the attraction and repulsion forces (in the nano-Newton range) between the bacterial cell and the metal surface in aqueous media were quantified. Results show that the bacterial adhesion force to aluminum is the highest among the metals investigated, whereas the one to copper is the lowest. The bacterial adhesion forces to metals are influenced by both the electrostatic force and metal surface hydrophobicity. It is also found that the physiological properties of the bacterium, namely the bacterial surface charges and hydrophobicity, also have influence on the bacteria-metal interaction. The adhesion to the metals by Pseudomonas sp. and D. desulfuricans was greater than by the marine SRB isolate. The cell-cell interactions show that there are strong electrostatic repulsion forces between bacterial cells. Cell probe atomic force microscopy has provided some useful insight into the interactions of bacterial cells with the metal surfaces.     

The role of polymer compatibility in the adhesion between surfaces saturated with modified dextrans

Wet and dry adhesion between dextran-coated surfaces were measured aiming to understand the influence of polymer compatibility. The wet adhesion measurements were performed using the atomic force microscope (AFM) colloidal probe technique whereas the dry adhesion measurements were performed using the micro adhesion measurement apparatus (MAMA). Two types of dextrans were used, one cationically modified dextran (DEX) and one that was both cationically and hydrophobically modified (HDEX), leading to three different combinations of polymer-coated surfaces; (1) DEX:DEX, (2) HDEX:DEX, and (3) HDEX:HDEX. DEX increased dry adhesion more than HDEX did, which likely is due to differences in the ability to form specific interactions, especially hydrogen bonding. HDEX gave strong wet adhesion, probably due to its poorer solvency, while DEX contributed to reducing the wet adhesion due to its hydrophilicity. All combinations showed a steric repulsion on approach in aqueous media. Furthermore, when HDEX was adsorbed on either or both surfaces a long range attractive force between the surfaces was detected outside this steric regime.     

Hydrophobic attraction as revealed by AFM force measurements and molecular dynamics simulation

Spherical calcium dioleate particles ( approximately 10 mum in diameter) were used as AFM (atomic force microscope) probes to measure interaction forces of the collector colloid with calcite and fluorite surfaces. The attractive AFM force between the calcium dioleate sphere and the fluorite surface is strong and has a longer range than the DLVO (Derjaguin-Landau-Verwey-Overbeek) prediction. The AFM force between the calcium dioleate sphere and the mineral surfaces does not agree with the DLVO prediction. Consideration of non-DLVO forces, including the attractive hydrophobic force and the repulsive hydration force, was necessary to explain the experimental results. The non-DLVO interactions considered were justified by the different interfacial water structures at calcite- and fluorite-water interfaces as revealed by the numerical computation experiments with molecular dynamics simulation.     

Fouling of reverse osmosis membranes by biopolymers in wastewater secondary effluent: Role of membrane surface properties and initial permeate flux

Reverse osmosis (RO) is being increasingly used in treatment of domestic wastewater secondary effluent for potable and non-potable reuse. Among other solutes, dissolved biopolymers, i.e., proteins and polysaccharides, can lead to severe fouling of RO membranes. In this study, the roles of RO membrane surface properties in membrane fouling by two model biopolymers, bovine serum albumin (BSA) and sodium alginate, were investigated. Three commercial RO membranes with different surface properties were tested in a laboratory-scale cross-flow RO system. Membrane surface properties considered include surface roughness, zeta potential, and hydrophobicity. Experimental results revealed that membrane surface roughness had the greatest effect on fouling by the biopolymers tested. Accordingly, modified membranes with smoother surfaces showed significantly lower fouling rates. When Ca²⁺ was present, alginate fouled RO membranes much faster than BSA. Considerable synergistic effect was observed when both BSA and alginate were present. The larger foulant particle sizes measured in the co-existence of BSA and alginate indicate formation of BSA-alginate aggregates, which resulted in greater fouling rates. Faster initial flux decline was observed at higher initial permeate flux even when the flux was measured against accumulative permeate volume, indicating a negative impact of higher operating pressure.     

Surface thermodynamics and adhesion forces governing bacterial transmission in contact lens related microbial keratitis

Contact lens induced microbial keratitis results from bacterial transmission from one surface to another. We investigated the adhesion forces of Pseudomonas aeruginosa, Staphylococci and Serratia to different contact lenses, lens cases and corneal surfaces using AFM, and applied a Weibull analysis on these adhesion forces to calculate bacterial transmission probabilities from lens case to corneas with a contact lens as an intermediate. Also a new surface thermodynamic parameter was introduced, the interfacial free energy of transmission, which in essence compares the interfacial free energies of bacterial adhesion, calculated from measured contact angles with liquids on the donating and receiving surfaces in the transmission process. Bacterial adhesion forces were generally strongest among all eight strains for the lens case (-6.5 to -12.0 nN) and corneas (-3.5 to -11.5 nN), while contact lenses (-0.6 to -13.1 nN) exerted slightly smaller adhesion forces. Consequently, bacterial transmission from lens case to contact lens yielded a smaller contribution in the final transmission than from contact lens to cornea. Bacterial transmission probabilities as derived from force analyses were higher when the interfacial free energies of transmission were more negative, which is in line with surface thermodynamic principles. Therewith this parameter could provide useful in analyzing other bacterial transmission phenomena between donating and receiving surfaces as well.     

Effect of surface proteins on Staphylococcus epidermidis adhesion and colonization on silicone

Shunt infections are one of the most serious complications in shunt implant surgery. Previous studies have suggested that cerebrospinal fluid (CSF) proteins could affect bacterial adhesion and subsequent shunt infection. A systematic study using immobilized protein on the surface of silane-modified silicone was conducted to determine how these modifications influenced Staphylococcus epidermidis adhesion and colonization. A comparison was also made with silicone having physically adsorbed protein. A colony-counting adhesion assay and scanning electron microscopy (SEM) were used to provide quantitative analysis of bacterial adhesion and semi-quantitative analysis of bacterial colonization, respectively. In order to determine the appropriate silanization process for effective protein immobilization, the effect of bovine serum albumin (BSA) immobilized on n-3-(trimethoxysilyl)propyl-ethylenediamine (AEAPS)/silicone, aminopropyltriethoxysilane (APTMS)/silicone, 3-(glycidyloxypropyl)trimethoxysilane (GPTMS)/silicone, and octadecyltrichlorosilane (OTS)/silicone on bacterial adhesion was investigated. Upon identifying that OTS is the most effective silane, different types of proteins, including: BSA, human serum albumin (HSA), gamma-globulin, and fibrinogen were immobilized on OTS/silicone by a photo-immobilization method. Immobilized protein on modified silicone surfaces was found to be stable in saline for 30 days, while physically adsorbed protein showed instability within hours as determined by contact angle measurements and X-ray photoelectron spectroscopy (XPS). For HSA/OTS/silicone, BSA/OTS/silicone, gamma-globulin/OTS/silicone, fibrinogen/OTS/silicon, and physically absorbed BSA on silicone, the contact angles were 78.5 degrees, 80.7 degrees, 78.9 degrees, 81.3 degrees, and 96.5 degrees; and the amount of nitrogen content was found to be 4.6%, 5.0%, 5.6%, 7.2%, and 3.2%, respectively. All protein immobilized on OTS/silicone surfaces significantly reduced bacterial adhesion by around 75% compared to untreated silicone, while physically adsorbed BSA on silicone reduced by only 29.4%, as determined by colony-counting adhesion assay. However, there was no significant difference on bacterial adhesion among the different types of proteins immobilized on OTS/silicone. Minimizing bacterial adhesion and colonization can be attributed to the increased concentration of -NH2 group, and stability and more hydrophilic nature of the protein/OTS/silicone surfaces.