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During the drying of inkjet printed droplets, the solute particles (IgG-Alexa-635 molecules) in the drop may distribute unevenly on the substrate, resulting in a "coffee-stain" spot morphology. In our study, we investigated the influence of the relative humidity on the distribution of inkjet printed fluorophore labeled IgG molecules on a polystyrene substrate. A theoretical model for an evaporating droplet was developed in order to predict the changes in the spot diameter, height and volume of a drying droplet. An experiment was performed where a sessile droplet was monitored using a CCD camera installed on a goniometer and good agreement was found between the experimental results and simulation data. We also compared the predicted morphology for an inkjet-printed microarray spot with the experimental results where IgG molecules were printed for various relative humidities. The spot morphology of the dried spots was analyzed by a confocal laser microscopy. At a lower relative humidity (i.e.

Item does not contain fulltext, Biofilms causing biomaterial-associated infection resist antibiotic treatment and usually necessitate the replacement of infected implants. Here we relate bacterial adhesion forces and the antibiotic susceptibility of biofilms on uncoated and polymer brush-coated silicone rubber. Nine strains of Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa adhered more weakly to brush-coated silicone rubber (-0.05 +/- 0.03 to -0.51 +/- 0.62 nN) than to uncoated silicone rubber (-1.05 +/- 0.46 to -5.1 +/- 1.3 nN). Biofilms of weakly adhering organisms on polymer brush coatings remained in a planktonic state, susceptible to gentamicin, unlike biofilms formed on uncoated silicone rubber.

We have studied the formation and the stability of grafted block complex coacervate core micelles (C3Ms) in solution and the influence of grafted block C3M coatings on the adsorption of the proteins ß-lactoglobulin, bovine serum albumin, and lysozyme. The C3Ms consist of a grafted block copolymer PAA21-b-PAPEO14 (poly(acrylic acid)-b-poly(acrylate methoxy poly(ethylene oxide)), with a negatively charged PAA block and a neutral PAPEO block and a positively charged homopolymer P2MVPI (poly(N-methyl 2-vinyl pyridinium iodide). In solution, these C3Ms partly disintegrate at salt concentrations between 50 and 100 mM NaCl. Adsorption of C3Ms and proteins has been studied with fixed-angle optical reflectometry, at salt concentrations ranging from 1 to 100 mM NaCl. In comparison with the adsorption of PAA21-b-PAPEO14 alone adsorption of C3Ms significantly increases the amount of PAA21-b-PAPEO14 on the surface. This results in a higher surface density of PEO chains. The stability of the C3M coatings and their influence on protein adsorption are determined by the composition and the stability of the C3Ms in solution. A C3M-PAPEO14/P2MVPI43 coating strongly suppresses the adsorption of all proteins on silica and polystyrene. The reduction of protein adsorption is the highest at 100 mM NaCl (>90%). The adsorbed C3M-PAPEO14/P2MVPI43 layer is partly removed from the surface upon exposure to an excess of ß-lactoglobulin solution, due to formation of soluble aggregates consisting of ß-lactoglobulin and P2MVPI43. In contrast, C3M-PAPEO14/P2MVPI228 which has a fivefold longer cationic block enhances adsorption of the negatively charged proteins on both surfaces at salt concentrations above 1 mM NaCl. A single PAA21-b-PAPEO14 layer causes only a moderate reduction of protein adsorption.

We have studied the formation and the stability of ionomer complexes from grafted copolymers (GICs) in solution and the influence of GIC coatings on the adsorption of the proteins beta-lactoglobulin (beta-lac), bovine serum albumin (BSA), and lysozyme (Lsz) on silica and polysulfone. The GICs consist of the grafted copolymer PAA(28)-co-PAPEO(22) {poly(acrylic acid)-co-poly[acrylate methoxy poly(ethylene oxide)]} with negatively charged AA and neutral APEO groups, and the positively charged homopolymers: P2MVPI(43) [poly(N-methyl 2-vinyl pyridinium iodide)] and PAH center dot HCl160 [poly(allylamine hydrochloride)]. In solution, these aggregates are characterized by means of dynamic and static light scattering. They appear to be assemblies with hydrodynamic radii of 8 nm (GIC-PAPEO(22)/P2MVPI(43)) and 22 nm (GIC-PAPEO(22)/PAH center dot HCl160), respectively. The GICs partly disintegrate in solution at salt concentrations above 10 mM NaCl. Adsorption of GICs and proteins has been studied with fixed angle optical reflectometry at salt concentrations ranging from 1 to 50 mM NaCl. Adsorption of GICs results in high density PEO side chains on the surface. Higher densities were obtained for GICs consisting of PAH center dot HCl160 (1.6 divided by 1.9 chains/nm(2)) than of P2MVPI(43) (0.6 divided by 1.5 chains/nm(2)). Both GIC coatings strongly suppress adsorption of all proteins on silica (>90%); however, reduction of protein adsorption on polysulfone depends on the composition of the coating and the type of protein. We observed a moderate reduction of beta-lac and Lsz adsorption (>60%). Adsorption of BSA on the GIC-PAPEO(22)/ P2MVPI(43) coating is moderately reduced, but on the GIC-PAPEO(22)/ PAH center dot HCl160 coating it is enhanced.

Using a parallel-plate flow chamber, the hydrodynamic shear forces to prevent bacterial adhesion (F-prev) and to detach adhering bacteria (F-det) were evaluated for hydrophilic glass, hydrophobic, dimethyldichlorosilane (DDS)-coated glass and six different bacterial strains, in order to test the following three hypotheses. 1. A strong hydrodynamic shear force to prevent adhesion relates to a strong hydrodynamic shear force to detach an adhering organism. 2. A weak hydrodynamic shear force to detach adhering bacteria implies that more bacteria will be stimulated to detach by passing an air-liquid interface (an air bubble) through the flow chamber. 3. DLVO (Derjaguin, Landau, Verwey, Overbeek) interactions determine the characteristic hydrodynamic shear forces to prevent adhesion and to detach adhering micro-organisms as well as the detachment induced by a passing air-liquid interface. F-prev varied from 0.03 to 0.70 pN, while F-det varied from 0.31 to over 19.64 pN, suggesting that after initial contact, strengthening of the bond occurs. Generally, it was more difficult to detach bacteria from DIDS-coated glass than from hydrophilic glass, which was confirmed by air bubble detachment studies. Calculated attractive forces based on the DLVO theory (F-DLVO) towards the secondary interaction minimum were higher on glass than on DIDS-coated glass. In general, all three hypotheses had to be rejected, showing that it is important to distinguish between forces acting parallel (hydrodynamic shear) and perpendicular (DLVO, air-liquid interface passages) to the substratum surface.

Adhesion and residence-time-dependent desorption of two Staphylococcus aureus strains with and without fibronectin (Fn) binding proteins (FnBPs) on Fn-coated glass were compared under flow conditions. To obtain a better understanding of the role of Fn-FnBP binding, the adsorption enthalpies of Fn with staphylococcal cell surfaces were determined using isothermal titration calorimetry (ITC). Interaction forces between staphylococci and Fn coatings were measured using atomic force microscopy (AFM). The strain with FnBPs adhered faster and initially stronger to an Fn coating than the strain without FnBPs, and its Fn adsorption enthalpies were higher. The initial desorption was high for both strains but decreased substantially within 2 s. These time scales of staphylococcal bond ageing were confirmed by AFM adhesion force measurement. After exposure of either Fn coating or staphylococcal cell surfaces to bovine serum albumin (BSA), the adhesion of both strains to Fn coatings was reduced, suggesting that BSA suppresses not only nonspecific but also specific Fn-FnBP interactions. Adhesion forces and adsorption enthalpies were only slightly affected by BSA adsorption. This implies that under the mild contact conditions of convective diffusion in a flow chamber, adsorbed BSA prevents specific interactions but does allow forced Fn-FnBP binding during AFM or stirring in ITC. The bond strength energies calculated from retraction force-distance curves from AFM were orders of magnitude higher than those calculated from desorption data, confirming that a penetrating Fn-coated AFM tip probes multiple adhesins in the outermost cell surface that remain hidden during mild landing of an organism on an Fn-coated substratum, like that during convective diffusional flow.

We introduce a procedure for determining shear forces at the balance between attachment and detachment of bacteria under flow. This procedure can be applied to derive adhesion forces in weak-adherence systems, such as polymer brush coatings, which are currently at the center of attention for their control of bacterial adhesion and biofilm formation.

The antigen I/II family of surface proteins is expressed by most oral streptococci, including Streptococcus mutans, and mediates specific adhesion to, among other things, salivary films and extracellular matrix proteins. In this study we showed that antigen I/II-deficient S. mutans isogenic mutant IB03987 was nearly unable to adhere to laminin films under flow conditions due to a lack of specific interactions (0.8 x 106 and 1.1 x 106 cells cm¿2 at pH 5.8 and 6.8, respectively) compared with parent strain LT11 (21.8 x 106 and 26.1 x 106 cells cm¿2). The adhesion of both the parent and mutant strains was slightly greater at pH 6.8 than at pH 5.8. In addition, atomic force microscopy (AFM) experiments demonstrated that the parent strain experienced less repulsion when it approached a laminin film than the mutant experienced. Upon retraction, combined specific and nonspecific adhesion forces were stronger for the parent strain (up to ¿5.0 and ¿4.9 nN at pH 5.8 and 6.8, respectively) than for the mutant (up to ¿1.5 and ¿2.1 nN), which was able to interact only through nonspecific interactions. Enthalpy was released upon adsorption of laminin to the surface of the parent strain but not upon adsorption of laminin to the surface of IB03987. A comparison of the adhesion forces in AFM with the adhesion forces reported for specific ligand-receptor complexes resulted in the conclusion that the number of antigen I/II binding sites for laminin on S. mutans LT11 is on the order of 6 x 104 sites per organism and that the sites are probably arranged along exterior surface structures, as visualized here by immunoelectron microscopy.

Most bacterial strains adhere poorly to poly(ethylene oxide) (PEO)-brush coatings, with the exception of a Pseudomonas aeruginosa strain. The aim of this study was to find factors determining whether P. aeruginosa strains do or do not adhere to a PEO-brush coating in a parallel plate flow chamber. On the basis of their adhesion, a distinction could be made between three adhesive and three non-adhesive strains of P. aeruginosa, while bacterial motilities and zeta potentials were comparable for all six strains. However, water contact angles indicated that the adhesive strains were much more hydrophobic than the non-adhesive strains. Furthermore, only adhesive strains released surfactive extracellular substances, which may be engaged in attractive interactions with the PEO chains. Atomic force microscopy showed that the adhesion energy, measured from the retract curves of a bacterial-coated cantilever from a brush coating, was significantly more negative for adhesive strains than for non-adhesive strains (P

Conformational characteristics and the adsorption behavior of endo-ß-1,3-glucanase from the hyperthermophilic microorganism Pyrococcus furiosus were studied by circular dichroism, steady-state and time-resolved fluorescence spectroscopy, and calorimetry in solution and in the adsorbed state. The adsorption isotherms were determined on two types of surfaces: hydrophobic Teflon and hydrophilic silica particles were specially designed so that they do not interact with light and therefore do not interfere with spectroscopic measurements. We present the most straightforward method to study structural features of adsorbed macromolecules in situ using common spectroscopic techniques. The enzyme was irreversibly adsorbed and immobilized in the adsorbed state even at high temperatures. Adsorption offered further stabilization to the heat-stable enzyme and in the case of adsorption on Teflon its denaturation temperature was measured at 133°C, i.e., the highest experimentally determined for a protein. The maintenance of the active conformation and biological function particularly at high temperatures is important for applications in biocatalysis and biotechnology. With this study we also suggest that nature may employ adsorption as a complementary mode to maintain structural integrity of essential biomolecules at extreme conditions of temperature.

Variations in surface hydrophobicity of six Lactobacillus strains with and without an S-layer upon changes in ionic strength are derived from contact angle measurements with low- and high-ionic-strength aqueous solutions. Cell surface hydrophobicity changed in response to changes in ionic strength in three out of the six strains, offering these strains a versatile mechanism to adhere to different surfaces. The dynamic behavior of the cell surface hydrophobicity could be confirmed for two selected strains by measuring the interaction force between hydrophobic and hydrophilic tips with use of atomic force microscopy.

Application of physico-chemical models to describe bacterial adhesion to surfaces has hitherto only been partly successful due to the structural and chemical heterogeneities of bacterial surfaces, which remain largely unaccounted for in macroscopic physico-chemical characterizations of the cell surfaces. In this study, the authors attempted to correlate microscopic adhesion of a collection of nine Streptococcus mitis strains to the negatively charged, hydrophilic silicon nitride tip of an atomic force microscope (AFM) with macroscopic adhesion of the strains to a negatively charged, hydrophilic glass in a parallel-plate flow chamber. The repulsive force probed by AFM upon approach of the tip to a bacterial cell surface ranged from 1·7 to 7·7 nN depending on the strain considered and was found to correspond to an activation barrier, governing initial, macroscopic adhesion of the organisms to the glass surface. Moreover, maximum distances at which attractive forces were probed by the AFM upon retraction of the tip (120 to 1186 nm) were related to the area blocked by an adhering bacterium, i.e. the distance kept between adhering bacteria. Bacterial desorption could not be related to adhesive forces as probed by the AFM, possibly due to the distinct nature of the desorption process occurring in the parallel-plate flow chamber and the forced detachment in AFM.

Atomic force microscopy (AFM) has emerged as a powerful technique for mapping the surface morphology of biological specimens, including bacterial cells. Besides creating topographic images, AFM enables us to probe both physicochemical and mechanical properties of bacterial cell surfaces on a nanometer scale. For AFM, bacterial cells need to be firmly anchored to a substratum surface in order to withstand the friction forces from the silicon nitride tip. Different strategies for the immobilization of bacteria have been described in the literature. This paper compares AFM interaction forces obtained between Klebsiella terrigena and silicon nitride for three commonly used immobilization methods, i.e., mechanical trapping of bacteria in membrane filters, physical adsorption of negatively charged bacteria to a positively charged surface, and glutaraldehyde fixation of bacteria to the tip of the microscope. We have shown that different sample preparation techniques give rise to dissimilar interaction forces. Indeed, the physical adsorption of bacterial cells on modified substrata may promote structural rearrangements in bacterial cell surface structures, while glutaraldehyde treatment was shown to induce physicochemical and mechanical changes on bacterial cell surface properties. In general, mechanical trapping of single bacterial cells in filters appears to be the most reliable method for immobilization.

Poly(ethylene oxide) (PEO)-brushes are generally recognized as protein-repellent surfaces, and although a role in discouraging microbial adhesion has been established for some strains and species, no study exists on the effects of PEO-brushes on a large variety of bacterial and yeast strains. In this paper, a PEO-brush has been covalently attached to glass and silica by reaction in a polymer melt. Subsequently, the presence of a PEO-brush was demonstrated using contact angle measurements, X-ray photoelectron spectroscopy and ellipsometry. For five bacterial (Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus salivarius, Escherichia coli and Pseudomonas aeruginosa) and two yeast strains (Candida albicans and Candida tropicalis), adhesion to PEO-brushes was compared with adhesion to bare glass in a parallel plate flow chamber. The initial deposition rates of Sta. epidermidis, Sta. aureus and Str. salivarius to glass were relatively high, between 2400 and 2600 cm-2 s-1, while E. coli and P. aeruginosa deposited much more slowly. The initial deposition rates of the yeasts to glass were 144 and 444 cm-2 s-1 for C. albicans GB 1/2 and C. tropicalis GB 9/9, respectively. Coating of the glass surface with a PEO-brush yielded more than 98 % reduction in bacterial adhesion, although for the more hydrophobic P. aeruginosa a smaller reduction was observed. For both yeast species adhesion suppression was less effective than for the bacteria and here too the more hydrophobic C. tropicalis showed less reduction than the more hydrophilic C. albicans. The PEO-brush had a thickness of 22 nm in water, as inferred from ellipsometry. Assuming that on bare glass the adhered micro-organisms are positioned only a few nanometers away from the surface and that the brush keeps them at a distance of 22 nm, it is calculated that the brush yields a sevenfold attenuation of the Lifshitz–Van der Waals attraction to the surface between the micro-organisms and the surface. Decreased Li

Preservation of biological functioning of proteins during immobilisation is of special interest in various biomedical and biotechnical applications. In industry physical adsorption of immunoglobulins (IgGs) onto solid surfaces is still the predominant immobilisation procedure because it is relatively easy to perform. Physical adsorption, however, often results in an undesired loss of biological activity. This loss of activity may be caused by changes in the specific folding (the conformation) of the IgG or by a reduced accessibility of the antigen binding sites by blocking, for instance when IgG molecules are adsorbed with their antigen binding sites oriented towards the sorbent surface.To control the orientation and conformation of adsorbed IgG molecules we studied the interactions involved in physical adsorption of IgG molecules on solid surfaces. Our goal was to optimize the biological activity of adsorbing IgG molecules. For this, we introduced a new method to achieve oriented physical adsorption of IgG. This concept is based on the anisodimensionality of IgG molecules and resembles 'molecular sieving' on the sorbent surface. The 'sieve' is created by preadsorbed molecules that form a steric barrier preventing adsorption at some sites, but leaving patches of uncovered surface area. The open areas in the preadsorbed layer can be tuned in such a way that only the smaller part of anisodimensional molecules can enter and adsorb. In the case of 19G this means that only the Fc part can adsorb to the surface and thereby forcing the larger antigen binding parts (F(ab) 2 ), directed towards the solution and, hence, accessible to bind antigens. A 'sieve' formed either with preadsorbed IgG molecules or with triblock copolymers of poly(ethylene oxide), PEO, and poly(propylene oxide), PPO, of the type PEOPPO-PEO was proven to yield a higher specific biological activity of subsequently adsorbing IgG.In brief, the effect of a 'molecular sieve' on IgG adsorption is essentially

The surface chemical composition of whole cells and isolated cell walls of four coryneform bacteria and of a Bacillus brevis strain has been determined by X-ray photoelectron spectroscopy (XPS). The XPS data were converted into concentrations of model compounds: peptides, polysaccharides, and hydrocarbonlike compounds. The composition of the surface of B. brevis differed markedly from that of coryneforms: the peptide concentration was about twice higher in the former case, which is attributed to the presence of an S-layer at the cell surface; in contrast, the surface of coryneforms was rich in hydrocarbonlike compounds (about 40%), which was concomitant with a high water contact angle. The peptide surface concentration of the isolated cell walls of the five strains deduced from XPS data fitted well with the total peptide content determined by biochemical analysis, which supports the validity of XPS to determine the overall macromolecular composition of the bacterial cell surface. Compared to biochemical analysis of isolated cell walls, XPS analysis of whole cells provides information which concerns directly the cell surface (2- to 5-nm-thick layer) and is less subject to alteration via losses of cell wall constituents or contamination by intracellular compounds.

Bacterial cells are ubiquitous in natural environments and also play important roles in domestic and industrial processes. They are found either suspended in the aqueous phase or attached to solid particles. The adhesion behaviour of bacteria is influenced by the physico-chemical properties of their cell surfaces, such as hydrophobicity and cell wall charge. The charge in the bacterial wall originates from carboxyl, phosphate and amino groups. The degree of dissociation of these anionic and cationic groups is determined by the pH and the activity of the surrounding electrolyte solution. Almost all bacterial cells are negatively charged at neutral pH, because the number of carboxyl and phosphate groups is generally higher than that of the amino groups. The presence of the charged cell wall groups leads to the spontaneous formation of an electrical double layer. The purpose of the present investigation is to elucidate the structure of the electrical double layer of bacterial cell surface. Such a study serves at least two goals. It allows the quantification of electrostatic interactions in the adhesion process and it contributes to gain better insight into the availability of (in)organic compounds for bacterial cells.The characteristics of the electrical double layer of bacterial cell surfaces have been revealed by applying a combination of experimental techniques, which include: chemical cell wall analysis, potentiometric proton titration and electrokinetic studies such as micro-electrophoresis, static conductivity and dielectric dispersion measurements.For the present study five Gram-positive bacterial strains, including four coryneforms and a Bacillus brevis, have been selected. Cell walls of these bacterial strains have been isolated and were subsequently subjected to chemical analyses and proton titration studies. Both methods provide information on the number of carboxyl, phosphate and amino groups.The chemical analysis of isolated cell walls involves the quantit

Proteolytic enzymes in liquid detergents suffer from lack of stability in the sense that activity diminishes with time. Although the phenomenon could be attributed to several factors, the influence of colloidal surfaces on the enzymatic stability was investigated. Besides the types of surfaces that are present in the usual detergent systems, other surfaces have been also studied. The enzyme under investigation was wild-type Savinase TM, which belongs to the class of microbial alkaline serine proteinases. This thesis contains a set of experiments and discussions on the interactions, viz. adsorption and inactivation, between Savinase and several types of surfaces.In order to understand the mechanism of adsorption of Savinase from aqueous solution, adsorption from solution was achieved by stagnation point flow towards an oxidized silicon strip. The adsorbed amount was monitored continuously as a function of time by reflectometry. In order to prevent autodigestion during the experiments, the enzyme was irreversibly inhibited by reaction of the active site with a small inhibitor. The adsorbed amount was monitored as a function of time. The experimental variables were the amphipathicity of the solid surface, the pH, the ionic strength, the type of monovalent ions used and the protein concentration. It was concluded that electrostatic interactions dominate the adsorption of Savinase on a hydrophilic surface and that unfolding of the protein molecule does not take place. On the other hand, for a hydrophobic surface, adsorption is dictated by unfolding of the protein structure and/or hydrophobic dehydration.Another type of surface that was relevant for this study, was a negatively charged model surface, carrying chemically grafted short poly(ethylene) oxide chains. To that end, a method was developed to prepare charge- stabilized polystyrene latex particles, carrying these moieties. The latices had a surface charge and possessed on the surface a PEO-350/methacrylate molecule

SummaryAdsorption of immunoglobulin G (IgG) is a common step in the production of immunological tests and biosensors. The use of IgG in these applications stems from its ability to specifically bind all kinds of molecules (antigens). In these tests the IgG molecules are immobilised onto solid surfaces, which is necessary to make visible the IgG-antigen binding. Upon adsorption, however, the IgG molecules may lose their ability to bind antigens. If the antigen binding sites are located close to the sorbent surface, this could reduce the accessibility for antigens. A second problem which may occur upon adsorption is related to structural rearrangements of the protein which could also cause a reduction in the antigen binding capacity.The purpose of the present work was to acquire more insight into the role of the various interactions between IgG molecules and sorbent surfaces on the adsorption behaviour and, especially, how these interactions determine the adsorbed state of the IgG molecules.After a general introduction in Chapter 1, in Chapter 2 an overview is given of the physical properties and some general aspects of the adsorption behaviour of IgG.The different types of protein-sorbent interactions are systematically studied by performing experiments under various system conditions. The influence of the sorbent surface hydrophobicity is investigated using both a hydrophobic and a hydrophilic surface. Chapter 3 describes the adsorption of the proteins onto three polymer latices. Two of these latices are hydrophobic, one being positively and the other negatively charged and the third latex is negatively charged and hydrophilic. The adsorption experiments described in Chapters 4, 5, and 6 are performed using a hydrophilic silica and a hydrophobic methylated surface. Electrostatic interactions are varied by choosing various values for the pH and ionic strength. The experiments are performed using two monoclonal IgGs directed against the pregnancy hormone human chorion

Interactions between bacteria and solid surfaces strongly influence the behaviour of bacteria in natural and engineered ecosystems. Many biofilm reactors and terrestrial environments are porous media. The purpose of the research presented in this thesis is to gain a better insight into the basic mechanims of bacterial adhesion and transport in such systems. This knowledge is essential for bacterial adhesion science in general, and important for practical applications such as the bioremediation of contaminated soils, sediments and groundwaters and the treatment of industrial waste streams in fixed bed biofilm reactors.Model systemsAssessment of cell-substratum interactions in bacterial adhesion require materials with defined properties and an experimental set-up that allows a reproducible quantification of the transport of bacterial cells from bulk liquid to substratum. We performed our studies with model systems and under welldefined conditions in which these prerequisites are met.Characteristics of solids and bacteria . Negatively charged teflon and glass were used as the model substrata. Eight coryneform bacteria and four pseudomonads were selected. These organisms have different negative cell surface charges at pH 7 (chapter 2). The hydrophobicity of solids and bacteria were determined by measuring the contact angle of drops of water on flat pieces of solid and on dried bacterial lawns. Glass is hydrophilic and teflon extremely hydrophobic. The hydrophobicities of dried bacteria varied between strongly hydrophilic and extremely hydrophobic. However, evidence was found that the drying of the bacteria changes their surface properties, especially in the case of amphiphilic cell-coatings. Therefore, a further characterization of the bacterial cell-surfaces was performed under hydrated conditions by measuring the isoelectric point of the bacterial strains (Chapter 4). An isoelectric point smaller than 2.8 correlated with the presence of anionic polysaccharides on the

Enzymes are proteins with the capacity of catalysing various reactions. Nowadays two types of enzymes, proteases and lipases, are available for use in detergent formulations for household and industrial laundry washing. Proteases are capable of catalysing the hydrolysis of proteins while lipases enable the hydrolysis of glycerol esters, the main component in fats and non-mineral oils. In this study, two enzymes each representing one of these categories were examined: Savinase TM, an extracellular serine protease of the subtilisin family from Bacillus lentus and Lipolase TM, an extracellular lipase from the thermophilic fungus Humicola Lanuginosa S-38. To improve the soil- and stain removal in laundry washing the enzyme has to adsorb onto the soiled fabric and hydrolyse one or more of the stain components. This thesis describes a study on the mechanisms by which these enzymes adsorb at various solid-liquid interfaces from buffered solutions in the absence of other components that are found in common detergents.Chapter 1 gives a general introduction. It contains an overview of the physico-chemical characteristics of the two enzymes and a summary of the present insight into the protein adsorption. In Chapter 2 the investigation of the adsorption measurement techniques for Savinase is reported together with methods used to inhibit the enzyme with the small organic inhibitor PMSF (phenylmethanesulfonyl fluoride) to suppress autolysis.Several techniques are known to measure the protein concentration in solution and, hence, to determine the adsorption. However, the measurement of a proteolytic enzyme, such as Savinase, at such low concentrations as used in laundry washing systems proved to be a serious complicating factor. Proteolytic enzymes hydrolyses proteins and as the enzyme itself is a protein it is capable of self-destruction or cannibalism, so-called autolysis. Of the four well known methods for measurement of the protein concentration only two proved to give relia

The subject of this thesis is proteins at interfaces. The main purpose of the work was to acquire more insight into the mechanism of adsorption of Bovine Serum Albumin (BSA) and monoclonal Immuno gamma Globulins (IgG's). both individually and in competition. Another aim was to achieve optimum conditions for the colloidal stability of polystyrene (PS) latices coated with these proteins, a feature that is important In the development of diagnostic test systems.First, in chapter 2 . a literature overview is given with special emphasis on (1) the adsorption of protein mixtures and (ii) the dynamic aspects of protein adsorption. The reason for studying the latter is that there are many indications that protein adsorption is to a certain extent irreversible. Hence, the ultimate properties of a protein adsorbate may partly be determined by its history. The structure stability of a protein molecule is considered to be one of the most Important parameters influencing its (competitive) adsorption behaviour. The heterogeneity of an adsorbed protein layer can also be related to this intrinsic property of the protein. The 'reversibility' aspects are reflected in the extents of desorption, exchange and/or displacement. The molecules that arrive the latest on the surface will be more easily removed compared to those adsorbing in a more initial stage, having more time to adapt themselves optimally.In chapter 3 single adsorption of BSA and monoclonal IgG's on differently charged PS latices is described. Monoclonal IgG's having different isoelectric: points were used, allowing us to systematically investigate the electrostatic aspects of Interaction. Much attention is paid to the proper purification and characterization of the materials used. The main finding in this chapter is the occurrence of maximum protein adsorption as a function of pH around the isoelectricity of the protein-latex complex, rather than that of the solute protein. An important aspect in the overall protein adsor