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Two blue multicopper oxidases (MCOs) (viz. Trametes hirsuta laccase (ThLc) and Myrothecium verrucaria bilirubin oxidase (MvBOx)) were immobilized on bare polycrystalline gold (Au) surfaces by direct adsorption from both dilute and concentrated enzyme solutions. The adsorption was studied in situ by means of null ellipsometry. Moreover, both enzyme-modified and bare Au electrodes were investigated in detail by atomic force microscopy (AFM) as well as electrochemically. When adsorbed from dilute solutions (0.125 and 0.25 mg mL⁻¹ in the cases of ThLc and MvBOx, respectively), the amounts of enzyme per unit area were determined to be ca. 1.7 and 4.8 pmol cm⁻², whereas the protein film thicknesses were determined to be 29 and 30 Å for ThLc and MvBOx, respectively. A well-pronounced bioelectrocatalytic reduction of molecular oxygen (O₂) was observed on MvBOx/Au biocathodes, whereas this was not the case for ThLc-modified Au electrodes (i.e., adsorbed ThLc was catalytically inactive). The initially observed apparent k(cat)(app) values for adsorbed MvBOx and the enzyme in solution were found to be very close to each other (viz. 54 and 58 s⁻¹, respectively (pH 7.4, 25 °C)). However, after 3 h of operation of MvBOx/Au biocathodes, kcatapp dropped to 23 s⁻¹. On the basis of the experimental results, conformational changes of the enzymes (in all likelihood, their flattening on the Au surface) were suggested to explain the deactivation of MCOs on the bare Au electrodes.

The salivary pellicle is a thin acellular organic film that forms on any type of surface upon exposure to saliva. The role of the pellicle is manifold, and it plays an important role in the maintenance of oral health. Its functions include not only substratum protection and lubrication, but also remineralization and hydration. It also functions as a diffusion barrier and possesses buffering ability. Not only the function, but also the formation, composition and stability of the pellicle are known to be highly influenced by the physicochemical properties of both substrata and ambient media. In this chapter, we discuss these aspects of salivary pellicles, an area where research has boomed in the past years partly because of the application of experimental techniques often reserved for more traditional surface science studies.

The objective of this investigation was to monitor the adsorption of antibodies to polystyrene surfaces using ellipsometry. Commercial polystyrene slides used for solid state diagnostics were selected as substrates and the adsorption of three different antibodies (human IgG, bovine IgG and goat anti-human IgG) were evaluated. Based on theoretical models describing the ellipsometric data, it was concluded that the adsorption of antibodies should result in layers that are sufficiently thick to be able to monitor the adsorption in terms of adsorbed amount and thickness of the layer with a reasonable precision. The experimental results confirmed this assumption and values of 2.0-2.3 mg/m(2) were detected for the adsorbed amount with a corresponding thickness of 10-16 nm. It was furthermore found that the antibodies bound irreversibly with respect to rinsing with protein-free solutions. In additional experiments, the consecutive incubation of human IgG and anti-human IgG was investigated. These results showed that, on average, approximately half of the surface immobilized anti-human IgG molecules are capable of binding to human IgG during its incubation. From the consecutive binding experiments it could also be concluded that antibodies present in the polyclonal anti-human IgG preparation were capable of binding to around four different epitopes on the human IgG. A final set of experiments addressed the stability of adsorbed human IgG layers with respect to drying and incubation with surfactant. The results revealed that the adsorbed antibody layer is relatively resistant to these treatments., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Friction force spectroscopy (FFS) has been applied to study the tribological properties of β- and κ-casein layers on hydrophobic substrates in aqueous solutions. Nanometer-sized imaging tips were employed. This allowed exerting and determining the high pressures needed to remove the layers and registering the topographic evolution during this process. Both β- and κ-casein layers showed similar and not particularly high initial frictional responses (friction coefficient of ∼1 when measured with a silicon nitride tip). The pressures needed to remove the layers were of the same order of magnitude for both proteins, ∼10(8) Pa, but slightly higher for those composed of β-casein. The technique has also shown to be useful in studying the two-dimensional lateral diffusion of the proteins and the wear on the layers they form.

The objective of the investigation was to study how different surface cleaning procedures affect the subsequent adsorption of bovine serum albumin (BSA) on silica. Ellipsometry was used to monitor the adsorbed amount and thickness of the protein films in situ at physiological buffer conditions. Plasma treatment was found to effectively reduce the amount of BSA on silica and on surfaces cleaned with hydrogen peroxide solutions (RCA method) adsorption was essentially absent. On clean silica surfaces the ellipsometric data furthermore suggest that a depletion zone is present at the surface where the BSA concentration is lower than in the bulk solution. Additional experiments showed that calcium did not promote the adsorption of BSA and that the adsorption of human serum albumin (HSA) is essentially absent on clean silica surfaces in agreement with the results obtained for BSA. The presented work contributes to the general understanding of protein adsorption as well as provides information on how to control serum albumin adsorption on silica based biomedical devices., (Copyright 2009 Elsevier Inc. All rights reserved.)

Water sorption isotherms of proteins are usually interpreted with such models as BET or GAB that imply the formation of multilayers at solid-gas interface. However, this approach is not applicable to globular proteins such as humid lysozyme where a solid-gas interface does not exist. Another popular approach is the D'Arcy-Watt model, where besides the formation of multilayers the heterogeneity of energies of sorption sites of proteins is taken into account. Here we present sorption calorimetric data on the hydration of lysozyme that confirms the existence of the heterogeneity. The magnitude of the heterogeneity is, however, lower than one can expect on the basis of the existence of a solid-gas interface. Moreover, the calorimetric data show a strong enthalpy-entropy compensation that leads to almost constant effective free energy of hydration in the activity range normally used for fitting the data to sorption models. This allows the use of the Langmuir equation for the fitting of the initial part of the sorption isotherm of lysozyme. Assuming the formation of a monolayer of water at the protein-protein interface, one can estimate the size of the lysozyme molecules from the sorption isotherm. The result of this estimation is in good agreement with the structural data on lysozyme, which supports the presented approach.

A method to coat hydrophobic surfaces with lipid molecules in a reproducible manner and in which the lipid molecules are resistant to detergent washings, would benefit the development of new ELISA assays. This work presents different approaches to build 1,2-dioleolyl-sn-glycero-3-phosphocholine (DOPC) layers doped with a monosialoganglioside (GM1) supported on silica surfaces, which are stable toward buffer rinsing and washing with surfactant (Tween 20). The three methods employed were: method 1, coadsorption of DOPC:GM1 (0-10 mol%) with the surfactant n-dodecyl-beta-D-maltoside (DDM) from micellar solutions, with successive adsorption and rinsing steps; method 2, vesicle fusion from DOPC: GM1 (0-10 mol%) liposomes; and method 3, deposition of GM1 from organic solvent (chloroform) and exposure to an aqueous environment (hydration method). The vesicle fusion method was also tested in polystyrene surfaces. Cholera toxin subunit B (CTB) was used to detect the presence of GM1 on the formed layers. The results indicated that the vesicle fusion was the only method that was successful in creating stable mono- and bilayers onto hydrophobized and hydrophilic silica, respectively. The mixed micellar solution method was suitable for creating pure lipid (DOPC) monolayers but the incorporation of GM1 in the micelles led to monolayers which were very unstable with respect to buffer rinsing. The hydration method led to monolayers of GM1 that were partly rinsed off by a continuous buffer flow. Adsorption of CTB was found to be proportional to the amount of GM1 present in the liposomes. The amount of CTB adsorbed onto the lipid bilayers was roughly the double as the one determined on the monolayers with the same liposome compositions. The vesicle fusion method was also able to create monolayers of pure DOPC and DOPC:10 mol% GM1 on the polystyrene surfaces.

The hydration of two different polymorphs of microcrystalline cellulose (cellulose I and II), as well as the hydration of amorphous cellulose was studied using water sorption calorimetry, gravimetric water vapor sorption, nitrogen sorption, and X-ray powder diffraction. Amorphous cellulose was prepared by means of ball-milling of microcrystalline cellulose (MCC). Whereas X-ray data showed the untreated MCC to consist of cellulose I, the amorphous cellulose was found to recrystallize into cellulose II after contact with water or water vapor at relative humidities (RHs) above 90%. Sorption isotherms show an increase of water sorption in the sequence cellulose I

Films formed from saliva on surfaces are important for the maintenance of oral health and integrity by protection against chemical and/or biological agents. The aim of the present study was to investigate adsorbed amounts, thickness, and structure of films formed from human whole saliva on alumina surfaces by means of in situ ellipsometry, neutron reflectivity, and atomic force microscopy. Alumina (Al2O3, synthetic sapphire) is a relevant and interesting substrate for saliva adsorption studies as it has an isoelectric point close to that of tooth enamel. The results showed that saliva adsorbs rapidly on alumina. The film could be modeled in two layers: an inner and dense thin region that forms a uniform layer and an outer, more diffuse and thicker region that protrudes toward the bulk of the solution. The film morphology described a uniformly covering dense layer and a second outer layer containing polydisperse adsorbed macromolecules or aggregates.

Structural properties of the acidic proline rich protein PRP-1 of salivary origin in bulk solution and adsorbed onto a negatively charged surface have been studied by Monte Carlo simulations. A simple model system with focus on electrostatic interactions and short-ranged attractions among the uncharged amino acids has been used. In addition to PRP-1, some mutants were considered to assess the role of the interactions in the systems. Contrary to polyelectrolytes, the protein has a compact structure in salt-free bulk solutions, whereas at high salt concentration the protein becomes more extended. The protein adsorbs to a negatively charged surface, although its net charge is negative. The adsorbed protein displays an extended structure, which becomes more compact upon addition of salt. Hence, the conformational response upon salt addition in the adsorbed state is the opposite as compared to that in bulk solution. The conformational behavior of PRP-1 in bulk solution and at charged surfaces as well as its propensity to adsorb to surfaces with the same net charge are rationalized by the block polyampholytic character of the protein. The presence of a triad of positively charged amino acids in the C-terminal was found to be important for the adsorption of the protein.

We have studied hydration (and dehydration) of thermally denatured hen egg lysozyme using sorption calorimetry. Two different procedures of thermal denaturation of lysozyme were used. In the first procedure the protein was denatured in an aqueous solution at 90 degrees C, in the other procedure a sample that contained 20% of water was denatured at 150 degrees C. The protein denatured at 90 degrees C showed very similar sorption behavior to that of the native protein. The lysozyme samples denatured at 150 degrees C were studied at several temperatures in the range of 25-60 degrees C. In the beginning of sorption, the sorption isotherms of native and denatured lysozyme are almost identical. At higher water contents, however, the denatured lysozyme can absorb a greater amount of water than the native protein due to the larger number of available sorption sites. Desorption experiments did not reveal a pronounced hysteresis in the sorption isotherm of denatured lysozyme (such hysteresis is typical for native lysozyme). Despite the unfolded structure, the denatured lysozyme binds less water than does the native lysozyme in the desorption experiments at water contents up to 34 wt %. Glass transitions in the denatured lysozyme were observed using both differential scanning calorimetry and sorption calorimetry. Partial molar enthalpy of mixing of water in the glassy state is strongly exothermic, which gives rise to a positive temperature dependence of the water activity. The changes of the free energy of the protein induced by the hydration stabilize the denatured form of lysozyme with respect to the native form.