The adsorption of blood proteins, serum albumin (BSA), immunoglobulin G (IgG) and fibrinogen (FGN), onto model SiO2 planar surfaces coated with poly-L-lysine/heparin multilayers (PLL/HEP) has been investigated by means of ellipsometry and quartz crystal microbalance with dissipation. Aiming at the development of low fouling coatings, this study has been focused on the effects that the number of layers and the type of polyelectrolyte present on the topmost layer have on the adsorption of these proteins. The three proteins interact with PLL-ended coatings whereas HEP-ended coatings prevent the adsorption of both BSA and IgG and induce a decrease in the adsorbed amount of FGN, down to 0.4 mg/m(2) for three bilayers, as the number of PLL/HEP bilayers increases. These results suggest that heparin-ended multilayers prevent protein adsorption, which is an indicative of good blood compatibility. As a consequence we propose that PLL/HEP coatings could be used for the development of vascular medical devices. (C) 2016 Elsevier Inc. All rights reserved.

To characterise bioelectrocatalytic oxygen reduction at gold nanoparticles (AuNPs) modified with Trametes hirsuta laccase (ThLc) combined electrochemical and quartz crystal microbalance measurements have been used. The electrodes with different degrees of AuNP-monolayer coverage, theta, have been studied. In every case of theta close to theoretically possible 44 ThLc molecules adsorbed at 22 nm diameter AuNP. The bioelectrocatalytic current was recalculated down to the current at a single AuNR Unexpectedly, the current at a single AuNP was higher when theta was higher. The maximum current reached at a single AuNP was 31.10(-18) A which corresponds to the enzyme turnover (k(cat)) 13 s(-1). This rate is lower than the homogeneous ThLc turnover (190 s(-1)) suggesting partial denaturation of ThLc upon adsorption or that some ThLc are not in DET contact with the electrode surface

The formation of salivary films onto different surfaces relevant in dental research like titania, hydroxyapatite, gold, zirconia, silica, and hydrophobized silica has been studied by means of QCM-D. Human whole saliva (HWS), and sterile filtered HWS (sHWS) both diluted in water to a final concentration of 25% (v/v) were used. Main differences between the salivary films formed from the two saliva types were observed with the help of ΔD vs Δf plots where sHWS samples showed an almost linear adsorption regime for most of the surfaces whereas most of the HWS samples had a marked multi-regime nature indicating that the former ones are homogenous and the later are heterogeneous supporting previous data on a multi-phase adsorption process. The films with highest shear elastic modulus, μ > 105 N m−2, shear viscosity, η ∼ 3 × 10−3 N s m−2, and lowest thickness (∼10 nm) were formed for both types of saliva onto hydroxyapatite and for sHWS on titania. Furthermore, the ratio between the loss, G″, and the storage modulus, G′, indicates that these films have a solid-like behavior (G″/G′ ≤ 0.5). In contrast, for the remaining surfaces the adsorbed films show higher d values and are also characterized by low μ ∼ 104 N m−2, η ∼ 10−3 N s m−2, and by high ratios, G″/G′ > 2, that indicate a fluid like behavior. These observations might be expected to have influence on the lubricating properties of the salivary films. The SDS induced elutability also indicates a different interaction strength and composition of the adsorbed films and is likely associated with the ease by which these surfaces can be cleaned. Our results suggest that, among the relevant materials, zirconia and titania would yield the more lubricious films whereas hydroxyapatite will be the most easily cleaned.

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 kcatapp 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.

We show in this work how systems formed by phosphoproteins on calcium phosphate surfaces can be directly characterized, in real time, in liquid medium, without the need for elution or labeling. Specifically, we show how this is possible by applying three different techniques: ellipsometry, quartz crystal microbalance with dissipation, and atomic force microscopy-based friction force spectroscopy. We apply these techniques to study two different model systems, i.e. those formed upon the adsorption of two model phosphoproteins (κ- and β-casein) on hydroxyapatite (HA) surfaces. Information on the kinetics of adsorption, surface excess, viscoelasticity, water content, thickness of the layers, and protein-surface interaction is provided. Results indicate that both phosphoproteins form homogeneous elastic highly hydrated monolayers on the HA surfaces, the strength of β-casein layers being higher by approximately a factor of 4. Based on the experimental results, models for the conformation of κ- and β-casein molecules adsorbed on HA surfaces are proposed.

In nearly all papers concerning enzyme–nanoparticle based bioelectronic devices, it is stated that the presence of nanoparticles on electrode surfaces per se enhances bioelectrocatalysis, although the reasons for that enhancement are often unclear. Here, we report detailed experimental evidence that neither an overpotential of bioelectrocatalysis, nor direct electron transfer and bioelectrocatalytic reaction rates for an adsorbed enzyme depend on the size of nanoparticles within the range of 20–80 nm, i.e. for nanoparticles that are considerably larger than the enzyme molecules.

We present a novel methodology for the study of proteinaceous films based on the friction force spectroscopy operation mode of the atomic force microscope. It provides information both on the strength at the nanoscale level and on the lateral diffusion properties of these systems. The usefulness of the data generated by this methodology are shown through its application to the study of different types of monolayers of model proteins, as well as to the study of the more complex and heterogeneous salivary films.

According to the amyloid hypothesis, abnormal processing of the β-amyloid precursor protein in Alzheimer's disease patients increases the production of β-amyloid toxic peptides, which, after forming highly aggregated fibrillar structures, lead to extracellular plaques formation, neuronal loss and dementia. However, a great deal of evidence has point to intracellular small oligomers of amyloid peptides, probably transient intermediates in the process of fibrillar structures formation, as the most toxic species. In order to study the amyloid-DNA interaction, we have selected here three different forms of the amyloid peptide: Aβ1-40, Aβ25-35 and a scrambled form of Aβ25-35. Surface Plasmon Resonance was used together with UV-visible spectroscopy, Electrophoresis and Electronic Microscopy to carry out this study. Our results prove that, similarly to the full length Aβ1-42, all conformations of toxic amyloid peptides, Aβ1-40 and Aβ25-35, may bind DNA. In contrast, the scrambled form of Aβ25-35, a non-aggregating and nontoxic form of this peptide, could not bind DNA. We conclude that although the amyloid-DNA interaction is closely related to the amyloid aggregation proneness, this cannot be the only factor which determines the interaction, since small oligomers of amyloid peptides may also bind DNA if their predominant negatively charged amino acid residues are previously neutralized.