MUC5B mucin films under mechanical confinement: A combined neutron reflectometry and atomic force microscopy study.

HYPOTHESIS: Among other functions, mucins hydrate and protect biological interfaces from mechanical challenges. Mucins also attract interest as biocompatible coatings with excellent lubrication performance. Therefore, it is of high interest to understand the structural response of mucin films to mechanical challenges. We hypothesized that this could be done with Neutron Reflectometry using a novel sample environment where mechanical confinement is achieved by inflating a membrane against the films. EXPERIMENTS: Oral MUC5B mucin films were investigated by Force Microscopy/Spectroscopy and Neutron Reflectometry both at solid-liquid interfaces and under mechanical confinement. FINDINGS: NR indicated that MUC5B films were almost completely compressed and dehydrated when confined at 1 bar. This was supported by Force Microscopy/Spectroscopy investigations. Force Spectroscopy also indicated that MUC5B films could withstand mechanical confinement by means of steric interactions for pressures lower than âˆ¼ 0.5 bar i.e., mucins could protect interfaces from mechanical challenges of this magnitude while keeping them hydrated. To investigate mucin films under these pressures by means of the employed sample environment for NR, further technological developments are needed. The most critical would be identifying or developing more flexible membranes that would still meet certain requirements like chemical homogeneity and very low roughness.

Effect of nonionic and amphoteric surfactants on salivary pellicles reconstituted in vitro.

Surfactants are important components of oral care products. Sodium dodecyl sulfate (SDS) is the most common because of its foaming properties, taste and low cost. However, the use of ionic surfactants, especially SDS, is related to several oral mucosa conditions. Thus, there is a high interest in using non-ionic and amphoteric surfactants as they are less irritant. To better understand the performance of these surfactants in oral care products, we investigated their interaction with salivary pellicles i.e., the proteinaceous films that cover surfaces exposed to saliva. Specifically, we focused on pentaethylene glycol monododecyl ether (C12E5) and cocamidopropyl betaine (CAPB) as model nonionic and amphoteric surfactants respectively, and investigated their interaction with reconstituted salivary pellicles with various surface techniques: Quartz Crystal Microbalance with Dissipation, Ellipsometry, Force Spectroscopy and Neutron Reflectometry. Both C12E5 and CAPB were gentler on pellicles than SDS, removing a lower amount. However, their interaction with pellicles differed. Our work indicates that CAPB would mainly interact with the mucin components of pellicles, leading to collapse and dehydration. In contrast, exposure to C12E5 had a minimal effect on the pellicles, mainly resulting in the replacement/solubilisation of some of the components anchoring pellicles to their substrate.

A comparison between the structures of reconstituted salivary pellicles and oral mucin (MUC5B) films.

HYPOTHESIS: Salivary pellicles i.e., thin films formed upon selective adsorption of saliva, protect oral surfaces against chemical and mechanical insults. Pellicles are also excellent aqueous lubricants. It is generally accepted that reconstituted pellicles have a two-layer structure, where the outer layer is mainly composed of MUC5B mucins. We hypothesized that by comparing the effect of ionic strength on reconstituted pellicles and MUC5B films we could gain further insight into the pellicle structure. EXPERIMENTS: Salivary pellicles and MUC5B films reconstituted on solid surfaces were investigated at different ionic strengths by Force Spectroscopy, Quartz Crystal Microbalance with Dissipation, Null Ellipsometry and Neutron Reflectometry. FINDINGS: Our results support the two-layer structure for reconstituted salivary pellicles. The outer layer swelled when ionic strength decreased, indicating a weak polyelectrolyte behavior. While initially the MUC5B films exhibited a similar tendency, this was followed by a drastic collapse indicating an interaction between exposed hydrophobic domains. This suggests that mucins in the pellicle outer layer form complexes with other salivary components that prevent this interaction. Lowering ionic strength below physiological values also led to a partial removal of the pellicle inner layer. Overall, our results highlight the importance that the interactions of mucins with other pellicle components play on their structure.

Sensing by wireless reading Ag/AgCl redox conversion on RFID tag: universal, battery-less biosensor design.

Massive integration of biosensors into design of Internet-of-Things (IoT) is vital for progress of healthcare. However, the integration of biosensors is challenging due to limited availability of battery-less biosensor designs. In this work, a combination of nanomaterials for wireless sensing of biological redox reactions is described. The design exploits silver nanoparticles (AgNPs) as part of the RFID tag antenna. We demonstrate that a redox enzyme, particularly, horseradish peroxidase (HRP), can convert AgNPs into AgCl in the presence of its substrate, hydrogen peroxide. This strongly changes the impedance of the tag. The presented example exploits gold nanoparticle (AuNP)-assisted electron transfer (ET) between AgNPs and HRP. We show that AuNP is a vital intermediate for establishing rapid ET between the enzyme and AgNPs. As an example, battery-less biosensor-RFID tag designs for H2O2 and glucose are demonstrated. Similar battery-less sensors can be constructed to sense redox reactions catalysed by other oxidoreductase enzymes, their combinations, bacteria or other biological and even non-biological catalysts. In this work, a fast and general route for converting a high number of redox reaction based sensors into battery-less sensor-RFID tags is described.

Time-resolved small-angle neutron scattering as a probe for the dynamics of lipid exchange between human lipoproteins and naturally derived membranes.

Atherosclerosis is the main killer in the western world. Today's clinical markers include the total level of cholesterol and high-/low-density lipoproteins, which often fails to accurately predict the disease. The relationship between the lipid exchange capacity and lipoprotein structure should explain the extent by which they release or accept lipid cargo and should relate to the risk for developing atherosclerosis. Here, small-angle neutron scattering and tailored deuteration have been used to follow the molecular lipid exchange between human lipoprotein particles and cellular membrane mimics made of natural, "neutron invisible" phosphatidylcholines. We show that lipid exchange occurs via two different processes that include lipid transfer via collision and upon direct particle tethering to the membrane, and that high-density lipoprotein excels at exchanging the human-like unsaturated phosphatidylcholine. By mapping the specific lipid content and level of glycation/oxidation, the mode of action of specific lipoproteins can now be deciphered. This information can prove important for the development of improved diagnostic tools and in the treatment of atherosclerosis.

Impact of molecular linker size on physicochemical properties of assembled gold nanoparticle mono-/multi-layers and their applicability for functional binding of biomolecules.

In this work the impact of molecular inter-linker size on gold nanoparticle (AuNP) mono-/multilayer structural properties, density and homogeneity has been investigated. These characteristics are of great importance for functional binding of biomolecules. Positively charged high or low molecular weight inter-linkers, poly-L-lysine (PLL) or N-(6-mercapto)hexylpyridinium (MHP), were used to attach negatively charged AuNPs on a planar gold surface as well as to further interlink into a multilayer structure via layer-by-layer deposition. The inter-particle interaction within the assembled AuNP films was adjusted by the ionic strength in the AuNPs dispersions. The AuNP layer density and structural/viscoelastic properties were evaluated by the quartz crystal microbalance with dissipation (QCM-D) technique. The validity of the commercial Voigt model, specifically developed for quantitative QCM-D data analysis of homogeneous viscoelastic films, was evaluated by a model independent analysis when comparing the assembled AuNP films with a homogeneous layer of a mucin from bovine submaxillary glands. Both AuNP mono- and multilayers, attached/interlinked via long flexible PLL molecules assembled to denser and more soft/viscous structures compared to those interlinked by short MHP compounds. Thus, PLL-interlinked AuNP mono-/multilayer structures were further investigated as a platform for laccase enzyme functional adsorption via qualitative assessment of bioelectrochemical characteristics of the enzyme.

Effects of Hydration on Structure and Phase Behavior of Pig Gastric Mucin Elucidated by SAXS.

In this work small-angle X-ray scattering (SAXS) was used to study hydration and temperature-induced changes of pig gastric mucin (PGM) within the entire concentration range. The scattering is interpreted as originating from PGM fiber-like structures that adopt rod-like bottle-brush conformation in dilute solutions. On the basis of the knowledge about molecular structure of mucins and SAXS data for dilute solutions, we propose a theoretical model for predicting mucin conformation in solution and calculate the corresponding scattering profile. This bottle-brush model comprises a protein backbone with carbohydrate side chains and corresponding structural parameters, such as grafting distance and lengths of the backbone and side chains. It describes the experimental PGM data from dilute solutions in the full q range very well. It furthermore suggests that the carbohydrate side chains are grafted with a regular separation of around 5 nm and a length of 14 nm. The cross-section size with a radius of about 1 nm is also in accordance with the size of the carbohydrate units. Structuring of PGM solutions at higher concentrations was investigated by analyzing semidilute and concentrated PGM samples. Starting at about 20 wt %, Bragg peaks become clearly visible indicating a more ordered mucin system. In very dehydrated and fully dry mucin samples these peaks are not present indicating lack of long-range order. The SAXS data show that the structural change occurring at about 80 wt % mucin and 25 °C corresponds to a glass transition in agreement with our previous calorimetric results. Temperature also has an effect on the phase behavior of mucin. At intermediate levels of hydration, a phase transition is observed at about 60-70 °C. The main Bragg peak appears to split in two, indicating formation of a different structure at elevated temperatures. These findings are used to improve the PGM-water phase diagram.

Reversible Self-Assembled Monolayers (rSAMs) as Robust and Fluidic Lipid Bilayer Mimics.

Lipid bilayers, forming the outer barrier of cells, display a wide array of proteins and carbohydrates for modulating interfacial biological interactions. Formed by the spontaneous self-assembly of lipid molecules, these bilayers feature liquid crystalline order, while retaining a high degree of lateral mobility. Studies of these dynamic phenomena have been hampered by the fragility and instability of corresponding biomimetic cell membrane models. Here, we present the construct of a series of oligoethylene glycol-terminated reversible self-assembled monolayers (rSAMs) featuring lipid-bilayer-like fluidity, while retaining air and protein stability and resistance. These robust and ordered layers were prepared by simply immersing a carboxylic acid-terminated self-assembled monolayer into 5-50 µM aqueous ω-(4-ethylene glycol-phenoxy)-α-(4-amidinophenoxy)decane solutions. It is anticipated that this new class of robust and fluidic two-dimensional biomimetic surfaces will impact the design of rugged cell surface mimics and high-performance biosensors.

Reversible Self-Assembled Monolayers (rSAMs): Adaptable Surfaces for Enhanced Multivalent Interactions and Ultrasensitive Virus Detection.

We report on the design of pH-switchable monolayers allowing a reversible and ordered introduction of affinity reagents on sensor surfaces. The principal layer building blocks consist of α-(4-amidinophenoxy)alkanes decorated at the ω-position with affinity ligands. These spontaneously self-assemble on top of carboxylic acid terminated SAMs to form reversible homo or mixed monolayers (rSAMs) that are tunable with respect to the nature of the head group, layer order and stability while featuring pH responsiveness and the dynamic nature of noncovalent build assemblies. We show that this results in a range of unique biosensor features. As a first example a sialic acid rSAM featuring strong lectin affinity is here used to sense hemagglutinin and influenza virus (H5N1) at the pM and fM level by in situ ellipsometry in a fully reversible fashion. We believe that the rSAM concept will find widespread use in surface chemistry and overall for boosting sensitivity in affinity biosensors.

Ex-Vivo Force Spectroscopy of Intestinal Mucosa Reveals the Mechanical Properties of Mucus Blankets.

Mucus is the viscous gel that protects mucosal surfaces. It also plays a crucial role in several diseases as well as in mucosal drug delivery. Because of technical limitations, mucus properties have mainly been addressed by in-vitro studies. However, this approach can lead to artifacts as mucus collection can alter its structure. Here we show that by using an implemented atomic force microscope it is possible to measure the interactions between micro-particles and mucus blankets ex-vivo i.e., on fresh excised mucus-covered tissues. By applying this method to study the small intestine, we were able to quantify the stiffness and adhesiveness of its mucus blanket at different pH values. We also demonstrate the ability of mucus blankets to bind and attract particles hundreds of µm away from their surface, and to trap and bury them even if their size is as big as 15 µm.

Poly-l-lysine/heparin multilayer coatings prevent blood protein adsorption.

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 0.4mg/m2 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.

Structural and Mechanical Properties of Thin Films of Bovine Submaxillary Mucin versus Porcine Gastric Mucin on a Hydrophobic Surface in Aqueous Solutions.

The structural and mechanical properties of thin films generated from two types of mucins, namely, bovine submaxillary mucin (BSM) and porcine gastric mucin (PGM) in aqueous environment were investigated with several bulk and surface analytical techniques. Both mucins generated hydrated films on hydrophobic polydimethylsiloxane (PDMS) surfaces from spontaneous adsorption arising from their amphiphilic characteristic. However, BSM formed more elastic films than PGM at neutral pH condition. This structural difference was manifested from the initial film formation processes to the responses to shear stresses applied to the films. Acidification of environmental pH led to strengthening the elastic character of BSM films with increased adsorbed mass, whereas an opposite trend was observed for PGM films. We propose that this contrast originates from that negatively charged motifs are present for both the central and terminal regions of BSM molecule, whereas a similar magnitude of negative charges is localized at the termini of PGM molecule. Given that hydrophobic motifs acting as an anchor are also localized in the terminal region, electrostatic repulsion between anchoring units of PGM molecules on a nonpolar PDMS surface leads to weakening of the mechanical integrity of the films.

Diffusion through Pig Gastric Mucin: Effect of Relative Humidity.

Mucus covers the epithelium found in all intestinal tracts, where it serves as an important protecting barrier, and pharmaceutical drugs administrated by the oral, rectal, vaginal, ocular, or nasal route need to penetrate the mucus in order to reach their targets. Furthermore, the diffusion in mucus as well as the viscosity of mucus in the eyes, nose and throat can change depending on the relative humidity of the surrounding air. In this study we have investigated how diffusion through gels of mucin, the main protein in mucus, is affected by changes in ambient relative humidity (i.e. water activity). Already a small decrease in water activity was found to give rise to a significant decrease in penetration rate through the mucin gel of the antibacterial drug metronidazole. We also show that a decrease in water activity leads to decreased diffusion rate in the mucin gel for the fluorophore fluorescein. This study shows that it is possible to alter transport rates of molecules through mucus by changing the water activity in the gel. It furthermore illustrates the importance of considering effects of the water activity in the mucosa during development of potential pharmaceuticals.

In Situ Potentiometry and Ellipsometry: A Promising Tool to Study Biofouling of Potentiometric Sensors.

In situ potentiometry and null ellipsometry was combined and used as a tool to follow the kinetics of biofouling of ion-selective electrodes (ISEs). The study was performed using custom-made solid-contact K(+)-ISEs consisting of a gold surface with immobilized 6-(ferrocenyl)hexanethiol as ion-to-electron transducer that was coated with a potassium-selective plasticized polymer membrane. The electrode potential and the ellipsometric signal (corresponding to the amount of adsorbed protein) were recorded simultaneously during adsorption of bovine serum albumin (BSA) at the surface of the K(+)-ISEs. This in situ method may become useful in developing sensors with minimized biofouling.

Interaction of Polyelectrolytes with Salivary Pellicles on Hydroxyapatite Surfaces under Erosive Acidic Conditions.

The modification of acidic beverage formulations with food-approved, nonhazardous substances with antierosive properties has been identified as a key strategy for counteracting the prevalence of dental erosion, i.e., the acid-induced dissolution of hydroxyapatite (HA, the main mineral component of tooth surfaces). While many of such substances have been reported, very little is known on how they interact with teeth and inhibit their acid-induced dissolution. With the aim of filling this gap in knowledge, we have studied under acidic conditions the interaction between two polyelectrolytes of differing ionic character, carboxymethyl cellulose (CMC) and chitosan, and saliva-coated hydroxyapatite, i.e., a model for the outer surface of teeth. These studies were performed by means of ellipsometry, quartz crystal microbalance with dissipation monitoring, and atomic force microscopy. We also studied, by means of pH variations, how dissolution of saliva-coated HA is affected by including these polyelectrolytes in the erosive solutions. Our results confirm that salivary films protect HA from acid-induced dissolution, but only for a limited time. If the acid is modified with CMC, this polyelectrolyte incorporates into the salivary films prolonging in time their protective function. Eventually, the CMC-modified salivary films are removed from the HA surfaces. From this moment, HA is continuously coated with CMC, but this offers only a weak protection against erosion. When the acid is modified with the cationic chitosan, the polyelectrolyte adsorbs on top of the salivary films. Chitosan-modified salivary films are also eventually replaced by bare chitosan films. In this case both coatings offer a similar protection against HA dissolution, which is nevertheless notably higher than that offered by CMC.

Anti-adherence and bactericidal activity of sphingolipids against Streptococcus mutans.

This study evaluated the anti-biofilm activity of sphingosine, phytosphingosine (PHS), and sphinganine for: (i) anti-adherence activity on hydroxyapatite (HA) surfaces; and (ii) bactericidal activity on different Streptococcus mutans phenotypes (i.e. planktonic cells and cells from a disrupted biofilm). For this, HA discs treated with sphingolipids were incubated with S. mutans and the number of adherent cells was evaluated by both culture and confocal microscopy. Sphinganine strongly inhibited bacterial adherence by 1000-fold compared with an untreated surface. Phytosphingosine and sphingosine inhibited bacterial adherence by eight- and five-fold, respectively, compared with an untreated surface. On saliva-coated HA, sphinganine and PHS inhibited bacterial adherence by 10-fold. Bactericidal activity of sphingolipids was evaluated by culture. For biofilms, the strongest bactericidal activity was exhibited by sphingosine compared with PHS and sphinganine. At a concentration of 12.5 µg ml(-1) , PHS and sphingosine were profoundly effective against planktonic and disrupted biofilms; and sphinganine reduced the number of cells in planktonic form by 100-fold and those derived from a disrupted biofilm by 1000-fold. Atomic force microscopy studies suggested that mechanical stability does not appear to be a factor relevant for anti-fouling activity. The results suggest that sphingolipids may be used to control oral biofilms, especially those loaded with S. mutans.

Textile-based sampling for potentiometric determination of ions.

Potentiometric sensing utilizing textile-based micro-volume sampling was applied and evaluated for the determination of clinically (Na(+), K(+), Cl(-)) and environmentally (Cd(2+), Pb(2+) and pH) relevant analytes. In this technological design, calibration solutions and samples were absorbed into textiles while the potentiometric cells (ion-selective electrodes and reference electrode) were pressed against the textile. Once the liquid, by wicking action, reached the place where the potentiometric cell was pressed onto the textile, hence closing the electric circuit, the potentiometric response was obtained. Cotton, polyamide, polyester and their blends with elastane were applied for micro-volume sampling. The textiles were found to influence the determination of pH in environmental samples with pH close to neutral and Pb(2+) at low analyte concentrations. On the other hand, textile-based micro-volume sampling was successfully applied in measurements of Na(+) using solid-contact sodium-selective electrodes utilizing all the investigated textiles for sampling. It was found that in order to extend the application of textile-based sampling toward environmental analysis of ions it will be necessary to tailor the physio-chemical properties of the textile materials. In general, textile-based sampling opens new possibilities for direct chemical analysis of small-volume samples and provide a simple and low-cost method to screen various textiles for their effects on samples to identify which textiles are the most suitable for on-body sensing.

Affinity states of biocides determine bioavailability and release rates in marine paints.

A challenge for the next generation marine antifouling (AF) paints is to deliver minimum amounts of biocides to the environment. The candidate AF compound medetomidine is here shown to be released at very low concentrations, ie ng ml(-1) day(-1). Moreover, the release rate of medetomidine differs substantially depending on the formulation of the paint, while inhibition of barnacle settlement is independent of release to the ambient water, ie the paint with the lowest release rate was the most effective in impeding barnacle colonisation. This highlights the critical role of chemical interactions between biocide, paint carrier and the solid/aqueous interface for release rate and AF performance. The results are discussed in the light of differential affinity states of the biocide, predicting AF activity in terms of a high surface affinity and preserved bioavailability. This may offer a general framework for the design of low-release paint systems using biocides for protection against biofouling on marine surfaces.

Sphingoid bases inhibit acid-induced demineralization of hydroxyapatite.

Calcium hydroxyapatite (HAp), the main constituent of dental enamel, is inherently susceptible to the etching and dissolving action of acids, resulting in tooth decay such as dental caries and dental erosion. Since the prevalence of erosive wear is gradually increasing, there is urgent need for agents that protect the enamel against erosive attacks. In the present study we studied in vitro the anti-erosive effects of a number of sphingolipids and sphingoid bases, which form the backbone of sphingolipids. Pretreatment of HAp discs with sphingosine, phytosphingosine (PHS), PHS phosphate and sphinganine significantly protected these against acid-induced demineralization by 80 ± 17%, 78 ± 17%, 78 ± 7% and 81 ± 8%, respectively (p < 0.001). On the other hand, sphingomyelin, acetyl PHS, octanoyl PHS and stearoyl PHS had no anti-erosive effects. Atomic force measurement revealed that HAp discs treated with PHS were almost completely and homogeneously covered by patches of PHS. This suggests that PHS and other sphingoid bases form layers on the surface of HAp, which act as diffusion barriers against H(+) ions. In principle, these anti-erosive properties make PHS and related sphingosines promising and attractive candidates as ingredients in oral care products.

Influence of substratum hydrophobicity on salivary pellicles: organization or composition?

Different physico-chemical properties (eg adsorption kinetics, thickness, viscoelasticity, and mechanical stability) of adsorbed salivary pellicles depend on different factors, including the properties (eg charge, roughness, wettability, and surface chemistry) of the substratum. Whether these differences in the physico-chemical properties are a result of differences in the composition or in the organization of the pellicles is not known. In this work, the influence of substratum wettability on the composition of the pellicle was studied. For this purpose, pellicles eluted from substrata of different but well-characterized wettabilities were examined by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that substratum hydrophobicity did not have a major impact on pellicle composition. In all substrata, the major pellicle components were found to be cystatins, amylases and large glycoproteins, presumably mucins. In turn, interpretation of previously reported data based on the present results suggests that variations in substratum wettability mostly affect the organization of the pellicle components.