Patrick A. Rühs

Simultaneous control of pH and ionic strength during interfacial rheology of β-lactoglobulin fibrils adsorbed at liquid/liquid Interfaces.

Proteins can aggregate as amyloid fibrils under denaturing and destabilizing conditions such as low pH (2) and high temperature (90 °C). Fibrils of β-lactoglobulin are surface active and form adsorption layers at fluid-fluid interfaces. In this study, β-lactoglobulin fibrils were adsorbed at the oil-water interface at pH 2. A shear rheometer with a bicone geometry set up was modified to allow subphase exchange without disrupting the interface, enabling the investigation of rheological properties after adsorption of the fibrils, as a function of time, different pH, and ionic strength conditions. It is shown that an increase in pH (2 to 6) leads to an increase of both the interfacial storage and loss moduli. At the isoelectric point (pH 5-6) of β-lactoglobulin fibrils, the maximum storage and loss moduli are reached. Beyond the isoelectric point, by further increasing the pH, a decrease in viscoelastic properties can be observed. Amplitude sweeps at different pH reveal a weak strain overshoot around the isoelectric point. With increasing ionic strength, the moduli increase without a strain overshoot. The method developed in this study allows in situ subphase exchange during interfacial rheological measurements and the investigation of interfacial ordering.

Shear and dilatational linear and nonlinear subphase controlled interfacial rheology of β-lactoglobulin fibrils and their derivatives

This work presents a linear and nonlinear interfacial rheological characterization of viscoelastic protein adsorption layers formed by β-lactoglobulin fibrils, β-lactoglobulin peptides, and native β-lactoglobulin (called monomers) at the water–oil interface at pH 2. The fibril and peptide solution presented a similar surface density, whereas β-lactoglobulin monomers lower the interfacial tension more efficiently. The interfacial tension/dilatational rheology response to drop area amplitude sweeps showed pronounced differences, as the β-lactoglobulin fibrils and monomer react nonlinear at high frequencies and area strains, an effect not observed for β-lactoglobulin peptides. Step strain experiments in combination with frequency sweeps present the material response: In the low frequency regime, β-lactoglobulin peptides and β-lactoglobulin monomers can be characterized by the behavior of irreversibly adsorbed molecules. At high frequencies, both peptides and monomers behaved like reversibly adsorbed molecule...

In-Situ Quantification of the Interfacial Rheological Response of Bacterial Biofilms to Environmental Stimuli

Understanding the numerous factors that can affect biofilm formation and stability remain poorly understood. One of the major limitations is the accurate measurement of biofilm stability and cohesiveness in real-time when exposed to changing environmental conditions. Here we present a novel method to measure biofilm strength: interfacial rheology. By culturing a range of bacterial biofilms on an air-liquid interface we were able to measure their viscoelastic growth profile during and after biofilm formation and subsequently alter growth conditions by adding surfactants or changing the nutrient composition of the growth medium. We found that different bacterial species had unique viscoelastic growth profiles, which was also highly dependent on the growth media used. We also found that we could reduce biofilm formation by the addition of surfactants or changing the pH, thereby altering the viscoelastic properties of the biofilm. Using this technique we were able to monitor changes in viscosity, elasticity and surface tension online, under constant and varying environmental conditions, thereby providing a complementary method to better understand the dynamics of both biofilm formation and dispersal.

Bridging the gap between the nanostructural organization and macroscopic interfacial rheology of amyloid fibrils at liquid interfaces.

The interfacial behavior of proteins and protein aggregates such as fibrils influences the bulk behavior of multiphase systems in foods, pharmaceuticals, and other technological applications. Additionally, it is an important factor in some biological processes such as the accumulation of amyloid fibrils at biological membranes in neurodegenerative diseases. Here, using β-lactoglobulin fibrils as a model system, we cover a large range of characteristic measuring length scales by combining atomic force microscopy, passive probe particle tracking, tensiometry, interfacial shear, and dilatational rheology in order to correlate the intricate structure of fibril-laden interfaces with their macroscopic adsorption kinetics and viscoelasticity. A subtle change in solution pH provokes pronounced changes in interfacial properties such as alignment, entanglement, multilayer formation, and fibril fracture, which can be resolved and linked across the various length scales involved.

Studying bacterial hydrophobicity and biofilm formation at liquid-liquid interfaces through interfacial rheology and pendant drop tensiometry.

Bacterial adsorption to interfaces is a key factor in biofilm formation. One major limitation to understanding biofilm formation and development is the accurate measurement of bacterial cell adhesion to hydrophobic interfaces. With this study, bacterial attachment and biofilm growth over time at water-oil interface was monitored through interfacial rheology and tensiometry. Five model bacteria (Pseudomonas putida KT2442, Pseudomonas putida W2, Salmonella typhimurium, Escherichia coli, and Bacillus subtilis) were allowed to adsorb at the water-oil interface either in their non-growing or growing state. We found that we were able to observe the initial kinetics of bacterial attachment and the transient biofilm formation at the water-oil interface through interfacial rheology and tensiometry. Electrophoretic mobility measurements and bacterial adhesion to hydrocarbons (BATH) tests were performed to characterize the selected bacteria. To validate interfacial rheology and tensiometry measurements, we monitored biofilm formation utilizing both confocal laser scanning microscopy and light microscopy. Using this combination of techniques, we were able to observe the elasticity and tension development over time, from the first bacterial attachment up to biofilm formation.

Adhesion Potential of Intestinal Microbes Predicted by Physico-Chemical Characterization Methods.

Bacterial adhesion to epithelial surfaces affects retention time in the human gastro-intestinal tract and therefore significantly contributes to interactions between bacteria and their hosts. Bacterial adhesion among other factors is strongly influenced by physico-chemical factors. The accurate quantification of these physico-chemical factors in adhesion is however limited by the available measuring techniques. We evaluated surface charge, interfacial rheology and tensiometry (interfacial tension) as novel approaches to quantify these interactions and evaluated their biological significance via an adhesion assay using intestinal epithelial surface molecules (IESM) for a set of model organisms present in the human gastrointestinal tract. Strain pairs of Lactobacillus plantarum WCFS1 with its sortase knockout mutant Lb. plantarum NZ7114 and Lb. rhamnosus GG with Lb. rhamnosus DSM 20021T were used with Enterococcus faecalis JH2-2 as control organism. Intra-species comparison revealed significantly higher abilities for Lb. plantarum WCSF1 and Lb. rhamnosus GG vs. Lb. plantarum NZ7114 and Lb. rhamnosus DSM 20021T to dynamically increase interfacial elasticity (10−2 vs. 10−3 Pa*m) and reduce interfacial tension (32 vs. 38 mN/m). This further correlated for Lb. plantarum WCSF1 and Lb. rhamnosus GG vs. Lb. plantarum NZ7114 and Lb. rhamnosus DSM 20021T with the decrease of relative hydrophobicity (80–85% vs. 57–63%), Zeta potential (-2.9 to -4.5 mV vs. -8.0 to -13.8 mV) and higher relative adhesion capacity to IESM (3.0–5.0 vs 1.5–2.2). Highest adhesion to the IESM collagen I and fibronectin was found for Lb. plantarum WCFS1 (5.0) and E. faecalis JH2-2 (4.2) whereas Lb. rhamnosus GG showed highest adhesion to type II mucus (3.8). Significantly reduced adhesion (2 fold) to the tested IESM was observed for Lb. plantarum NZ7114 and Lb. rhamnosus DSM 20021T corresponding with lower relative hydrophobicity, Zeta potential and abilities to modify interfacial elasticity and tension. Conclusively, the use of Zeta potential, interfacial elasticity and interfacial tension are proposed as suitable novel descriptive and predictive parameters to study the interactions of intestinal microbes with their hosts.

Interfacial localization of nanoclay particles in oil-in-water emulsions and its reflection in interfacial moduli

The localization of nanoclay particles dispersed in the oil phase of a model oil-in-water emulsion depends on the wetting property of layered nanoparticles. Investigation at a single droplet interface shows that nanoclay is located at different interfacial regions depending on the hydrophilic property of the nanoclay surface. Hydrophobic nanoclays do not present Pickering phenomena at the interface and hardly form an interfacial layer. Hydrophilic nanoclay particles quickly move to the interface and form a Pickering interface with a high interfacial shear modulus. With surfactant, poor hydrophilic nanoclays can be located at the interface due to improvement of the wetting behavior caused by the surfactants dissolved in the aqueous continuous phase. With ionic molecules changing the wetting behavior of particles, the interfacial localization of nanoclays can be controlled and improve the mechanical property of emulsion.

Stabilization mechanism of double emulsions made by microfluidics

The stability of double emulsions is crucial for their application as delivery systems and microcapsule templates. However, this stability is often challenged by many molecular species present in customized formulations and by the fast dynamics when microfluidic emulsification processes are used. With the help of designed single emulsion experiments, particle contact angle measurements and interfacial rheology, we investigate the stabilization mechanisms of typical double emulsion formulations containing colloidal particles in the middle oil phase and surfactants in the continuous aqueous phase. In contrast to the inefficient stabilization with conventional surfactants, we find that colloidal particles and surface active polymers are able to quickly form a strong elastic film at the oil–water interface that prevents rupture of the thin fluid separating adjacent droplets, thus providing an efficient means to stabilize double emulsions within the short timescales of microfluidic processes.

3D printing of bacteria into functional complex materials

3D printing of bacteria-laden hydrogels enables the digital fabrication of complex functional materials. Despite recent advances to control the spatial composition and dynamic functionalities of bacteria embedded in materials, bacterial localization into complex three-dimensional (3D) geometries remains a major challenge. We demonstrate a 3D printing approach to create bacteria-derived functional materials by combining the natural diverse metabolism of bacteria with the shape design freedom of additive manufacturing. To achieve this, we embedded bacteria in a biocompatible and functionalized 3D printing ink and printed two types of “living materials” capable of degrading pollutants and of producing medically relevant bacterial cellulose. With this versatile bacteria-printing platform, complex materials displaying spatially specific compositions, geometry, and properties not accessed by standard technologies can be assembled from bottom up for new biotechnological and biomedical applications.

Strong Microcapsules with Permeable Porous Shells Made through Phase Separation in Double Emulsions

Microcapsules for controlled chemical release and uptake are important in many industrial applications but are often difficult to produce with the desired combination of high mechanical strength and high shell permeability. Using water-oil-water double emulsions made in microfluidic devices as templates, we developed a processing route to obtain mechanically robust microcapsules exhibiting a porous shell structure with controlled permeability. The porous shell consists of a network of interconnected polymer particles that are formed upon phase separation within the oil phase of the double emulsion. Porosity is generated by an inert diluent incorporated in the oil phase. The use of undecanol and butanol as inert diluents allows for the preparation of microcapsules covering a wide range of shell-porosity and force-at-break values. We found that the amount and chemical nature of the diluent influence the shell porous structure by changing the mechanism of phase separation that occurs during polymerization. In a proof-of-concept experiment, we demonstrate that the mechanically robust microcapsules prepared through this simple approach can be utilized for the on-demand release of small molecules using a pH change as exemplary chemical trigger.