Protein film formation on cell culture surfaces investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy.

Conventional cell culture surfaces typically consist of polystyrene, with or without surface modifications created through plasma treatment or protein/peptide coating strategies. Other polymers such as fluorinated ethylene propylene are increasingly being implemented in the design of closed cell culture vessels, for example to facilitate the production of cells for cancer immunotherapy. Cultured cells are sensitive to culture vessel material changes through different mechanisms including cell-surface interactions, which are in turn dependent on the amount, type, and conformation of proteins adsorbed on the surface. Here, we investigate the protein deposition from cell culture medium onto untreated polystyrene and fluoropolymer surfaces using quartz crystal microbalance with dissipation monitoring and atomic force microscopy. Both of these non-polar surfaces showed comparable protein deposition kinetics and resulted in similar mechanical and topographical film properties. At protein concentrations found in typical serum-free media used to culture dendritic cells, two deposition phases can be observed. The protein layers form within the first few minutes of contact with the cell culture medium and likely consist almost exclusively of albumin. It is indicated that initial protein film formation will be completed prior to cell settling and initial cell contact will be established with the secondary protein layer. The structural properties of the protein film surface will strongly depend on the albumin concentration in the medium and presumably be less affected by the chemical composition of the cell culture surface.

Evaluating the Cell Membrane Penetration Potential of Lipid-Soluble Compounds Using Supported Phospholipid Bilayers.

Supported phospholipid bilayers (SPBs) are promising models for studying the passive penetration of lipid-soluble compounds into cells and cell membranes. A widely used tool to characterize molecular SPB interactions is the quartz crystal microbalance with dissipation monitoring (QCM-D). As QCM-D provides access to the mass density of supported membranes, it is well-suited to examine surface adsorption and membrane disruption phenomena. In the present study, we report on a novel approach to characterize SPB interactions with low molecular weight lipid-soluble substances. SPBs were formed on a silica-coated QCM-D crystal, exposed to various phenolic compounds (vanillin, gallic acid, and protocatechualdehyde), and subjected to linear temperature variation. While the exposure of the SPBs to the phenolic compounds did not result in detectable mass density changes, we observed noticeable alterations in their gel-fluid phase transitions. It was found that QCM-D can detect small variations in a SPB's main transition temperature (≪1 °C) and further resolve compound-specific lipid interactions. The acoustic sensing technique thus offers great potential for the use of supported membranes as stable and versatile model systems to study the transport of lipid-soluble substances into phospholipid bilayers and to assess their interactions therein.

Partitioning and Accumulation of Perfluoroalkyl Substances in Model Lipid Bilayers and Bacteria.

Perfluoroalkyl substances (PFAS) are ubiquitous and persistent environmental contaminants, yet knowledge of their biological effects and mechanisms of action is limited. The highest aqueous PFAS concentrations are found in areas where bacteria are relied upon for functions such as nutrient cycling and contaminant degradation, including fire-training areas, wastewater treatment plants, and landfill leachates. This research sought to elucidate one of the mechanisms of action of PFAS by studying their uptake by bacteria and partitioning into model phospholipid bilayer membranes. PFAS partitioned into bacteria as well as model membranes (phospholipid liposomes and bilayers). The extent of incorporation into model membranes and bacteria was positively correlated to the number of fluorinated carbons. Furthermore, incorporation was greater for perfluorinated sulfonates than for perfluorinated carboxylates. Changes in zeta potential were observed in liposomes but not bacteria, consistent with PFAS being incorporated into the phospholipid bilayer membrane. Complementary to these results, PFAS were also found to alter the gel-to-fluid phase transition temperature of phospholipid bilayers, demonstrating that PFAS affected lateral phospholipid interactions. This investigation compliments other studies showing that sulfonated PFAS and PFAS with more than seven fluorinated carbons have a higher potential to accumulate within biota than carboxylated and shorter-chain PFAS.

Optimizing Bacteriophage Surface Densities for Bacterial Capture and Sensing in Quartz Crystal Microbalance with Dissipation Monitoring.

Surface immobilized bacteriophages (phages) are increasingly used as biorecognition elements on bacterial biosensors (e.g., on acoustical, electrical, or optical platforms). The phage surface density is a critical factor determining a sensor's bacterial binding efficiencies; in fact, phage surface densities that are too low or too high can result in significantly reduced bacterial binding capacities. Identifying an optimum phage surface density is thus crucial when exploiting the bacteriophages' bacterial capture capabilities in biosensing applications. Herein, we investigated surface immobilization of the Pseudomonas aeruginosa specific E79 (tailed) phage and the Salmonella Typhimurium specific PRD1 (nontailed) phage and their subsequent bacterial capture abilities using quartz crystal microbalance with dissipation monitoring (QCM-D). The QCM-D was used in two experimental setups: (i) a conventional setup and (ii) a combined setup with ellipsometry. Both setups were exploited to link the phages' immobilization behaviors to their bacterium capture efficiency. While E79 displayed characteristic optima in both the mechanical (QCM-D) and the optical (ellipsometry) data that coincided with its specific bacterial capture optimum, no optima were observed during PRD1 immobilization. The characteristic optima suggests that the E79 phage undergoes a surface rearrangement event that changes the hydration state of the phage film, thereby impairing the E79 phage's ability to capture bacteria. However, the absence of such optima during deposition of the nontailed PRD1 phage suggests that other mechanisms may also lead to reduced bacterial capture by surface immobilized bacteriophages.

Toward More Free-Floating Model Cell Membranes: Method Development and Application to Their Interaction with Nanoparticles.

Identifying the mechanisms of nanoparticle (NP) interactions with cell membranes is key to understanding potential NP cytotoxicity and applications as nanocarriers for targeted drug delivery. To elucidate these mechanisms of interaction, supported phospholipid bilayers (SPBs) are commonly used as models of cell membranes. However, SPBs are soft thin films, and, as such, their properties can be significantly affected by the underlying substrate. Free-floating cell membranes would be best modeled by weakly adhered SPBs; thus, we propose a method for tailoring the interfacial interaction of an electrically charged SPB-substrate system based on modulations in the solution chemistry. Using the dissipation signal of the quartz crystal microbalance with dissipation monitoring (QCM-D), we show that the method can be used to tailor SPB-substrate interactions without the loss of its structural integrity. To demonstrate the application of the method, SPBs are exposed to cationic and anionic polystyrene latex NPs. These studies reveal that the bilayer response to the modulations in the interfacial interaction with its underlying substrate can be used as a sensitive tool to probe the integrity of SPBs upon exposure to NPs. As expected, anionic NPs tend to impart no significant damage to the anionic bilayers, whereas cationic NPs can be detrimental to bilayer integrity. This is the first report of a QCM-D based method to probe bilayer integrity following exposure to NPs. Importantly, the degree of SPB interaction with its underlying substrate is shown to be a critical factor in the kinetics of bilayer disruption by cationic NPs, whereby weakly adhered bilayers are prone to significantly faster breakup. Since free-floating cell membranes are better represented by a weakly adhered SPB, the results of this work critically influence paradigms in experimental studies involving SPBs as models for cell membranes.

Direct detection of the gel-fluid phase transition of a single supported phospholipid bilayer using quartz crystal microbalance with dissipation monitoring.

Supported phospholipid bilayers (SPBs) are valuable models for fundamental studies of biological membranes and their interaction with biologically relevant solutes or particles. Herein, we demonstrate the capability of the quartz crystal microbalance with dissipation monitoring (QCM-D) to directly detect the gel-fluid phase transition of a SPB. The approach involves comparison of the frequency response of a bare and a bilayer-coated QCM-D crystal during linear temperature variation. Phase transition results in a change of the resonance frequency that coincides directly with the accompanied change in bilayer thickness detected by ellipsometry. Experiments performed at different heating rates further demonstrate the use of dissipation monitoring to determine the phase transition temperature based on the temperature-induced viscosity changes of the ambient medium in the immediate environment of the bilayer. Unlike other methods, the proposed approach enables precise determination of the phase transition of a SPB without the need for thermal equilibration of the measurement chamber and, thus, has great potential for sensitive detection of structural and/or compositional changes of the bilayer.

Linking aggregation of Aspergillus niger spores to surface electrostatics: a theoretical approach.

The effect of medium pH on conidial aggregation during submerged cultivation of Aspergillus niger is considered to originate from the electrostatic surface properties of the spores. As previously shown, these properties are greatly influenced by the presence of a melanin-containing surface coating covering the outer spore wall layer. The present study was designed to elucidate the impact of such a coating on the spores' surface potential and their electrostatic repulsion under acidic conditions. A Poisson-Boltzmann model was proposed and potential profiles across the surface coating of noninteracting and interacting spores were calculated. The surface potentials thus obtained were in line with the observed pH dependence of the zeta potential. This dependence was consistent with the outcome of aggregation experiments. Apparently contradictory results regarding the zeta potential and the aggregation behavior of the spores were obtained when the ionic strength was varied. However, both of these observations could be explained by the model.

The role of initial spore adhesion in pellet and biofilm formation in Aspergillus niger.

Fungi grow on a great variety of organic and inorganic materials. Colony establishment and growth on solid surfaces require adhesion of spores and hyphae to the substrate, while cell-to-cell interactions among spores and/or hyphae are a prerequisite for the development of three-dimensional mycelial structures such as pellets or biofilms. Surface adherence has been described as a two-step process, comprised of the initial attachment of ungerminated conidia followed by further adhesion of the forming germ tubes and growing hyphae. In the present study, we analyzed the contribution of adhesion of ungerminated spores to pellet and biofilm formation in Aspergillus niger. Mutants deficient in melanin biosynthesis were constructed by the deletion of the alb1 gene, encoding a polyketide synthase essential for pigment biosynthesis. Δalb1 conidia have an altered surface structure and changed physicochemical surface properties. Spore aggregation in liquid culture as well as spore surface attachment differ between the wild type and the mutant in a pH-dependent manner. In liquid culture further pellet formation is unaffected by altered spore-spore interactions, indicating that germ tube and hyphal adherence can compensate for deficiencies in the initial step of spore attachment. In contrast, under conditions promoting adhesion of Δalb1 conidia to polymer surfaces the mutant forms more stable biofilms than the wild type, suggesting that initial spore adhesion supports sessile growth.

On the origin of the electrostatic surface potential of Aspergillus niger spores in acidic environments.

The electrostatic surface potential of fungal spores is generally regarded as potentially influencing spore aggregation and pellet formation in submerged cultures of filamentous fungi. Spores of Aspergillus niger are typically characterized by negative zeta potentials over a wide range of pH values. In this study, this particular behavior is ascribed to the presence of an extensive melanin coating. It is proposed on the basis of zeta potential and pigment extraction experiments that this outermost layer affects the pH-dependent surface potential in two manners: (i) by the addition of negative charges to the spore surface and (ii) by the pH-dependent release of melanin pigment. Chemical analyses revealed that deprotonation of melanin-bound carboxyl groups is most probably responsible for pigment release under acidic conditions. These findings were incorporated into a simple model which has the ability to qualitatively explain the results of zeta potential experiments and, moreover, to provide the basis for quantitative investigations on the role of electrostatics in spore aggregation.

Determination of adhesion between single Aspergillus niger spores in aqueous solutions using an atomic force microscope.

The interaction force between single cells in contact is of high interest in various interdisciplinary fields of biotechnology, for instance, in cultivation or biofilm formation. A method for the determination of adhesion forces between two single Aspergillus niger spores in different aqueous solutions was established in this study. Adhesion force distributions were determined at three different sodium chloride concentrations and two different pH values using an atomic force microscope (AFM). It was pointed out that adhesion data can be described by log-normal density functions, of which corresponding parameters have been estimated. Using the knowledge of distribution shape, the influence of the environmental condition on the mean values of adhesion force could be studied quantitatively. The highest value of 0.95 nN was observed at pH 2.5 and an ionic strength of 0.5 mol L(-1). Decreasing the ionic strength to 0.05 mol L(-1) decreases the adhesion force mean for about 25%. Increasing the pH value to pH 5 at a sodium chloride concentration of 0.154 mol L(-1) entails a decrease of adhesion from 0.88 to 0.56 nN. These results qualitatively agree with the absolute value of the expected surface potential of Aspergillus niger spores, which is much higher at pH 5 and should take more effect at lower concentrations of counterions.