Jagoba Iturri

Study of the multilayer assembly and complex formation of poly(diallyldimethylammonium chloride) (PDADMAC) and poly(acrylic acid) (PAA) as a function of pH

The assembly of polyelectrolyte multilayers and the formation of complexes in a solution of poly(diallyldimethylammonium chloride) (PDADMAC) and poly(acrylic acid) (PAA) have been studied as a function of pH. Quartz Crystal Microbalance with Dissipation (QCM-D) in combination with Ellipsometry shows that PAA–PDADMAC multilayers display a supralinear growth pattern at pH 3 with a corresponding change in the frequency response of more than 650 Hz for 17 polyelectrolyte layers. Atomic Force Microscopy (AFM) confirmed the formation of a 200 nm thick film. For pH values higher than 6, assembly is drastically reduced resulting in values below 150 Hz in the QCM-D for the same number of layers. Isothermal Titration Calorimetry (ITC) and Dynamic Light Scattering (DLS) were used to study complex formation. At pH 3, an exothermic heat of complex formation of −2.11 kcal mol−1 was measured. With increasing pH, the heat of complex formation has been seen to decrease. At pH 10, the heat of complex formation tends to be more endothermic and is approximately 0 at pH 13. The addition of urea to PAA at pH 13 resulted in an exothermic heat of complex formation of 1.41 kcal mol−1 during titration with PDADMAC, highlighting the role of hydrogen bonding with water of PAA in governing the interaction between PAA and PDADMAC.

Specific ζ-Potential Response of Layer-by-Layer Coated Colloidal Particles Triggered by Polyelectrolyte Ion Interactions

The zeta-potential of PSS/PAH and PSS/PDADMAC coated silica particles was studied in the presence of ClO4(-) and H2PO4(-) salts. In the presence of ClO4(-), layer-by-layer (LbL) coated silica particles with PDADMAC as the top layer show a reversal in the surface charge with increasing salt concentration but remain positive in phosphate solutions. LbL particles with PAH as the top layer become, however, negative in the presence of H2PO4(-) but retain their positive charge in the presence of ClO4(-). Charge reversal was explained by specific interaction of ClO4(-) ions with the quaternary amine groups and of H2PO4(-) with the primary amines through hydrogen bonding. Atomic force microscopy (AFM) and quartz crystal microbalance with dissipation (QCM-D) were employed to study the corresponding layer stability on planar surfaces.

Bioinspired Orientation-Dependent Friction

Spatular terminals on the toe pads of a gecko play an important role in directional adhesion and friction required for reversible attachment. Inspired by the toe pad design of a gecko, we study friction of polydimethylsiloxane (PDMS) micropillars terminated with asymmetric (spatular-shaped) overhangs. Friction forces in the direction of and against the spatular end were evaluated and compared to friction forces on symmetric T-shaped pillars and pillars without overhangs. The shape of friction curves and the values of friction forces on spatula-terminated pillars were orientation-dependent. Kinetic friction forces were enhanced when shearing against the spatular end, while static friction was stronger in the direction toward the spatular end. The overall friction force was higher in the direction against the spatula end. The maximum value was limited by the mechanical stability of the overhangs during shear. The aspect ratio of the pillar had a strong influence on the magnitude of the friction force, and its contribution surpassed and masked that of the spatular tip for aspect ratios of >2.

Spherical Polyelectrolyte Brushes Constant Zeta Potential with Varying Ionic Strength: An Electrophoretic Study Using a Hairy Layer Approach

The ζ-potential of spherical brushes of poly[2-(methacryloyloxy)ethyl]trimethylammonium chloride (PMETAC) and poly(potassium sulfopropyl methacrylate) (PSPM) was measured as a function of ionic strength. The ζ-potential of PMETAC brushes varies from +30 mV at 10 mM NaCl to +20 mV at 200 mM, and the ζ-potential of PSPM changes from −30 mV to −25 mV, at their respective ionic strengths. This unusual weak dependence of the ζ-potential on ionic strength is quantitatively explained on the basis of the responsiveness of brushes toward changes in the ionic strength as well as taking into account the specific hydrodynamics of the hairy brush solution interface. The electric potential distribution is described in the framework of the Debye–Huckel approximation. An analytical equation describing the dependence of the ζ-potential of polyelectrolyte brushes on ionic strength is provided.

Synchronized cell attachment triggered by photo-activatable adhesive ligands allows QCM-based detection of early integrin binding

The Quartz Crystal Microbalance with dissipation (QCM-D) technique was applied to monitor and quantify integrin-RGD recognition during the early stages of cell adhesion. Using QCM-D crystals modified with a photo-activatable RGD peptide, the time point of presentation of adhesive ligand at the surface of the QCM-D crystal could be accurately controlled. This allowed temporal resolution of early integrin-RGD binding and the subsequent cell spreading process, and their separate detection by QCM-D. The specificity of the integrin-RGD binding event was corroborated by performing the experiments in the presence of soluble cyclicRGD as a competitor, and cytochalasin D as inhibitor of cell spreading. Larger frequency change in the QCM-D signal was observed for cells with larger spread area, and for cells overexpressing integrin αvβ3 upon stable transfection. This strategy enables quantification of integrin activity which, in turn, may allow discrimination among different cell types displaying distinct integrin subtypes and expression levels thereof. On the basis of these findings, we believe the strategy can be extended to other photoactivatable ligands to characterize cell membrane receptors activity, a relevant issue for cancer diagnosis (and prognosis) as other several pathologies.

Investigating cell‐substrate and cell–cell interactions by means of single‐cell‐probe force spectroscopy

Cell adhesion forces are typically a mixture of specific and nonspecific cell‐substrate and cell–cell interactions. In order to resolve these phenomena, Atomic Force Microscopy appears as a powerful device which can measure cell parameters by means of manipulation of single cells. This method, commonly known as cell‐probe force spectroscopy, allows us to control the force applied, the area of interest, the approach/retracting speed, the force rate, and the time of interaction. Here, we developed a novel approach for in situ cantilever cell capturing and measurement of specific cell interactions. In particular, we present a new setup consisting of two different half‐surfaces coated either with recrystallized SbpA bacterial cell surface layer proteins (S‐layers) or integrin binding Fibronectin, on which MCF‐7 breast cancer cells are incubated. The presence of a clear physical boundary between both surfaces benefits for a quick detection of the region under analysis. Thus, quantitative results about SbpA‐cell and Fibronectin‐cell adhesion forces as a function of the contact time are described. Additionally, the importance of the cell spreading in cell–cell interactions has been studied for surfaces coated with two different Fibronectin concentrations: 20 μg/mL (FN20) and 100 μg/mL (FN100), which impact the number of substrate receptors. Microsc. Res. Tech. 80:124–130, 2017.

Stick–Slip Friction of PDMS Surfaces for Bioinspired Adhesives

Friction plays an important role in the adhesion of many climbing organisms, such as the gecko. During the shearing between two surfaces, periodic stick-slip behavior is often observed and may be critical to the adhesion of gecko setae and gecko-inspired adhesives. Here, we investigate the influence of short oligomers and pendent chains on the stick-slip friction of polydimethylsiloxane (PDMS), a commonly used material for bioinspired adhesives. Three different stick-slip patterns were observed on these surfaces (flat or microstructured) depending on the presence or absence of oligomers and their ability to diffuse out of the material. After washing samples to remove any untethered oligomeric chains, or after oxygen plasma treatment to convert the surface to a thin layer of silica, we decouple the contributions of stiffness, oligomers, and pendant chains to the stick-slip behavior. The stick phase is mainly controlled by the stiffness while the amount of untethered oligomers and pendant chains available at the contact interface defines the slip phase. A large amount of oligomers and pendant chains resulted in a large slip time, dominating the period of stick-slip motion.

Effect of the Concentration of Cytolytic Protein Cyt2Aa2 on the Binding Mechanism on Lipid Bilayers Studied by QCM-D and AFM

Bacillus thuringiensis is known by its insecticidal property. The insecticidal proteins are produced at different growth stages, including the cytolytic protein (Cyt2Aa2), which is a bioinsecticide and an antimicrobial protein. However, the binding mechanism (and the interaction) of Cyt2Aa2 on lipid bilayers is still unclear. In this work, we have used quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM) to investigate the interaction between Cyt2Aa2 protein and (cholesterol-)lipid bilayers. We have found that the binding mechanism is concentration dependent. While at 10 μg/mL, Cyt2Aa2 binds slowly on the lipid bilayer forming a compliance protein/lipid layer with aggregates, at higher protein concentrations (100 μg/mL), the binding is fast, and the protein/lipid layer is more rigid including holes (of about a lipid bilayer thickness) in its structure. Our study suggests that the protein/lipid bilayer binding mechanism seems to be carpet-like at low protein concentrations and pore forming-like at high protein concentrations.

Analysis of Responsive Polymer Films Using Surface Acoustic Waves

The Surface Acoustic Wave (SAW) technique is applied for the first time to quantify the properties of a responsive polymer brush layer. Using a single SAW chip, the response of five different brush compositions to several pH changes was monitored in parallel in a single run. These results were compared with QCM-D studies on the same system. SAW exhibited two remarkable advantages against QCM-D: (i) multiplexing capability, which allowed considerable reduction in experimental time and expenses (1/8 reduction of experimental time, 1/5 in the number of chips, and 1/10 in solvent consumption in our case), and (ii) higher sensitivity in both mass and viscosity change than QCM-D (4-5 times higher in our systems). Our results demonstrate the suitability and advantages of the SAW technology for application in polymer science, in particular for the study of the compositional effects in responsive thin layers.

In Vitro Characterization of the Two-Stage Non-Classical Reassembly Pathway of S-Layers

The recombinant bacterial surface layer (S-layer) protein rSbpA of Lysinibacillus sphaericus CCM 2177 is an ideal model system to study non-classical nucleation and growth of protein crystals at surfaces since the recrystallization process may be separated into two distinct steps: (i) adsorption of S-layer protein monomers on silicon surfaces is completed within 5 min and the amount of bound S-layer protein sufficient for the subsequent formation of a closed crystalline monolayer; (ii) the recrystallization process is triggered—after washing away the unbound S-layer protein—by the addition of a CaCl2 containing buffer solution, and completed after approximately 2 h. The entire self-assembly process including the formation of amorphous clusters, the subsequent transformation into crystalline monomolecular arrays, and finally crystal growth into extended lattices was investigated by quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM). Moreover, contact angle measurements showed that the surface properties of S-layers change from hydrophilic to hydrophobic as the crystallization proceeds. This two-step approach is new in basic and application driven S-layer research and, most likely, will have advantages for functionalizing surfaces (e.g., by spray-coating) with tailor-made biological sensing layers.