Ferritin-supported lipid bilayers for triggering the endothelial cell response.

Hybrid nanoassemblies of ferritin and silica-supported lipid bilayers (ferritin-SLBs) have been prepared and tested for the adhesion, spreading and proliferation of retinal microvascular endothelial cells (ECs). Lipid membranes with varying surface charge were obtained by mixing cationic 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC) with zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at increasing POPC/POEPC ratios. The supported bilayer formation and their subsequent interaction processes with ferritin were studied at the pH of 7.4 at different protein concentrations, by using the quartz crystal microbalance with dissipation monitoring and by atomic force microscopy. Both kinetics and viscoelastic parameters of the protein-lipid membrane interface were scrutinized, as well as surface coverage. Phase-contrast optical microscopy analyses of the ferritin-SLBs substrates after their interaction with endothelial cells evidenced the highest cell adhesion (2-4h of incubation time) and proliferation (from 24h to 5 days) for the membranes of POPC/POEPC (75:25 ratio). Moreover, ferritin increased both cell adhesion and proliferation in comparison to control glass (respectively 1.5- and 1.75-fold) as well as proliferation in comparison to bare POPC/POEPC (95:5 ratio) (2 fold). Results are very promising in the goal of modulating the endothelial cell response through the interplay of viscoelastic/charge properties of the solid-supported membranes and the SLB-conditioned ferritin activity.

Bone response to surface-modified titanium implants: studies on the early tissue response to implants with different surface characteristics.

In a series of experimental studies, the bone formation around systematically modified titanium implants is analyzed. In the present study, three different surface modifications were prepared and evaluated. Glow-discharge cleaning and oxidizing resulted in a highly stoichiometric TiO2 surface, while a glow-discharge treatment in nitrogen gas resulted in implants with essentially a surface of titanium nitride, covered with a very thin titanium oxide. Finally, hydrogen peroxide treatment of implants resulted in an almost stoichiometric TiO2, rich in hydroxyl groups on the surface. Machined commercially pure titanium implants served as controls. Scanning Auger Electron Spectroscopy, Scanning Electron Microscopy, and Atomic Force Microscopy revealed no significant differences in oxide thickness or surface roughness parameters, but differences in the surface chemical composition and apparent topography were observed. After surface preparation, the implants were inserted in cortical bone of rabbits and evaluated after 1, 3, and 6 weeks. Light microscopic evaluation of the tissue response showed that all implants were in contact with bone and had a large proportion of newly formed bone within the threads after 6 weeks. There were no morphological differences between the four groups. Our study shows that a high degree of bone contact and bone formation can be achieved with titanium implants of different surface composition and topography.

Plasma oxidized polyhydroxymethylsiloxane--a new smooth surface for supported lipid bilayer formation.

A novel substrate for preparation of supported lipid bilayers (SLBs), smooth at the subnanometer scale and of variable thickness from ten to several hundred nanometers, was developed by surface oxidation of spin-coated poly(hydroxymethylsiloxane) (PHMS) films. The deposited polymeric thin films were modified by a combination of oxygen plasma and thermal treatment (PHMS(ox)), in order to convert the outermost surface layer of the polymer film to a stable SiO(2) film, suitable for SLB formation. The hydrophilic, SiO(2)-like surfaces were characterized by XPS, wetting angle, ellipsometry, and AFM. Lipid bilayers were formed on this surface using the well-known vesicle adsorption-rupture-fusion process, usually performed on glass or vapor-deposited SiO(2). Reproducible formation of homogeneous SLBs of different compositions (POPC, DOEPC, and POPC/DOPS) was demonstrated on the new SiO(2) surface by quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance (SPR), and optical reflectometry measurements. The SLB formation kinetics on the PHMS(ox)-coated sensors showed very similar characteristics, for all investigated PHMS thicknesses, as on reference sensors coated with vapor-deposited SiO(2). The good adhesive properties of the PHMS to gold allows for the preparation of thin PHMS(ox) layers compatible with SPR. The much smaller roughness at the nanometer scale of the PHMS(ox) surfaces, compared to standard vapor-deposited SiO(2)-coated sensors, makes them advantageous for AFM and optical experiments and promising for patterning. To benefit optical experiments with the PHMS(ox) surfaces, it was also investigated how the PHMS film thickness influences the SPR and reflectometry responses upon SLB formation.

Transport effects in the electrooxidation of methanol studied on nanostructured Pt/glassy carbon electrodes.

Transport effects in the methanol oxidation reaction (MOR) were investigated using nanostructured Pt/glassy carbon (GC) electrodes and, for comparison, a polycrystalline Pt electrode. The nanostructured Pt/GC electrodes, consisting of a regular array of catalytically active cylindrical Pt nanostructures with 55 +/- 10 nm in diameter and different densities supported on a planar GC substrate, were fabricated employing hole-mask colloidal lithography (HCL). The MOR measurements were performed under controlled transport conditions in a thin-layer flow cell interfaced to a differential electrochemical mass spectrometry (DEMS) setup. The measurements reveal a distinct variation in the MOR activity and selectivity (product distribution) with Pt nanostructure density and with electrolyte flow rate, showing an increasing overall activity, reflected by a higher Faradaic reaction current, as well as a pronounced increase of the turnover frequency for CO(2) formation and of the CO(2) current efficiency with decreasing flow rate and increasing Pt coverage. These findings are discussed in terms of the "desorption-readsorption-reaction" model introduced recently (Seidel et al. Faraday Discuss. 2008, 140, 67). Finally, consequences for applications in direct methanol fuel cells are outlined.

Diffusion-limited kinetics of adsorption of biomolecules on supported nanoparticles.

We derive general equations describing the diffusion-limited kinetics of irreversible adsorption of biomolecules on nanoparticles, fabricated on a flat surface, in the case of no hydrodynamic flow in the solution. Under such conditions, the gradients in the concentration of biomolecules occur near the surface, while in more remote regions the gradients may or may not be significant depending on the surface concentration and size of nanoparticles and the bulk concentration of biomolecules. The equations obtained make it possible to understand the conditions of realization of various regimes of adsorption.

Transport effects in the oxygen reduction reaction on nanostructured, planar glassy carbon supported Pt/GC model electrodes.

The role of transport and re-adsorption processes on the oxygen reduction reaction (ORR), and in particular on its selectivity was studied using nanostructured model electrodes consisting of arrays of Pt nanostructures of well-defined size and separation on a planar glassy carbon (GC) substrate. The electrochemical measurements were performed under controlled transport conditions in a double-disk electrode thin-layer flow-cell configuration; the model electrodes were fabricated by colloidal lithography techniques, yielding Pt nanostructures of well defined and controlled size and density (diameter: 140 or 85 nm, height: 20 or 10 nm, separation: from 1-2 to more than 10 diameters). The nanostructured model electrodes were characterized by scanning electron microscopy and electrochemical probing of the active surface area (via the hydrogen adsorption charge). The electrocatalytic measurements revealed a pronounced variation of the hydrogen peroxide yield, which increases by up to two orders of magnitude with increasing separation and decreasing size of the Pt nanostructures. Similar, though less pronounced effects were observed upon varying the electrolyte flow and thus the mass transport characteristics. These effects are discussed in a reaction model which includes (i) direct reduction to H(2)O on the Pt surface and (ii) additional H(2)O(2) formation and desorption on both Pt and carbon surfaces and subsequent partial re-adsorption and further reduction of the H(2)O(2) molecules on the Pt surface.

Mesoscopic mass transport effects in electrocatalytic processes.

The role of mesoscopic mass transport and re-adsorption effects in electrocatalytic reactions was investigated using the oxygen reduction reaction (ORR) as an example. The electrochemical measurements were performed on structurally well-defined nanostructured model electrodes under controlled transport conditions in a thin-layer flow cell. The electrodes consist of arrays of Pt ultra-microelectrodes (nanodisks) of defined size (diameter approximately 100 nm) separated on a planar glassy carbon (GC) substrate, which were fabricated employing hole-mask colloidal lithography (HCL). The measurements reveal a distinct variation in the ORR selectivity with Pt nanodisk density and with increasing electrolyte flow, showing a pronounced increase of the H2O2 yield, by up to 65%, when increasing the flow rate from 1 to 30 microL s(-1). These results are compared with previous findings and discussed in terms of a reaction model proposed recently (A. Schneider et al., Phys. Chem. Chem. Phys., 2008, 10, 1931), which includes (i) direct reduction to H2O on the Pt surface and (ii) additional H2O2 formation and desorption on both Pt and carbon surfaces and subsequent partial re-adsorption and further reduction of the H2O2 molecules on the Pt surface. The potential of model studies on structurally defined catalyst surfaces and under well-defined mass transport conditions in combination with simulations for the description of electrocatalytic reactions is discussed.

Supported phospholipid bilayers as a platform for neural progenitor cell culture.

Supported phospholipid bilayers constitute a biomimetic platform for cell behavior studies and a new approach to the design of cell culture substrates. Phosphocholine bilayers are resistant to cell attachment, but can be functionalized with bioactive molecules to promote specific cell interactions. Here, we explore phosphocholine bilayers, functionalized with the laminin-derived IKVAV pentamer, as substrates for attachment, growth, and differentiation of neural progenitor cells (AHPs). By varying peptide concentration (0-10%), we discovered a strongly nonlinear relationship between cell attachment and IKVAV concentration, with a threshold of 1% IKVAV required for attachment, and saturation in cell binding at 3% IKVAV. This behavior, together with the 10-fold reduction in cell attachment when using a jumbled peptide sequence, gives evidence for a specific interaction between IKVAV and its AHP cell-surface receptor. After 8 days in culture, the peptide-functionalized bilayers promoted a high degree of cell cluster formation. This is in contrast to the predominant monolayer growth, observed for these cells on the standard laminin coated growth substrates. The peptide-functionalized bilayer did not induce differentiation levels over those observed for the laminin coated substrates. These results are promising in that peptide-functionalized bilayers can allow attachment and growth of stem cells without induction of differentiation.

Simulation of methane oxidation on Pt.

The authors present a generic model of CH4 oxidation on Pt with the emphasis on the role of surface-oxide formation. The latter process is treated in terms of the theory of first-order phase transitions. The corresponding Monte Carlo simulations indicate that the surface-oxide formation may result in stepwise features in the reaction kinetics. Specifically, with increasing CH4 pressure and/or decreasing O2 pressure, the model predicts a sharp transition from a low-reactive state with the surface completely covered by oxide to a high-reactive state with the surface covered by chemisorbed oxygen. In the former case, the reaction is first order in CH4 and zero order in O2. In the latter case, both reaction orders are positive. All these findings help in interpreting available experiments.

A novel cell force sensor for quantification of traction during cell spreading and contact guidance.

In this work, we present a ridged, microfabricated, force sensor that can be used to investigate mechanical interactions between cells exhibiting contact guidance and the underlying cell culture substrate, and a proof-of-function evaluation of the force sensor performance. The substrates contain arrays of vertical pillars between solid ridges that were microfabricated in silicon wafers using photolithography and deep reactive ion etching. The spring constant of the pillars was measured by atomic force microscopy. For time-lapse experiments, cells were seeded on the pillared substrates and cultured in an on-stage incubator on a microscope equipped with reflected differential interference contrast optics. Endothelial cells (ECs) and fibroblasts were observed during attachment, spreading, and migration. Custom image analysis software was developed to resolve cell borders, cell alignment to the pillars and migration, displacements of individual pillars, and to quantify cell traction forces. Contact guidance classification was based on cell alignment and movement angles with respect to microfabricated ridges, as well as cell elongation. In initial investigations made with the ridged cell force sensor, we have observed contact guidance in ECs but not in fibroblast cells. A difference in maximal amplitude of mechanical forces was observed between a contact-guided and non-contact-guided, but mobile, EC. However, further experiments are required to determine the statistical significance of this observation. By chance, we observed another feature of cell behavior, namely a reversion of cell force direction. The direction of forces measured under rounded fibroblast cells changed from outwards during early cell attachment to inwards during further observation of the spreading phase. The range of forces measured under fibroblasts (up to 138 nN) was greater than that measured in EC (up to 57 nN), showing that the rigid silicon sensor is capable of resolving a large range of forces, and hence detection of differences in traction forces between cell types. These observations indicate proof-of-function of the ridged cell force sensor to induce contact guidance, and that the pillared cell force sensor constructed in rigid silicon has the necessary sensitivity to detect differences in traction force vectors between different cell phenotypes and morphologies.

A multitechnique study of liposome adsorption on Au and lipid bilayer formation on SiO2.

We have used a new setup for parallel quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance (SPR) measurements to measure the detailed kinetics of vesicle-to-bilayer transformation on SiO2 and vesicle adsorption on Au, respectively. The combination of SPR and QCM-D, complemented by atomic force microscopy measurements, has enabled a complete, time-resolved separation of vesicle and bilayer coverages, and thus, for the first time, allowed precise quantification of the critical surface coverage of vesicles needed for rupture. We furthermore demonstrate and quantify a previously undetected vesicle-size- and concentration-dependent loss of lipid material during the later stages of the process.

Imaging and manipulation of adsorbed lipid vesicles by an AFM tip: experiment and Monte Carlo simulations.

Single lipid vesicles adsorbed on SiO(2) were manipulated using an atomic force microscope (AFM) operated in contact mode. For large force setpoints, single vesicles were either pushed sideways or ruptured by the tip, depending on the tip type (sharp or blunt) used, while for small force setpoints the vesicles were imaged by the tip. To extend the interpretation of and to guide the experiment, we have developed a generic model of the vesicle-tip-substrate system and performed Monte Carlo simulations, addressing the influence of force setpoint and tip speed and shape on the type of imaging or manipulation observed. Specifically, we have explored AFM-image height and width variations versus force setpoint, typical AFM images for small and large force setpoints, tip-induced vesicle strain versus force setpoint, typical vesicle shapes during pushing for different tip speeds, and the details of vesicle rupture induced by the tip.

Transient and steady state CO oxidation kinetics on nanolithographically prepared supported Pd model catalysts: experiments and simulations.

Applying molecular-beam methods to a nanolithographically prepared planar PdSiO2 model catalyst, we have performed a detailed study of the kinetics of CO oxidation. The model catalyst was prepared by electron-beam lithography, allowing individual control of particle size and position. The sample was structurally characterized by atomic force microscopy and scanning electron microscopy before and after reaction. In the kinetic experiments, the O-rich and CO-rich regimes were investigated systematically with respect to their transient and steady-state behaviors, both under bistable and monostable reaction conditions. Separate molecular beams were used in order to supply the reactants, allowing individual control over the reactant fluxes. The desorbing CO2 was detected by both angle-resolved and angle-integrated mass spectrometries. The experimental results were analyzed using different types of microkinetic models, including a detailed reaction-diffusion model, which takes into account the structural parameters of the catalyst as well as scattering of the reactants from the support. The model quantitatively reproduces the results as a function of the reactant fluxes and the surface temperature. Various kinetic effects observed are discussed in detail on the basis of the model. Specifically, it is shown that under conditions of limited oxygen mobility, the switching behavior between the kinetic regimes is largely driven by the surface mobility of CO.

Local reaction rates and surface diffusion on nanolithographically prepared model catalysts: experiments and simulations.

Combining molecular beam methods and angular resolved mass spectrometry, we have studied the angular distribution of desorbing products during CO oxidation on a planar Pd/silica supported model catalyst. The model catalyst was prepared by means of electron beam lithography, allowing individual control of particle size, position, and aspect ratio, and was characterized by atomic force microscopy and scanning electron microscopy before and after reaction. In the experiment, both oxygen and CO rich regimes were investigated using separate molecular beams for the two reactants. This allows exploration of diffusion effects of reactants on the particles and of shadowing and backscattering phenomena. A reaction-diffusion model was developed in order to extract information about local reaction rates on the surface of the catalyst nanoparticles. The model takes into account the structural parameters of the catalyst as well as the backscattering of the reactants and products from the support. It allows a quantitative description of the experimental data and provides a detailed understanding of temperature and reactant flux dependent effects. Moreover, information on the surface mobility of oxygen under steady-state reaction conditions could be obtained by comparison with the experimental results.

Two-dimensional hydration shells of alkali metal ions at a hydrophobic surface.

We study the hydration shell formation of alkali metal ions at a graphite surface. Two-dimensional shell structures are found in the initial stage of hydration, in contrast to the three-dimensional structures in bulk water and clusters. Comparison of vibrational spectra with experiments identifies the shell structures and the thermally induced transition from the first to the second shell. We also found intriguing competition between hydration and ion-surface interaction, leading to different solvation dynamics between K and Na. Implications of these results in ionic processes at interfaces are elaborated.

Simulations of temperature dependence of the formation of a supported lipid bilayer via vesicle adsorption.

Recent experimental investigations of the kinetics of vesicle adsorption in solution on SiO2 demonstrate a thermally activated transition from adsorbed intact vesicles to a supported lipid bilayer. Our Monte Carlo simulations clarify the mechanism of this process. The model employed is an extension of the model used earlier to describe vesicle adsorption at room temperature. Specifically, it includes limitations of the adsorption rate by vesicle diffusion in the solution, and adsorption- and lipid-membrane-induced rupture of arriving and already adsorbed vesicles. Vesicles and lipid molecules, formed after rupture of vesicles, are considered immobile. With these ingredients, the model is able to quantitatively reproduce the temperature-dependent adsorption kinetics, including a higher critical surface concentration of intact vesicles for lower temperatures, and the apparent activation energy for the vesicle-to-bilayer transition E(a) approximately 5 kcal/mol.

Fluctuations and bistabilities on catalyst nanoparticles.

We show that coverage fluctuations on catalyst particles can drastically alter their macroscopic catalytic behavior. Scrutinizing the occurrence of kinetic bistabilities, it is demonstrated by molecular beam experiments on model catalysts that macroscopically observable bistabilities vanish completely with decreasing particle size, as previously predicted by theory. The effect is attributed to fluctuation-induced transitions between two kinetic reaction regimes, with a transition rate controlled by both particle size and surface defects. These results suggest that fluctuation-induced effects represent a general phenomenon affecting the reaction kinetics on nanostructured surfaces.

Kinetics of protein aggregation with formation of unreactive intermediates.

Irreversible protein aggregation resulting in formation and deposition of insoluble fibrils or amorphous precipitates is usually assumed to occur via sequential attachment of monomers to soluble intermediates. We complement this scheme by slow conversion of the intermediates to a relatively stable form so that they do not react with monomers but can be trapped by precipitates. For reasonable values of parameters, our model predicts that the aggregation kinetics order may be between 2.0 and 2.5. In particular, the model can be used to explain the reaction order, 2.17 +/- 0.09, observed for aggregation of recombinant human granulocyte colony stimulating factor.

Chemically patterned, metal oxide based surfaces produced by photolithographic techniques for studying protein- and cell-surface interactions I: Microfabrication and surface characterization.

Chemical patterns on smooth wafer substrates comprising areas with two different metals have been produced by vacuum metal deposition and photolithographic techniques. The combination of metals has been chosen from the series titanium (Ti), aluminium (Al), vanadium (V) and niobium (Nb), producing patterns (dots and stripes with dimensions of 50, 100 and 150 micrometer) with one of the metals as the background and with the second metal (foreground pattern) deposited on the background metal. The structure and chemical composition of the patterned surfaces were evaluated by scanning electron microscopy, X-ray photoelectron spectroscopy and imaging time-of-flight secondary-ion mass spectrometry. The surfaces proved to be geometrically well defined with the expected surface-chemical composition, i.e. a surface oxide (passive) film essentially composed of TiO(2),Al(2)O(3),V(2)O(5), or Nb(2)O(5). Ti/Ti patterned surfaces were produced as controls and found to show no chemical composition contrast. The surface roughness of the pattern was greater than that of the background by a factor of 2-3, but was still extremely smooth with Ra<2nm. The patterns serve as model surfaces for studying in vitro the behaviour of cells as well as the adsorption of serum proteins on different metal oxides, which will be reported in a companion paper. These surfaces can be used to compare and contrast the response of osteoblasts to Ti and other alloy components, such as Al, V, or Nb, which are used in load-bearing medical implants.