Alessandro Podestà

The effect of surface nanometre-scale morphology on protein adsorption.

BACKGROUND Protein adsorption is the first of a complex series of events that regulates many phenomena at the nano-bio interface, e.g. cell adhesion and differentiation, in vivo inflammatory responses and protein crystallization. A quantitative understanding of how nanoscale morphology influences protein adsorption is strategic for providing insight into all of these processes, however this understanding has been lacking until now. METHODOLOGY/PRINCIPAL FINDINGS Here we introduce novel methods for quantitative high-throughput characterization of protein-surface interaction and we apply them in an integrated experimental strategy, to study the adsorption of a panel of proteins on nanostructured surfaces. We show that the increase of nanoscale roughness (from 15 nm to 30 nm) induces a decrease of protein binding affinity (<or=90%) and a relevant increase in adsorbed proteins (<or=500%) beyond the corresponding increase of specific area. We demonstrate that these effects are caused by protein nucleation on the surface, which is promoted by surface nanoscale pores. CONCLUSIONS/SIGNIFICANCE These results show that the adsorption of proteins depends significantly on surface nanostructure and that the relevant morphological parameter regulating the protein adsorption process is the nanometric pore shape. These new findings improve our understanding of the role of nanostructures as a biomaterial design parameter and they have important implications for the general understanding of cell behavior on nanostructured surfaces.

Quantitative Characterization of the Influence of the Nanoscale Morphology of Nanostructured Surfaces on Bacterial Adhesion and Biofilm Formation

Bacterial infection of implants and prosthetic devices is one of the most common causes of implant failure. The nanostructured surface of biocompatible materials strongly influences the adhesion and proliferation of mammalian cells on solid substrates. The observation of this phenomenon has led to an increased effort to develop new strategies to prevent bacterial adhesion and biofilm formation, primarily through nanoengineering the topology of the materials used in implantable devices. While several studies have demonstrated the influence of nanoscale surface morphology on prokaryotic cell attachment, none have provided a quantitative understanding of this phenomenon. Using supersonic cluster beam deposition, we produced nanostructured titania thin films with controlled and reproducible nanoscale morphology respectively. We characterized the surface morphology; composition and wettability by means of atomic force microscopy, X-ray photoemission spectroscopy and contact angle measurements. We studied how protein adsorption is influenced by the physico-chemical surface parameters. Lastly, we characterized Escherichia coli and Staphylococcus aureus adhesion on nanostructured titania surfaces. Our results show that the increase in surface pore aspect ratio and volume, related to the increase of surface roughness, improves protein adsorption, which in turn downplays bacterial adhesion and biofilm formation. As roughness increases up to about 20 nm, bacterial adhesion and biofilm formation are enhanced; the further increase of roughness causes a significant decrease of bacterial adhesion and inhibits biofilm formation. We interpret the observed trend in bacterial adhesion as the combined effect of passivation and flattening effects induced by morphology-dependent protein adsorption. Our findings demonstrate that bacterial adhesion and biofilm formation on nanostructured titanium oxide surfaces are significantly influenced by nanoscale morphological features. The quantitative information, provided by this study about the relation between surface nanoscale morphology and bacterial adhesion points towards the rational design of implant surfaces that control or inhibit bacterial adhesion and biofilm formation.

Evidence of Extended Solidlike Layering in [Bmim][NTf2] Ionic Liquid Thin Films at Room-Temperature

We report the direct observation of solidlike ordering at room temperature of thin films of [Bmim][NTf2] ionic liquid on mica, amorphous silica, and oxidized Si(110). A statistical quantitative analysis of atomic force microscopy topographies shows that on these surfaces [Bmim][NTf2] forms layered structures, characterized by a perpendicular structural periodicity of approximately 0.6 nm. Remarkably, even the highest structures, up to 50 nm high, behave solidlike against the AFM probe. Conversely, on highly oriented pyrolitic graphite the ionic liquid forms nanometer-sized, liquidlike domains. The results of this study are directly relevant for those applications where ILs are employed in form of thin films supported on solid surfaces, such as in microelectromechanical or microelectronic devices. More generally, they suggest that at the liquid/solid interface the structural properties of ILs can be far more complex than those depicted so far, and prompt new fundamental investigations of the forces that drive supported ILs through a liquidlike-to-solidlike transition.

Supercapacitors based on nanostructured carbon electrodes grown by cluster-beam deposition

Nanostructured carbon films have been grown at room temperature by supersonic cluster beam deposition. Due to a structure based on nanotube embryos and a porosity with grain sizes of a few tens of nanometers, these films have a highly accessible surface area needed for electrochemical applications such as supercapacitors. Films with a density of 1 g/cm3 show, in the dc regime, a specific capacitance per electrode of 75 F/g on a single-cell device with polycarbonate as the organic electrolyte. The resulting energy and power densities of cluster-assembled carbon electrodes are 76 Wh/kg and 506 kW/kg. The possibility of depositing nanostructured films over a large area on a variety of substrates makes cluster-beam deposition very interesting for the realization of supercapacitors.

Production and characterization of highly intense and collimated cluster beams by inertial focusing in supersonic expansions

Intense and collimated supersonic cluster beams have been produced by exploiting inertial focusing effects. To this goal we have developed and tested a novel focusing nozzle (focuser). Using this device with a pulsed microplasma cluster source we have obtained cluster beams with a divergence of 10 mrad and average densities of 3×1010 atoms/cm3 (2×1012 atoms/cm3 pulsed) corresponding to deposition rates of 2 nm/s at 300 mm distance from the source nozzle. With a focusing nozzle cluster thermal relaxation and mass distribution in a supersonic expansion can be controlled. We have measured the cluster transverse velocities, with extremely high precision, by characterizing the cluster beam deposition on a substrate by an atomic force microscope. Besides the relevance for the understanding of relaxation processes in expanding jets, the inertial focusing of clusters has several important consequences for the synthesis of nanostructured films with controlled structure and for all the experimental techniques requi...

The influence of the precursor clusters on the structural and morphological evolution of nanostructured TiO2 under thermal annealing

We have produced nanostructured titanium dioxide thin films by supersonic cluster beam deposition. The as-deposited films have a nanocrystalline or amorphous structure depending on the mass distribution of the precursor clusters. This can be controlled by aerodynamic separation effects typical of supersonic expansions. On thermal annealing at temperatures from 400 to 800 °C in ambient atmosphere, amorphous-to-anatase and anatase-to-rutile phase transitions have been observed. The nanostructure and microstructure evolution of the film upon annealing has been characterized by atomic force microscopy and transmission electron microscopy. The influence of the precursor clusters in the evolution of the film nanostructure at high temperatures has been demonstrated. This observation opens up new perspectives for batch fabrication of devices based on cluster-assembled materials.

Cluster beam microfabrication of patterns of three-dimensional nanostructured objects

This letter describes the use of supersonic cluster beam deposition (SCBD) through a stencil mask for the fabrication of patterns of cluster-assembled objects. Using carbon cluster beams, micrometer-size pillars and tips have been produced on a variety of substrates. SCBD is characterized by high deposition rates, high lateral resolution, and low temperature processing. Nanostructured objects can be produced with high aspect ratio and controlled shapes. Micropatterning with SCBD can be of interest for applications requiring the integration of cluster-assembled structures with microelectronic or micromechanical devices.

Cluster beam synthesis of nanostructured thin films

The use of clusters as elemental building blocks can open routes toward the fabrication of a new class of nanostructured solids and devices. We report the synthesis of nanostructured films using supersonic cluster beam deposition. A new type of cluster source based on microplasma ablation has been developed. This allows a substantial improvement in terms of deposition rate and control on cluster mass distribution. These achievements make supersonic cluster beams a useful tool in the arena of cluster assembling of materials. We have applied this technique to the growth of nanostructured carbon thin films. The structure and morphology of the films can be controlled by varying the cluster mass distribution prior to deposition. Deposition conditions affect the surface roughness and the onset of scale-invariant morphology on a dimension domain extending from the nanometer up to the micrometer. The cluster beam deposition method shows very promising features in view of the large scale growth of nanostructured f...

Early Events in Insulin Fibrillization Studied by Time-Lapse Atomic Force Microscopy

The importance of understanding the mechanism of protein aggregation into insoluble amyloid fibrils lies not only in its medical consequences, but also in its more basic properties of self-organization. The discovery that a large number of uncorrelated proteins can form, under proper conditions, structurally similar fibrils has suggested that the underlying mechanism is a general feature of polypeptide chains. In this work, we address the early events preceding amyloid fibril formation in solutions of zinc-free human insulin incubated at low pH and high temperature. Here, we show by time-lapse atomic force microscopy that a steady-state distribution of protein oligomers with a quasiexponential tail is reached within a few minutes after heating. This metastable phase lasts for a few hours, until fibrillar aggregates are observable. Although for such complex systems different aggregation mechanisms can occur simultaneously, our results indicate that the prefibrillar phase is mainly controlled by a simple coagulation-evaporation kinetic mechanism, in which concentration acts as a critical parameter. These experimental facts, along with the kinetic model used, suggest a critical role for thermal concentration fluctuations in the process of fibril nucleation.

Direct Microfabrication of Topographical and Chemical Cues for the Guided Growth of Neural Cell Networks on Polyamidoamine Hydrogels

Cell patterning is an important tool for organizing cells in surfaces and to reproduce in a simple way the tissue hierarchy and complexity of pluri-cellular life. The control of cell growth, proliferation and differentiation on solid surfaces is consequently important for prosthetics, biosensors, cell-based arrays, stem cell therapy and cell-based drug discovery concepts. We present a new electron beam lithography method for the direct and simultaneous fabrication of sub-micron topographical and chemical patterns, on a biocompatible and biodegradable PAA hydrogel. The localized e-beam modification of a hydrogel surface makes the pattern able to adsorb proteins in contrast with the anti-fouling surface. By also exploiting the selective attachment, growth and differentiation of PC12 cells, we fabricated a neural network of single cells connected by neuritis extending along microchannels. E-beam microlithography on PAA hydrogels opens up the opportunity of producing multifunctional microdevices incorporating complex topographies, allowing precise control of the growth and organization of individual cells.