G. Bongiorno

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.

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.

Electronic structure of cluster assembled nanostructured TiO2 by resonant photoemission at the Ti L2,3 edge

The electronic structure of cluster assembled nanostructured TiO(2) thin films has been investigated by resonant photoemission experiments with photon energies across the Ti L(2,3) edge. The samples were produced by supersonic cluster beam deposition with a pulsed microplasma cluster source. The valence band shows resonance enhancements in the binding energy region between 4 and 8 eV, populated by O 2p and hybridized Ti 3d states, and in the region about 1 eV below the Fermi level associated with defects related Ti 3d states. The data show that in as-deposited films Ti atoms are mainly fully (sixfolds) coordinated to oxygen atoms in octahedral symmetry and only a small fraction is in a broken symmetry environment. Since resonant photoemission is closely linked to the local electronic and structural configurations around the Ti atom, it is possible to correlate the resonant photoemission intensity and lineshape with the presence of defects of the films and with the degree of hybridization between the titanium and oxygen atoms.

Adhesive-free colloidal probes for nanoscale force measurements: Production and characterization

We describe novel approaches for the production and characterization of epoxy- and adhesive-free colloidal probes for atomic force microscopy (AFM). Borosilicate glass microspheres are strongly attached to commercial AFM cantilevers exploiting the capillary adhesion force due to the formation of a water meniscus, and then a thermal annealing of the sphere-cantilever system at a temperature slightly below the softening point of borosilicate glass. Controlling the wettability of the surfaces involved turned out to be a crucial element for the control of surface adhesion and for the implementation of a completely adhesive-free production method of colloidal probes. Moreover, we present a statistical characterization protocol of the probe dimensions and roughness based on the AFM inverse imaging of colloidal probes on spiked gratings. We have assessed the influence of defects of the grating on the characterization of the probe, and discussed the accuracy of our characterization technique in comparison to the methods based on scanning electron or optical microscopy, or on the manual analysis of AFM inverse images.

Core level spectroscopy of free titanium clusters in supersonic beams

Synchrotron radiation x-ray absorption spectroscopy (XAS) is one of the most powerful techniques to interrogate the local electronic structure and chemical status of bulk and nanostructured systems. The application of this technique to the study of size effects in free clusters of transition metal atoms would advance substantially fundamental knowledge of nano-objects and the tailoring of their magnetic and catalytic properties. To date core level spectroscopy of free transition metal clusters has been out of reach due to the lack of a cluster source able to produce clusters in the gas phase with a density suitable for synchrotron radiation sources. Here we demonstrate the XAS characterization of free titanium clusters in a supersonic molecular beam. We use a high-intensity cluster beam source coupled to a synchrotron beamline to investigate the size dependence of core level excitation of Tin clusters in the mass range 15<n<1000. The x-ray absorption of Tin evolves from a multi-peaked complex structure similar to that of Ti atoms towards spectra characterized by two main absorption features as in bulk titanium. The intensities and the fine structure of the spectra are size dependent showing regularities compatible with geometric shell closings and the presence of a structural transition at about 540 atoms/cluster.

sp hybridization in free carbon nanoparticles-presence and stability observed by near edge X-ray absorption fine structure spectroscopy

The presence and stability of sp hybridized atoms in free carbon nanoparticles was investigated by NEXAFS spectroscopy. The experiments show that a predominant fraction of carbon atoms is found in linear sp-chains and that conversion into sp(2) structures proceeds already at low temperature and in the gas phase.

Titanium fullerenoid oxides

Nanostructured titanium oxide films synthesized by supersonic cluster beam deposition were analyzed by high-resolution transmission electron microscopy. Nanoparticles were produced in a pulsed microplasma cluster source in the presence of He or helium-oxygen mixture as carrier gas. While films grown using He consist only of rutile and anatase TiO2 nanocrystals formed upon exposure to air, films grown with a He∕O mixture also contain isolated TiOx cages that closely resemble carbon fullerenes. The diameter of the cages ranges from about 0.9to2.7nm. A fraction of the cages have irregular shapes, possibly induced by oxygen vacancies. The TiOx fullerenoids grow in the gas phase, in a narrow temperature/pressure range within the cluster source, and are preserved through low-energy deposition.

High Throughput Tools for the Study of Protein-Nanostructured Surface Interaction

The aim of this review is to describe and to analyze the ingredients that are necessary in order to develop a robust and effective experimental approach for the high-throughput characterization of protein-nanostructured surface interaction. In the first part of this paper we review the nanostructured surface synthesis methods that are potentially able to create nanostructured inorganic surface libraries. In the second part, we address another fundamental aspect consisting in the availability of high-throughput proteins detection methods. We describe in details new emerging analytical tools compatible with nanostructured surfaces, analyzing different possible strategies, depending on the objective of the experiment and on the library format.

Accessing the fractal dimension of free clusters in supersonic beams

In this paper a method for the quantitative determination of a morphology descriptor of free clusters with complex nanostructure is presented and applied to transition metal nanoparticles produced by a pulsed vaporization source. The method, which is based on the low-pressure aerodynamic mobility of neutral particles, can be applied as a characterization tool to a broad class of gas-phase nanoparticle sources for on-line investigation of particle growth and for quantifying coalescence versus agglomerate aggregation. We report on the application of this method for the characterization of free titanium clusters produced by a pulsed microplasma cluster source in the size range of approximately 300–6000 atoms. The clusters have an open fractal-like structure, with the fractal dimension depending on their thermal history during growth and evolving towards softer aggregates for longer residence times where lower-temperature conditions characterize the growth environment.