Mauro Taborelli

Influence of surface and protein modification on immunoglobulin G adsorption observed by scanning force microscopy

Scanning force microscopy has been used successfully to produce images of individual protein molecules. However, one of the problems with this approach has been the high mobility of the proteins caused by the interaction between the sample and the scanning tip. To stabilize the proteins we have modified the adsorption properties of immunoglobulin G on graphite and mica surfaces. We have used two approaches: first, we applied glow discharge treatment to the surface to increase the hydrophilicity, favoring adhesion of hydrophilic protein molecules; second, we used the arginine modifying reagent phenylglyoxal to increase the protein hydrophobicity and thus enhance its adherence to hydrophobic surfaces. We used scanning force microscopy to show that the glow discharge treatment favors a more homogeneous distribution and stronger adherence of the protein molecules to the graphite surface. Chemical modification of the immunoglobulin caused increased aggregation of the proteins on the surface but did not improve the adherence to graphite. On mica, clusters of modified immunoglobulins were also observed and their adsorption was reduced. These results underline the importance of the surface hydrophobicity and charge in controlling the distribution of proteins on the surface.

Morphological difference between fibronectin sprayed on mica and on PMMA.

We have imaged with scanning force microscopy in air fibronectin (Fn) molecules sprayed on mica and on polymethylmetacrylate (PMMA), the latter being extensively used as biomaterial for implants. On mica we can observe small aggregates as well as individual molecules whose shape is influenced by the tip interaction during the scanning process, most of the isolated molecules showing a V-shape oriented in the scan direction. This indicates that the arms of the molecules are relatively free to move and the binding to the mica substrate is located near the disulfide bridge between the two subunits of the molecule. On the other side, when Fn molecules are sprayed on PMMA under the same conditions as for mica, we observe a thin network which we interpret as Fn molecules bound to each other. We relate our observation to the fact that mica is known to be strongly hydrophilic, which could reduce the Fn binding properties by interacting relatively strongly with molecules. On the other side, PMMA being hydrophobic, would interact less with molecules, leaving more binding sites for inter-molecular attachment.

Scanning force microscopy and cryo-electron microscopy of tobacco mosaic virus as a test specimen

In this study, tobacco mosaic virus (TMV) provides a resolution criterion for specimen preparation methods as well as for imaging parameters of the scanning force microscope (SFM). We present scanning force microscopic images of the virus embedded in 0.5% buffered phosphotungstic acid solution adsorbed on a freshly cleaved mica surface, and imaged under atmospheric conditions. Individual TMV particles were clearly identified with a characteristic shape of long rods of about 300 nm long and 60-70 nm in apparent width due to the geometric parameters of the tip. The structure of the virus was compared with cryo-electron microscopic data of vitrified suspensions observed to a resolution of 1.15 nm. Uncoated TMV particles were also deposited on evaporated titanium thin films and imaged by SFM.

Surface properties of electropolished titanium and vanadium

Abstract The surface topography, the chemical composition and the hydroxylation state are the surface properties playing a key role in the first stage of the biocompatibility process, namely the adsorption of water and proteins on the implant surface. To understand the very different tissue response to titanium and vanadium, we have measured the above-mentioned surface properties on similarly prepared Ti and V electropolished samples. Scanning force microscopy shows granular and homogeneous surfaces in both Ti and V samples, but with roughness twice as small in the case of V and with a lateral grain size of the order of 20–30 nm for Ti and of 80–100 nm for V. The surface chemical composition is strongly affected by thermal treatments, as revealed by Auger electron spectroscopy. On electropolished Ti, the surface segregation of Cl (originating from the electropolishing bath) occurs at 720 K and is well described by a purely diffusive model, i.e. Ficks law. For the segregation of S on Ti at higher temperature, we have extracted the energy of segregation and observed a rather strong influence of sulphur diffusion depending on the presence of chlorine on the surface. Finally, thermal desorption spectroscopy measurements indicate that water is mainly dissociated on hydroxyl groups on both Ti and V; the large amount of detected water indicates that it is deeply trapped inside the sample and not only chemisorbed on its surface.

Covalent immobilization of immunoglobulins G and Fab′ fragments on gold substrates for scanning force microscopy imaging in liquids

Scanning force microscopy offers the possibility of observing protein molecules under liquid environment. The main difficulty in obtaining reproducible images is given by the low adhesion of the molecules to the substrate. Physisorbed molecules are displaced by the scanning tip or are resuspended in the medium. We have therefore performed a covalent immobilization of immunoglobulin G (IgG) or its monovalent Fab′ fragment on gold surfaces thanks to thiol groups. For this purpose, multiple thiol groups were chemically introduced into the IgG molecule by treatment with Traut’s reagent. As an alternative, for a Fab′ fragment, we prepared molecules with a single thiol group located close to the C terminus of the truncated heavy chain. Both immobilization techniques enable us to observe clearly discernable individual molecules in liquid media. The grafting of Fab′ fragments on gold surface open new opportunities to study protein interactions.

Hydroxylation and crystallization of electropolished titanium surface

Surface properties and dissociative chemisorption of water on titanium-oxide surfaces are of particular interest for the biocompatibility of this material. Scanning force microscopy images of electropolished titanium samples and of thin films of titanium evaporated under vacuum show a similar topography with a grain size of 30 nm. Mass spectrometer thermal desorption spectroscopy displays two peaks for e/m = 18, at 380 and 520 K corresponding to adsorbed water molecules and hydroxyl groups, respectively. Auger spectra show the segregation toward the surface of chlorine from the metal-oxide interface upon heating at 720 K and of sulphur from the bulk after annealing at 950 K. The oxygen diffusion from the surface toward the bulk during the heating process induces a metallic behaviour of the surface layer which is revealed by tunneling spectroscopy: the semiconducting gap present in dI/dV curves for the air-exposed sample vanishes upon annealing. STM images of the annealed surface show the presence of tiny crystallites of a few nm in size.

Structural characterization of Ti90Al6V4 alloy and sulphur segregation

Abstract Ti90Al6V4 alloy is used in orthopaedic medical implant applications because of its biocompatibility, corrosion resistance and mechanical load resistance. We characterized the time evolution of the surface composition of TiAlV, the surface roughness and the microstructure, which influence the formation of a suitable tissue-implant interface. For this we used Auger electron spectroscopy, X-ray photoelectron spectroscopy, atomic force microscopy and X-ray diffraction. The impurity diffusion was accelerated by annealing in ultra high vacuum. We found sulphur segregation from the bulk to the surface. The value of the activation energy of 150 kJ/mol allows us to conclude that diffusion at room temperature is too slow to influence the composition during the implant lifetime. The microstructure of the alloy is equiaxed at room temperature and is modified by annealing above 950°C with the appearance of a lamellar phase. In the diffractograms after the treatments performed in our study we always observed only the α-phase peaks. During annealing in air the surface oxide thickens and increases its vanadium content compared to the bulk alloy. This finding is relevant for high temperature applications, but also for the machining treatments on devices applied at room temperature since the possible surface heating during wear processes could modify the surface composition.

Preparation of isolated biomolecules for SFM observations: T4 bacteriophage as a test sample

The T4 bacteriophage has been used to investigate protocols for the preparation of samples for scanning force microscopy in air, in order to obtaining reproducible images. The resolution of images and the distribution of bacteriophages on the substrate depends on the buffer type, its concentration, the surface treatment of substrate, and the method of deposition. The best imaging conditions for the phages require dilution in a volatile buffer at low ionic strength and adsorption onto hydrophilic surfaces. When imaging with the scanning force microscopy the quality of the images is influenced by the vertical and lateral forces applied on the sample and by the tip geometry.