Rippling of graphitic surfaces: a comparison between few-layer graphene and HOPG.

The surface structure of Few-Layer Graphene (FLG) epitaxially grown on the C-face of SiC has been investigated by TM-AFM in ambient air and upon interaction with dilute aqueous solutions of bio-organic molecules (l-methionine and dimethyl sulfoxide, DMSO). Before interaction with molecular solutions, we observe nicely ordered, three-fold oriented rippled domains, with a 4.7 ± 0.2 nm periodicity (small periodicity, SP) and a peak-to-valley distance in the range 0.1-0.2 nm. Upon mild interaction with the molecular solution, the ripple periodicity "relaxes" to 6.2 ± 0.2 nm (large periodicity, LP), while the peak-to-valley height increases to 0.2-0.3 nm. When additional energy is transferred to the system through sonication in solution, graphene planes are peeled off, as shown by quantitative analysis of Raman spectroscopy and X-ray photoelectron spectroscopy which indicate a neat reduction of thickness. Upon exfoliation rippled domains are no longer observed. In comparative experiments on cleaved HOPG, we could not observe ripples on pristine samples in ambient air, while LP ripples develop upon interaction with the molecular solutions. Recent literature on similar systems is not univocal regarding the interpretation of rippling. The ensemble of our comparative observations on FLG and HOPG can be hardly rationalized solely on the basis of the surface assembly of molecules, either organic molecules coming from the solution or adventitious species. We propose to consider rippling as the manifestation of the free-energy minimization of quasi-2D layers, eventually affected by factors such as interplanar stacking, and interactions with molecules and/or with the AFM tip.

Morphology, mechanical properties and viability of encapsulated cells.

The morphological and mechanical properties of encapsulated yeast cells (Saccharomyces cerevisiae) have been investigated by atomic force microscopy (AFM). Single living cells have been coated through the alternate deposition of oppositely charged polyelectrolyte (PE) layers. The properties of cells coated by different numbers of PE layers and from PE solutions of different ionic strength have been investigated. AFM imaging indicates an increase in PE coating stability when decreasing the solution ionic strength. The Young's moduli of the different examined systems have been evaluated through a quantitative analysis of force-distance curves by using the Hertz-Sneddon model. The analysis indicates an increase in hybrid system stiffness when lowering the ionic strength of the PE solution. An evaluation of the viability of encapsulated cells was obtained by confocal laser scanning microscopy (CLSM) measurements. CLSM analysis indicates that cells preserve their subcellular structure and duplication capability after encapsulation. By coupling AFM and CLSM data, a correlation between local stiffness and duplication rate was obtained.

A thermodynamic and structural study of myelin basic protein in lipid membrane models.

Myelin basic protein (MBP) is a major protein of the myelin membrane in the central nervous system. It is believed to play a relevant role in the structure and function of the myelin sheath and is a candidate autoantigen in demyelinating processes such as multiple sclerosis. MBP has many features typical of soluble proteins but is capable of strongly interacting with lipids, probably via a conformation change. Its structure in the lipid membrane as well as the details of its interaction with the lipid membrane are still to be resolved. In this article we study the interaction of MBP with Langmuir films of anionic and neutral phospholipids, used as experimental models of the lipid membrane. By analyzing the equilibrium surface pressure/area isotherms of these films, we measured the protein partition coefficient between the aqueous solution and the lipid membrane, the mixing ratio between protein and lipid, and the area of the protein molecules inserted in the lipid film. The penetration depth of MBP in the lipid monolayer was evaluated by x-ray reflectivity measurements. The mixing ratio and the MBP molecular area decrease as the surface pressure increases, and at high surface pressure the protein is preferentially located at the lipid/water interface for both anionic and neutral lipids. The morphology of MBP adsorbed on lipid films was studied by atomic force microscopy. MBP forms bean-like structures and induces a lateral compaction of the lipid surface. Scattered MBP particles have also been observed. These particles, which are 2.35-nm high, 4.7-nm wide, and 13.3-nm long, could be formed by protein-lipid complexes. On the basis of their size, they could also be either single MBP molecules or pairs of c-shaped interpenetrating molecules.

Self-assembled monolayers of organosulphur molecules bearing calix[4]arene moieties.

We investigate the self-assembly of modified calix[4]arene on gold surfaces. Calix[4]arene was modified through a reaction sequence which led to assembling of the crown-5 moiety and to the insertion of two thioether groups into the starting molecule. The so-obtained calix[4]arene-crown-5 bis(7-thiatridecyloxy) (hereafter called calix[4]arene) was in the stable 1,3-alternate conformation. The calix[4]arene/gold interface was investigated by means of spectroscopic ellipsometry (SE), scanning tunneling microscopy (STM) and cyclic voltammetry (CV). SE data indicate a layer thickness compatible with the formation of a monomolecular layer. This result is confirmed by STM imaging which shows the formation of a high density of small pits, one gold layer deep, a typical feature of self-assembled organosulphur monolayers on gold. CV measurements performed in presence of the [Ru(NH(3))(6)(2+/3+)] redox couple indicate a passivation of the metal electrode, resulting in a reduction of the redox current, after the layer deposition. CV has also been used to investigate the selectivity properties of calix[4]arene-covered gold electrodes by measuring the redox current decrease in the presence of different salt solutions. It is found that calix[4]arene-covered electrodes are able to complex K(+) and Ba(2+), while no complexation is observed in the case of Li(+), Na(+), Cs(+), Mg(2+) and Ca(2+).

Encapsulated yeast cells inside Paramecium primaurelia: a model system for protection capability of polyelectrolyte shells.

One of the most promising applications of encapsulated living cells is their use as protected transplanted tissue into the human body. A suitable system for the protection of living cells is the use of nano- or microcapsules of polyelectrolytes. These shells can be deposited easily on top of the cells by means of a layer-by-layer technique. An interesting feature of the capsules is the possibility to control their properties on a nanometre level, tuning their wall texture via the preparation conditions. Here we introduce a model system to test the protection ability of polyelectrolyte capsules. Common bakery yeast cells were encapsulated. They were coated with a fluorescently labelled shell at conditions known to guarantee cell survival, and the cell interior was stained with DAPI. The protozoan Paramecium primaurelia was incubated with this double-stained living yeast and visualized by means of two-photon excitation fluorescence microscopy. Cross-sections of the dye-stained material as well as autofluorescence of the fixed protozoan allowed us to follow the digestion of the coated yeast with time. Our investigation reveals that capsules prepared under these deposition conditions are permeable to lysosomal enzymes, leading to degradation of the yeast inside the intact capsules. Our preliminary results indicate the suitability of the introduced model as a test system of this permeability.

Confocal laser scanning microscopy to study formation and properties of polyelectrolyte nanocapsules derived from CdCO3 templates.

Three-dimensional confocal laser scanning microscopy (CLSM) was used as an essential investigation method to obtain information about the formation and morphological characteristics of nanocapsules. Nanocapsules are built by layer-by-layer deposition of alternatively charged polyelectrolytes on templates forming nanostructured hollow shells. CLSM is unique in allowing for monitoring of the core dissolution process in real time and for studying nanocapsule functioning in hydrated conditions within a three-dimensional and temporal framework. Since we are also interested in the identification of other possible templates, we briefly report on the use of yeast cells as biocolloidal cores monitored by means of two-photon microscopy. Here we focus our attention on the use of CdCO(3) crystals as template candidates for the preparation of stable capsules. Both cubic and spherical CdCO(3) cores have been produced. Cubic cores exhibit higher monodispersity and smaller size compared to spherical ones. Capsules templated on these cores have a higher surface-to-volume ratio that is valuable for applications related to drug delivery, functional properties of the shells and adsorption of proteins, and other biologically relevant molecules. Microsc. Res. Tech. 59:536-541, 2002.

An atomic force microscopy investigation of protein crystal surface topography.

Tapping mode atomic force microscopy was employed to study the surface structure of different protein crystals in a liquid environment. The (101) face of hen egg-white lysozyme crystals and the (111) face of horse spleen ferritin crystals were studied. On the (101) face of lysozyme crystals we observed islands delimitated by micro-steps and elongated in the [010] direction. The elongation direction coincides with the preferential growth direction predicted by a growth model reported in the literature. The islands observed on the ferritin (111) face are also delimitated by micro-steps but have circular symmetry. Sectioning of the images allowed us to measure the step heights. The surface free energy was estimated from the growth step morphology. Molecular resolution was achieved for ferritin crystals, showing a hexagonal surface packing, as expected for the molecular lattice of a (111) face in a fcc crystal.

Structure of rat tail tendon collagen examined by atomic force microscope.

The Atomic Force Microscope (AFM) was used to inspect collagen fibrils deposited on mica sheets at different fibrillogenesis times. Collagen was obtained from rat tail tendon fibers. Various fibril forms were observed, together with the characteristic periodic intra-fibril structure (D-bands). The fibril thickness, width, D-band periodicity and depth were measured and the statistical distribution of these parameters at 1, 2, 5, 10 and 15 days of in vitro fibril formation time was calculated. The fibrils showed an increasing size with time, but the band interval measure remained stable. The band depth, after an initial increase, exhibited a relative steadiness. The results indicate that AFM offers, at low resolution, images qualitatively similar to those obtained with electron microscopy, but with less manipulation of the sample. A quantitative evaluation of collagen structural features in the nanometer scale is made possible by AFM.