Jessem Landoulsi

Silane layers on silicon surfaces: mechanism of interaction, stability, and influence on protein adsorption.

In this work the mechanism of (3-aminopropyl)triethoxysilane (APTES) interaction with silicon surfaces is investigated at the molecular level. We studied the influence of experimental parameters such as time, temperature, and concentration on the quality of the APTES layer in terms of chemical properties, morphology, and stability in aqueous medium. This was achieved using a highly sensitive IR mode recently developed, grazing angle attenuated total reflection (GA-ATR). This technique provides structural information on the formed APTES layer. The topography of this layer was investigated by atomic force microscopy in aqueous medium. The hydrophilicity was also studied using contact angle measurement. Combining these techniques enables discussion of the mechanism of silane grafting. Considerable differences were observed depending on the reaction temperature, room temperature or 90 °C. The data suggest the presence of two adsorption sites with different affinities on the oxidized silicon layer. This also allows the optimal parameters to be established to obtain an ordered and stable silane layer. The adsorption of proteins on the APTES layer was achieved and monitored using in situ quartz crystal microbalance with dissipation monitoring and ex situ GA-ATR analyses.

Growth Mechanism of Confined Polyelectrolyte Multilayers in Nanoporous Templates.

We investigate the mechanism of polyelectrolyte multilayer (PEM) assembly in nanoporous templates with a view to synthesizing nanotubes or nanowires under optimal conditions. For this purpose, we focus on the effect of parameters related to the geometrical constraints (pore diameter), the size of the macromolecules (their molar mass and the ionic strength), and the interaction between the pore walls and the adsorbed chains (modulated by the ionic strength). Our results reveal the existence of two regimes in the mechanism of PEM growth: (i) the first regime is comparable to that observed on flat substrates, including the influence of ionic strength and (ii) the second regime, which is slower in terms of kinetics, results from the interconnection established between polyelectrolyte chains across the pores and leads to the formation of a dense gel. As a consequence, the diffusion of polyelectrolytes in nanopores becomes the controlling factor of PEM growth in this second regime. The dense gel, owing to its peculiar structure, enhances the formation of nanowires or of partially occluded nanotubes in some cases, depending on initial pore dimensions.

Synthesis of Collagen Nanotubes with Highly Regular Dimensions through Membrane-Templated Layer-by-Layer Assembly

Nanotubes made from a fibrillar protein, namely, collagen, were fabricated by a template-based method combined with layer-by-layer (LbL) deposition. The ability to incorporate collagen in LbL multilayered film was first demonstrated by in situ quartz crystal microbalance and ex situ ellipsometry on a flat model substrate, using poly(styrene sulfonate) (PSS) as polyanion. Collagen-based nanotubes were then fabricated by alternately immersing a polycarbonate membrane, used as template, in PSS and collagen aqueous solutions. Direct evidence for nanotube formation was obtained by dissolving the membrane and imaging the liberated (PSS/collagen)(n) nanostructures by scanning electron microscopy and by transmission electron microscopy. The proposed strategy constitutes a practical alternative to electrospinning as it allows a very good control over the dimensions (outside and inside diameters and length) of the resulting nanotubes. Besides their fundamental interest, collagen-based nanotubes are useful nano-objects for the creation of new nanostructured biomaterials with numerous potential applications in the biomedical field.

Chemical modifications of Au/SiO2 template substrates for patterned biofunctional surfaces.

The aim of this work was to create patterned surfaces for localized and specific biochemical recognition. For this purpose, we have developed a protocol for orthogonal and material-selective surface modifications of microfabricated patterned surfaces composed of SiO(2) areas (100 μm diameter) surrounded by Au. The SiO(2) spots were chemically modified by a sequence of reactions (silanization using an amine-terminated silane (APTES), followed by amine coupling of a biotin analogue and biospecific recognition) to achieve efficient immobilization of streptavidin in a functional form. The surrounding Au was rendered inert to protein adsorption by modification by HS(CH(2))(10)CONH(CH(2))(2)(OCH(2)CH(2))(7)OH (thiol-OEG). The surface modification protocol was developed by testing separately homogeneous SiO(2) and Au surfaces, to obtain the two following results: (i) SiO(2) surfaces which allowed the grafting of streptavidin, and subsequent immobilization of biotinylated antibodies, and (ii) Au surfaces showing almost no affinity for the same streptavidin and antibody solutions. The surface interactions were monitored by quartz crystal microbalance with dissipation monitoring (QCM-D), and chemical analyses were performed by polarization modulation-reflexion absorption infrared spectroscopy (PM-RAIRS) and X-ray photoelectron spectroscopy (XPS) to assess the validity of the initial orthogonal assembly of APTES and thiol-OEG. Eventually, microscopy imaging of the modified Au/SiO(2) patterned substrates validated the specific binding of streptavidin on the SiO(2)/APTES areas, as well as the subsequent binding of biotinylated anti-rIgG and further detection of fluorescent rIgG on the functionalized SiO(2) areas. These results demonstrate a successful protocol for the preparation of patterned biofunctional surfaces, based on microfabricated Au/SiO(2) templates and supported by careful surface analysis. The strong immobilization of the biomolecules resulting from the described protocol is advantageous in particular for micropatterned substrates for cell-surface interactions.

Ennoblement of stainless steel in the presence of glucose oxidase: Nature and role of interfacial processes

The ennoblement of the free corrosion potential (E(corr)) of AISI 316L stainless steel which did not occur in synthetic fresh water (SFW), was observed after introduction of glucose oxidase (Gox) and glucose, or of hydrogen peroxide (H(2)O(2)). The composition of the surface was monitored using AFM and XPS, a detailed XPS analysis being based on the discrimination between oxygen of organic and inorganic nature proposed in a previous study. In H(2)O(2) medium, the main changes regarding the inorganic phase were the increase of the oxygen concentration in the passive film, the increase of the molar concentration ratio of oxidized species Fe(ox)/Cr(ox) and the growth of nanoparticles, presumably made of ferric oxide/hydroxide. In Gox medium, no significant changes were observed in both oxygen concentration and Fe(ox)/Cr(ox) ratio, but the density of colloidal particles decreased, indicating a dissolution of Fe oxide/hydroxide under the influence of gluconate. In contrast with H(2)O(2), in SFW and Gox the amount of organic compounds increased due to the accumulation of polysaccharides and proteins. The influence of glucose oxidase on the ennoblement of stainless steel is not due to indirect effects of H(2)O(2) through the change of surface composition. The E(corr) ennoblement seems to be directly due to the presence of H(2)O(2) and to the electrochemical behavior of H(2)O(2) and related oxygen species. This consideration is important for understanding and controlling microbial influenced corrosion.

Enzyme immobilization on silane-modified surface through short linkers: fate of interfacial phases and impact on catalytic activity.

We investigated the mechanism of enzyme immobilization on silanized surfaces through coupling agents (cross-linkers) in order to understand the role of these molecules on interfacial processes and their effect on catalytic activity. To this end, we used a model multimeric enzyme (G6PDH) and several cross-linking molecules with different chemical properties, including the nature of the end-group (-NCO, -NCS, -CHO), the connecting chain (aliphatic vs aromatic), and geometrical constraints (meta vs para-disubstituted aromatics). There did not seem to be radical differences in the mechanism of enzyme adsorption according to the linker used as judged from QCM-D, except that in the case of DIC (1,4-phenylene diisocyanate) the adsorption occurred more rapidly. In contrast, the nature of the cross-linker exerted a strong influence on the amount of enzyme immobilized as estimated from XPS, and more unexpectedly on the stability of the underlying silane layer. DIC, PDC (1,4-phenylene diisothiocyanate), or GA (glutaraldehyde) allowed successful enzyme immobilization. When the geometry of the linker was changed from 1,4-phenylene diisothiocyanate to 1,3-phenylene diisothiocyanate (MDC), the silane layer was subjected to degradation, upon enzyme adsorption, and the amount of immobilized molecules was significantly lowered. TE (terephtalaldehyde) and direct enzyme deposition without cross-linker were similar to MDC. The organization of immobilized enzymes also depended on the immobilization procedure, as different degrees of aggregation were observed by AFM. A correlation between the size of the aggregates and the catalytic properties of the enzyme was established, suggesting that aggregation may enhance the thermostability of the multimeric enzyme, probably through a compaction of the 3D structure.

Ordered nanostructures on a hydroxylated aluminum surface through the self-assembly of fatty acids.

We investigate the mechanism of self-assembly of fatty acids (FA) and methyl oleate on an Al oxy-hydroxide surface with a view to deciphering the role and nature of interfacial processes (adsorption, chemical binding, molecular organization, etc.). For this purpose, we focus on parameters related to intrinsic properties of molecules, namely the level of unsaturation and the nature of the head group (carboxylic acid or ester). After the FA adsorption, the presence of coordinative bonded carboxylate species on the Al oxy-hydroxide surface is demonstrated by means of PM-IRRAS analysis. We observe that contact of methyl oleate with the surface leads to its chemical transformation through a saponification reaction. As a consequence, it binds to the surface in a manner similar to that for fatty acids. Through an innovative mode of atomic force microscopy (AFM), the organization of the adsorbed molecules is demonstrated. Our results reveal the existence of highly ordered nanostructures guided by the FA self-assembly. The size of these nanostructures was determined with accuracy, suggesting that it exceeds one FA monolayer. By contrast, no organization was observed with methyl oleate.

Probing peptide-inorganic surface interaction at the single molecule level using force spectroscopy

We investigate the interaction between D-Ala-D-Ala peptide and a stainless steel (SS) surface by AFM force spectroscopy with view to understand the role and nature of interfacial processes at the single molecule level. For this purpose, force-distance curves were recorded between the D-Ala-D-Ala modified tip and the SS surface in NaHCO(3)-enriched medium. The SS surface was prepared in a way that allows iron oxide species, presumably FeOOH, to be formed and remains stable during AFM measurements. Dynamic force measurements show that the unbinding force linearly increases with the logarithm of the loading rate, as generally observed for receptor–ligand complexes. Our results reveal also the existence of two regimes, suggesting the presence of multiple energy barriers in the energy landscape. From these dynamic force spectroscopy measurements, the kinetic off-rate constant is determined. An average unbinding force in the range of 50-300 pN is obtained, depending on the loading rate. Accordingly, in a medium in which the electrostatic interactions are not dominating, the binding mechanism of the peptide and SS surface cannot be attributed to covalent bonds and may be due to a combination of van der Waals and hydrogen bonds. Our findings open up new way to probe peptide-inorganic surface interactions and to understand the mechanism of peptide specific binding which is of particular interest in the design of hybrid materials.

Development of novel anti-Kv 11.1 antibody-conjugated PEG-TiO2 nanoparticles for targeting pancreatic ductal adenocarcinoma cells.

Titanium dioxide (TiO2) has been widely used in many nanotechnology areas including nanomedicine, where it could be proposed for the photodynamic and sonodynamic cancer therapies. However, TiO2 nanoformulations have been shown to be toxic for living cells. In this article, we report the development of a new delivery system, based on nontoxic TiO2 nanoparticles, further conjugated with a monoclonal antibody against a novel and easily accessible tumor marker, e.g., the Kv 11.1 potassium channel. We synthesized, by simple solvothermal method, dicarboxylic acid-terminated PEG TiO2 nanocrystals (PEG–TiO2 NPs). Anti-Kv 11.1 monoclonal antibodies (Kv 11.1-Mab) were further linked to the terminal carboxylic acid groups. Proper conjugation was confirmed by X-ray photoelectron spectroscopy analysis. Kv 11.1-Mab-PEG–TiO2 NPs efficiently recognized the specific Kv 11.1 antigen, both in vitro and in pancreatic ductal adenocarcinoma (PDAC) cells, which express the Kv 11.1 channel onto the plasma membrane. Both PEG TiO2 and Kv 11.1-Mab-PEG–TiO2 NPs were not cytotoxic, but only Kv 11.1-Mab-PEG–TiO2 NPs were efficiently internalized into PDAC cells. Data gathered from this study may have further applications for the chemical design of nanostructures to be applied for therapeutic purposes in pancreatic cancer.

nPEG-TiO2 Nanoparticles: A Facile Route to Elaborate Nanostructured Surfaces for Biological Applications

We report the synthesis of diacid-terminated PEG-functionalized cubic TiO(2) nanocrystals by a simple one-step solvothermal method, and their further use to form nanostructured surfaces for protein immobilization. The relevance and major interest of the so-obtained nanocrystals are the presence of terminal carboxylic acid groups at their surface, as confirmed by infrared analyses, in addition to the surrounding PEG chains, essential to avoid non specific interactions. These functional chemical groups were used to (i) immobilize the synthesized nanocubes on a cysteamine-modified Au surface, and to (ii) attach proteins via a presumable covalent link. AFM images show that the shapes and the narrow size distribution of the nanocubes, observed by TEM, were preserved after their immobilization on the modified Au surface. Moreover, the efficiency and specificity of antigen recognition were demonstrated using spectroscopic analyses. Our successful approach provides a versatile and facile way to elaborate specific and sensitive nanostructured surfaces for biosensors.