Multifunctional self-assembled nanoscale materials based on bio-matter are of special interests because of promising peculiar applications, like in optoelectronics, biosensors, photovoltaics. Among the existing carrier matrices conductive metal oxides (e.g. indium tin oxide, ITO), carbon nanotubes, graphenes, silicon (Si) are the most frequently used materials because of their unique characteristics such as good conductivity, good optical properties and excellent adhesion to substrates. In our work we combined purified photosynthetic reaction center protein (RC) and porous silicon (PSi) investigating the morphology and optoelectronic properties of the bio-nanocomposite material. FTIR spectroscopy, scanning electron microscopy and energydispersive X-ray spectroscopy indicated the binding of the protein to the PSi. Specular reflectance spectra showed a red shift in the characteristic interference peak of the PSi microcavity which was saturated at higher concentration of the protein. The binding was more effective if the functionalization was done by the Si-specific oligopeptide compared to the classical covalent binding via aminopropyl-triethoxysilane (APTES). Excitation by single saturation flashes indicated that the RC still exhibited photochemical turnover after the binding.

Investigating cell mechanics is of high interest because it includes several essential and fundamental cellular processes such as cell motion, deformation and interaction, as well as adhesion, division, shape maintenance and mechanical signals processing into biochemical ones. This type of studies can lead to a better understanding of mechanical properties of cells and cellular structures and their interactions with the environment can be also evaluated. Comparative studies on the mechanics of normal and malignant cells of the same type are of great importance as they can reveal new information about, and help understanding mechanisms of malignant transformation. In this work, we investigate the morphology and the elastic properties of live cultured, normal human mammalian epithelial cells (HMEC) and cancer breast epithelial cells (MCF7), using atomic force microscopy (AFM). We describe the different behavior of the two similar cell lines under curcumin treatment. The first changes in the HMEC cell morphology are observed after two hours long incubation with curcumin. A six-hour long treatment leaves the MCF7 cells morphology non-affected, but the microtubules of HMEC cells disassemble and form a ring-like organization circumscribing the nuclear area. The observed morphological changes were correlated to modifications in cell's mechanics via elasticity force-volume measurements. Curcumin treatment modified elasticity of the HMEC cells increasing the cell's average Young's modulus two- to three-fold, especially in the cytoplasmic area. Contrariwise, a slight decrease in the Young's modulus was noticed for the MCF7 cells, as they become softer due to the action of curcumin. This different response of the malignant and non-malignant cells under the same treatment might be of high importance in developing follow-up therapy against breast cancer. Moreover, an anticancer agent, the Cetuximab antibody that can interfere with Epidermal Growth Factor Receptor (EGFR) activity, was coupled to the AFM tip in order to evaluate the Cetuximab-EGFR binding forces on the surface of human epithelial carcinoma cells (A431) presenting an EGFR overexpression as in a large variety of tumors. The effect of mixed treatments, i.e. different therapeutic drugs in combination with the Cetuximab, was studied on the ligand-EGFR binding forces and on cell elasticity. Our results evidence the modification of the A431 cells' elasticity and of the binding forces between the EGFR receptor and the Cetuximab agent when the cells were treated with Cetuximab alone or in combination with other drugs, opening the way to the development of new approaches for epithelial carcinoma treatment.

Non-linear optical microscopy (NLOM) is an effective method to follow pathological processes involving collagen fibrosis or collagen destruction. In particular, the intensity of the second harmonic generation (SHG) signal depends on the structure and orientation of the collagen fibers. In this work, we have used two-photon fluorescence (2PEF) and SHG to investigate the dental caries process. The objective of this study is the characterization of the collagen network in sound and carious dentin using NLOM to study non decalcified teeth. Materials and methods: Longitudinal slices with thickness up to 0.5mm were prepared from freshly extracted teeth: 4 from a sound tooth without caries and 4 from a carious tooth. The samples were polished to 0.25 µm and cleaned up in an ultrasound alcohol bath for 5 min. Fluorescence microscopy, at a wavelength of 480nm, was performed on sound and carious dentin structures. Dentin collagen network images were provided by means of 2PEF and SHG, using an incident wavelength of 800nm on areas previously defined by fluorescence microscopy. Results and conclusion: We have visualized groups of collagen fibres that are constituents of the healthy dentin extracellular matrix and succeeded to differentiate between healthy and carious tooth dentin structure. In a carious lesion, the SHG signal is low and the collagen network is difficult to observe. The obtained 2PEF and SHG three-dimensional images of dentine reveal the tubule network. The visualization of the three dimensional structure of the samples and the optical sectioning capacity inherent to the NLOM technique is a powerful approach for non-invasive investigation of dentine structure and caries.

Cell spray deposition techniques for the needs of tissue engineering are being actively developed during the last few years. They permit easier deposition of cells on surfaces with complex configurations, creation of cell multilayers and three-dimensional cell cultures. Objectives: The aim of the present study was to optimize the parameters of cell spray deposition (cell concentration, cell environment during the spraying process, and functionalization of surfaces) in order to achieve better cell adhesion, improved cell vitality and cell proliferation on the surface. Methods: Two cell lines were used in the experiments: human pulp fibroblasts isolated from fleshly extracted teeth and immortalized oral keratinocytes, TERT-2 OKF-6 (BWH Cell Culture and Microscopy Core, Boston, MA, USA). The method of non-automatic nebulization in sterile conditions was used for cell deposition. Surfaces were functionalized prior to cell spraying with either different configurations of poly-lysine (PLL)/poly-glutamic acid (PGA) multilayers or with a collagen film (Collagen Type I from calf skin). Cell characterization was performed at 24, 72 hours and 7 days after deposition using fluorescence microscopy (Nikon TE 2000), FDA/PI vital cell coloration and atomic force microscopy (AFM- MFP3D Asylum Research) in tapping and/or contact mode Results: Fluorescence microscopy images recorded on the seventh day after cell deposition proved cell survival after the spraying and evidenced the pattern of cell vitality for this period. Cell surface imaging and force mapping via AFM in force-volume mode provided the characteristics and variations of cell morphology and cell elasticity. These measurements confirmed cell adhesion on the functionalized surfaces and enabled identifying the optimal surface functionalization for an improved cell adhesion and proliferation. Conclusion: Cell spraying is an innovative method for cell deposition onto different types of surfaces that can constitute a valuable technique in cell engineering.

Porous silicon (pSi) is a promising biomaterial that is non-toxic and bioresorbable. Surface modifications can offer control over the degradation rate and can also impart properties that promote cell adhesion. Coupling the capacities of Dental Pulp Stem Cells (DPSC) with the pSi properties is a promising tool in regenerative medicine. P-type silicon wafers were etched at a constant current density of 30 mA/cm2 or 300 mA/cm2. The samples were oxidized or hydrosililzed. The topography of surface modified pSi samples was analysed by scanning electronic microscope (SEM) and water contact angle measurement. Dental pulp cells were collected from healthy adults and analyzed by flow cytometry. Cells were incubated on pSi samples for either 4 hours or 24 hours. Cellular morphology on pSi was evaluated with fluorescein diacetate (FDA) staining. Cell proliferation was measured through acid phosphatase activity. After oxidation or hydrosililation, at either 30 or 300 mA/cm2, pSi wafers became clearly hydrophilic. SEM revealed a highly porous surface, with a mean size of pore of 10nm±2 for 30mA/cm2, and 21nm±3 for 300mA/cm2. With flow cytometry, cells were 17% CD34+ and 77% CD146+. Acid phosphatase assay showed that samples etched with 300mA/cm2 tend to offer a better adhesion for cells; the same tendency was observed for hydrosililation treatment. Cells presented the same morphology on pSi as on culture plate. Surface modification, by turning pSi from hydrophobic to hydrophilic, allows cell adhesion. The two tested sizes of pore and the two tested surface treatments allowed adhesion of DPSC. Cell morphology on pSi was similar to culture plates. pSi resorption time is influenced by surface modification and DPSC adhesion is possible on both surface modifications, highlighting an interest for cell/tissue graft. And the tunable size of pore might permit to incorporate growth factors or nutriments inside the scaffold.

We study the effect of different surface functionalization methods on the sensing performances of porous silicon (PSi) microcavities when used for detection of biomolecules. Previous research on porous silicon demonstrated versatility of these devices for sensor applications based on their photonic responses. The interface between biological molecules and the Si semiconductor surface is a key issue for improving biomolecular recognition in these devices. PSi microcavities were fabricated to reveal reflectivity pass-band spectra in the visible and near-infrared domain. To assure uniform infiltration of proteins the number of layers of Bragg mirrors was limited to five, the first layer being of high porosity. In one approach the devices were thermally oxidized and functionalized to assure covalent binding of molecules. Secondly, the as etched PSi surface was modified with adhesion peptides isolated via phage display technology and presenting high binding capacity for Si. Functionalization and molecular binding events were monitored via reflectometric interference spectra as shifts in the resonance peaks of the cavity structure due to changes in the refractive index when a biomolecule is attached to the large internal surface of PSi. Improved sensitivity is obtained due to the peptide interface linkers between the PSi and biological molecules compared to the silanized devices. We investigate the formation of peptide-Si interface layer via X-ray photoelectron spectroscopy, scanning tunneling microscopy and scanning electron microscopy.

The aim of this work was to investigate the possibilities to support device functionality that includes strongly confined and localized light emission and detection processes within nano/micro-structured semiconductors for biosensing applications. The interface between biological molecules and semiconductor surfaces, yet still under-explored is a key issue for improving biomolecular recognition in devices. We report on the use of adhesion peptides, elaborated via combinatorial phage-display libraries for controlled placement of biomolecules, leading to user-tailored hybrid photonic systems for molecular detection. An M13 bacteriophage library has been used to screen 1010 different peptides against various semiconductors to finally isolate specific peptides presenting a high binding capacity for the target surfaces. When used to functionalize porous silicon microcavities (PSiM) and GaAs/AlGaAs photonic crystals, we observe the formation of extremely thin (<1nm) peptide layers, hereby preserving the nanostructuration of the crystals. This is important to assure the photonic response of these tiny structures when they are functionalized by a biotinylated peptide layer and then used to capture streptavidin. Molecular detection was monitored via both linear and nonlinear optical measurements. Our linear reflectance spectra demonstrate an enhanced detection resolution via PSiM devices, when functionalized with the Si-specific peptide. Molecular capture at even lower concentrations (femtomols) is possible via the second harmonic generation of GaAs/AlGaAs photonic crystals when functionalized with GaAs-specific peptides. Our work demonstrates the outstanding value of adhesion peptides as interface linkers between semiconductors and biological molecules. They assure an enhanced molecular detection via both linear and nonlinear answers of photonic crystals.