Effect of a static magnetic field on Escherichia coli adhesion and orientation.

This preliminary study focused on the effect of exposure to 0.5 T static magnetic fields on Escherichia coli adhesion and orientation. We investigated the difference in bacterial adhesion on the surface of glass and indium tin oxide-coated glass when exposed to a magnetic field either perpendicular or parallel to the adhesion surface (vectors of magnetic induction are perpendicular or parallel to the adhesion surface, respectively). Control cultures were simultaneously grown under identical conditions but without exposure to the magnetic field. We observed a decrease in cell adhesion after exposure to the magnetic field. Orientation of bacteria cells was affected after exposure to a parallel magnetic field. On the other hand, no effect on the orientation of bacteria cells was observed after exposure to a perpendicular magnetic field.

Surface assembly on biofunctional magnetic nanobeads for the study of protein-ligand interactions.

One of the major challenges of proteomics today is to increase the power potential for the identification of as many proteins as possible and to characterize their interactions with specific free ligands (interactomics) or present on cell walls (cell marker), in order to obtain a global, integrated view of disease processes, cellular processes and networks at the protein level. The work presented here proposes the development of biofunctionalized magnetic nanobeads that might be used for interactomic investigations. The strategy consisted in immobilizing proteins via a non covalent technique that provides greater possibilities for the advent of faster, cheaper and highly miniaturizable protein analysis systems, in particular in situations where the amount of isolated protein is scarce (trace proteins). The advantage of the immobilization technique proposed here over more conventional covalent binding techniques is that it is versatile and universal (not protein specific) thus applicable to a wide range of proteins, in "mild" conditions that are non deleterious to the native structure and bioactivity of the immobilized protein. The feasibility of the technique was investigated using a model protein (streptavidin). The nanobeads were analyzed in size by light diffusion and transmission electronic spectroscopy, and in quantity of immobilized protein using a bioassay developed in the laboratory. Results are promising in that nanobeads exhibited good colloidal stability and surface concentrations in the monolayer range.

Evaluation of endothelial cell adhesion onto different protein/gold electrodes by EIS.

To study cell attachment to biomaterials, several proteins such as fibronectin, collagen IV, heparin, immunoglobulin G, and albumin have been deposited onto polystyrene adsorbed on a self-assembled monolayer (silane or thiol) on glass or gold, respectively. The different steps of this multilayer assembly have been characterized by electrochemical impedance spectroscopy (EIS). These data are compared to those of adhesion rate, viability percentage, and cytoskeleton labeling for a better understanding of the cell adhesion process to each protein. All the proteins are endothelial cell adhering biomolecules but not with the same features. A linear relationship has been established between adhesion rate and resistance of the endothelial cell/protein interface for all negatively charged proteins.

Electrical probing of endothelial cell behaviour on a fibronectin/polystyrene/thiol/gold electrode by Faradaic electrochemical impedance spectroscopy (EIS).

The electrochemical impedance spectroscopy (EIS) technique has been shown to be an effective tool for monitoring endothelial cell behaviour on a multilayer functionalised gold electrode. Polystyrene, a reproducible model substrate, is deposited as a thin layer on a thiol functionalised gold electrode. Fibronectin, a protein promoting endothelial cell adhesion, is then adsorbed on the polystyrene surface. The different steps of this multilayer assembly are characterized by Faradaic impedance. The charge transfer resistance and the capacitance for the total layer are modified at each step according to the electrical properties of each layer. This gives the endothelial cells' electrical state in terms of its resistive and capacitive properties. In this study, the endothelial cell layer presents a specific charge transfer resistance equal to 1.55 kOmega cm(2) with no large defects in the cell layer, and a specific capacitance equal to few microF cm(-2) explained by the existence of pseudopods. These electrical properties are correlated to the endothelial cell viability, adhesion and cytoskeleton organization.

Cytotoxicity of polyethyleneimine (PEI), precursor base layer of polyelectrolyte multilayer films.

Polyethyleneimine (PEI) is a synthetic polymer commonly used as precursor base layer in polyelectrolyte multilayer films. However, the biological properties of this cationic macromolecule are poorly understood. The aim of this experimental investigation was to evaluate in vitro the biocompatibility of PEI towards two different human cell lines. The experimental investigation was undertaken on pure titanium (Ti) and nickel-titanium (NiTi) alloy samples with an average surface roughness of Ra=0.3microm. A biological study was undertaken at day 0 (2h after seeding), day 2, day 4 and day 7 to observe the cellular response of fibroblasts and osteoblasts cell lines in terms of morphology, adhesion (as observed by scanning electron microscopy), and viability (Mosmann's test). The results showed that PEI can be successfully deposited onto Ti or NiTi alloy, but generates a detrimental cellular response on both substrates as illustrated by a decrease of both fibroblast and osteoblast adhesion and proliferation over a 7-day culture period. These results suggest that PEI is potentially cytotoxic and may not be biocompatible enough in clinical applications using high molecular weight. As a consequence, polyelectrolyte multilayer films, which are promising in prosthesis and implantology fields, could not be coated with PEI at a high molecular weight. A lower molecular weight should be considered or a more biocompatible molecular base as precursor layer of polyelectrolyte multilayer films would be better to use for a good human bio-integration.

Electrochemical impedance probing of DNA hybridisation on oligonucleotide-functionalised polypyrrole.

We report a new approach for detecting DNA hybridisation using non faradaic electrochemical impedance spectroscopy. The technique was applied to a system of DNA probes bearing amine groups that are immobilized by covalent grafting on a supporting polypyrrole matrix functionalised with activated ester groups. The kinetics of the attachment of the ss-DNA probe was monitored using the temporal evolution of the open circuit potential (OCP). This measurement allows the determination of the time necessary for the chemical reaction of ss-DNA probe into the polypyrrole backbone. The hybridisation reactions with the DNA complementary target and non complementary target were investigated by non faradaic electrochemical impedance spectroscopy. Results show a significant modification in the Nyquist plot upon addition of the complementary target whereas, in presence of the non complementary target, the Nyquist plot is not modified. The spectra, in the form of Nyquist plot, were analysed with the Randles circuit. The transfer charge resistance R(2) shows a linear variation versus the complementary target concentration. Sensitivity and detection limit (0.2nM) were determined and detection limit was lower of one order of magnitude than that obtained with the same system and measuring variation of the oxidation current at constant potential.

Study of mixed Langmuir-Blodgett films of immunoglobulin G/amphiphile and their application for immunosensor engineering.

Langmuir-Blodgett (LB) technique appears to be quite suitable for generating biospecific surfaces and it has potential application for fabricating biosensors. In this work, mixed Langmuir-Blodgett films of immunoglobulin G/amphiphile have been transferred onto hydrophobic silver surface previously modified by 1-octadecanethiol (ODT) SAMs. In order to obtain stable LB films, the influences of different parameters - type of amphiphile, surface pressure and pH - on the properties of mixed IgG/amphiphile monolayer, were investigated. Electrochemical properties of the engineered immunosensor have been measured by impedimetric spectroscopy. The immunosensor obtained exhibits a high sensitivity and a good specificity in a linear dynamic range from 200 to 1000 ng ml(-1).

Surface free energy and bacterial retention to saliva-coated dental implant materials--an in vitro study.

The aim of the present investigation was to compare the in vitro bacterial retention on saliva-coated implant materials (pure titanium grade 2 (cp-Ti) and a titanium alloy (Ti-6Al-4V) surfaces), presenting similar surface roughness, and to assess the influence of physico-chemical surface properties of bacterial strain and implant materials on in vitro bacterial adherence. Two bacterial strains (one hydrophilic strain and one hydrophobic strain) were used and the following were evaluated: bacterial cell adherence, SFE values as well as the Lifshitz-van-der Waals, the Lewis acid base components of SFE, the interfacial free energy and the non-dispersive interactions according to two complementary contact angle measurement methods: the sessile drop method and the captive bubble method. Our results showed similar patterns of adherent bacterial cells on saliva-coated cp-Ti and saliva-coated Ti-6Al-4V. These findings could suggest that bacterial colonization (i.e. plaque formation) is similar on saliva-coated cp-Ti and Ti-6Al-4V surfaces and indicate that both materials could be suitable for use as transgingival abutment or healing implant components. The same physico-chemical properties exhibited by saliva-coated cp-Ti and TA6V, as shown by the sessile drop method and the captive bubble method, could explain this similar bacterial colonisation. Therefore, higher values of total surface free energy of saliva-coated cp-Ti and saliva-coated TA6V samples (gamma(SV) approximately 65mJ/m(2)) were reported using the captive bubble method indicating a less hydrophobic character of these surfaces than with the sessile drop method (gamma(S) approximately 44.50mJ/m(2)) and consequently possible differences in oral bacterial retention according the theory described by Absolom et al. The number of adherent hydrophobic S. sanguinis cells was two-fold higher than that of hydrophilic S. constellatus cells. Our results confirm that physico-chemical surface properties of oral bacterial strains play a role in bacterial retention to implant materials in the presence of adsorbed salivary proteins.

Fibroblast cells: a sensing bioelement for glucose detection by impedance spectroscopy.

Modifying the electrical properties of fibroblasts against various glucose concentrations can serve as a basis for a new, original sensing device. The aim of the present study is to test a new biosensor based on impedancemetry measurement using eukaryote cells. Fibroblast cells were grown on a small optically transparent indium tin oxide semiconductor electrode. Electrochemical impedance spectroscopy (EIS) was used to measure the effect of D-glucose on the electrical properties of fibroblast cells. Further analyses of the EIS results were performed using equivalent circuits in order to model the electrical flow through the interface. The linear calibration curve was established in the range 0-14 mM. The specification of the biosensors was verified using cytochalasin B as an inhibitor agent of the glucose transporters. The nonreactivity to sugars other than glucose was demonstrated. Such a biosensor could be applied to a more fundamental study of cell metabolism.