Surface nanostructures for fluorescence probing of supported lipid bilayers on reflective substrates.

The fluorescence interference contrast (FLIC) effect prevents the use of fluorescence techniques to probe the continuity and fluidity of supported lipid bilayers on reflective materials due to a lack of detectable fluorescence. Here we show that adding nanostructures onto reflective surfaces to locally confer a certain distance between the deposited fluorophores and the reflecting surface enables fluorescence detection on the nanostuctures. The nanostructures consist of either deposited nanoparticles or epitaxial nanowires directly grown on the substrate and are designed such that they can support a lipid bilayer. This simple method increases the fluorescence signal sufficiently to enable bilayer fluorescence detection and to observe the recovery of fluorescence after photobleaching in order to assess lipid bilayer formation on any reflective surface.

Support of Neuronal Growth Over Glial Growth and Guidance of Optic Nerve Axons by Vertical Nanowire Arrays.

Neural cultures are very useful in neuroscience, providing simpler and better controlled systems than the in vivo situation. Neural tissue contains two main cell types, neurons and glia, and interactions between these are essential for appropriate neuronal development. In neural cultures, glial cells tend to overgrow neurons, limiting the access to neuronal interrogation. There is therefore a pressing need for improved systems that enable a good separation when coculturing neurons and glial cells simultaneously, allowing one to address the neurons unequivocally. Here, we used substrates consisting of dense arrays of vertical nanowires intercalated by flat regions to separate retinal neurons and glial cells in distinct, but neighboring, compartments. We also generated a nanowire patterning capable of guiding optic nerve axons. The results will facilitate the design of surfaces aimed at studying and controlling neuronal networks.

3D-nanostructured boron-doped diamond for microelectrode array neural interfacing.

The electrode material is a key element in the design of long-term neural implants and neuroprostheses. To date, the ideal electrode material offering high longevity, biocompatibility, low-noise recording and high stimulation capabilities remains to be found. We show that 3D-nanostructured boron doped diamond (BDD), an innovative material consisting in a chemically stable material with a high aspect ratio structure obtained by encapsulation of a carbon nanotube template within two BDD nanolayers, allows neural cell attachment, survival and neurite extension. Further, we developed arrays of 20-µm-diameter 3D-nanostructured BDD microelectrodes for neural interfacing. These microelectrodes exhibited low impedances and low intrinsic recording noise levels. In particular, they allowed the detection of low amplitude (10-20 µV) local-field potentials, single units and multiunit bursts neural activity in both acute whole embryonic hindbrain-spinal cord preparations and long-term hippocampal cell cultures. Also, cyclic voltammetry measurements showed a wide potential window of about 3 V and a charge storage capacity of 10 mC.cm(-2), showing high potentiality of this material for neural stimulation. These results demonstrate the attractiveness of 3D-nanostructured BDD as a novel material for neural interfacing, with potential applications for the design of biocompatible neural implants for the exploration and rehabilitation of the nervous system.

Fluid and highly curved model membranes on vertical nanowire arrays.

Sensing and manipulating living cells using vertical nanowire devices requires a complete understanding of cell behavior on these substrates. Changes in cell function and phenotype are often triggered by events taking place at the plasma membrane, the properties of which are influenced by local curvature. The nanowire topography can therefore be expected to greatly affect the cell membrane, emphasizing the importance of studying membranes on vertical nanowire arrays. Here, we used supported phospholipid bilayers as a model for biomembranes. We demonstrate the formation of fluid supported bilayers on vertical nanowire forests using self-assembly from vesicles in solution. The bilayers were found to follow the contours of the nanowires to form continuous and locally highly curved model membranes. Distinct from standard flat supported lipid bilayers, the high aspect ratio of the nanowires results in a large bilayer surface available for the immobilization and study of biomolecules. We used these bilayers to bind a membrane-anchored protein as well as tethered vesicles on the nanowire substrate. The nanowire-bilayer platform shown here can be expanded from fundamental studies of lipid membranes on controlled curvature substrates to the development of innovative membrane-based nanosensors.

Neurite outgrowth and synaptophysin expression of postnatal CNS neurons on GaP nanowire arrays in long-term retinal cell culture.

We have established long-term cultures of postnatal retinal cells on arrays of gallium phosphide nanowires of different geometries. Rod and cone photoreceptors, ganglion cells and bipolar cells survived on the substrates for at least 18 days in vitro. Glial cells were also observed, but these did not overgrow the neuronal population. On nanowires, neurons extended numerous long and branched neurites that expressed the synaptic vesicle marker synaptophysin. The longest nanowires (4 µm long) allowed a greater attachment and neurite elongation and our analysis suggests that the length of the nanowire per se and/or the adsorption of biomolecules on the nanowires may have been important factors regulating the observed cell behavior. The study thus shows that CNS neurons are amenable to gallium phosphide nanowires, probably as they create conditions that more closely resemble those encountered in the in vivo environment. These findings suggest that gallium phosphide nanowires may be considered as a material of interest when improving existing or designing the next generation of implantable devices. The features of gallium phosphide nanowires can be precisely controlled, making them suitable for this purpose.

Surface-assisted laser desorption-ionization mass spectrometry on titanium dioxide (TiO2) nanotube layers.

The paper reports on the use of a titanium oxide (TiO(2)) nanotube layer as a sensitive substrate for surface-assisted laser desorption-ionization mass spectrometry (SALDI-MS) of peptides and small molecules. The nanotube layers were prepared by electrochemical anodization of titanium foil. The optimized TiO(2) nanotubes morphology coupled to a controlled surface chemistry allowed desorption-ionization (D/I) of a peptide mixture (Mix1) with a detection limit of 10 femtomoles for the neurotensin peptide. The performance of the TiO(2) nanotubes for the D/I of small molecules was also tested for the detection of sutent, a small tyrosine kinase inhibitor, and verapamil. A detection limit of 50 fmol was obtained for these molecules, as compared to 500 fmol using classical matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). Both amorphous and anatase TiO(2) layers displayed a comparable performance for D/I of analyte molecules. In a control experiment, we have performed D/I of analyte molecules on a flat TiO(2) layer. The absence of signal emphasizes the role of the nanostructured substrate in the D/I process.

High sensitive matrix-free mass spectrometry analysis of peptides using silicon nanowires-based digital microfluidic device.

We present for the first time an electrowetting on dielectric (EWOD) microfluidic system coupled to a surface-assisted laser desorption-ionization (SALDI) silicon nanowire-based interface for mass spectrometry (MS) analysis of small biomolecules. Here, the transfer of analytes has been achieved on specific locations on the SALDI interface followed by their subsequent mass spectrometry analysis without the use of an organic matrix. To achieve this purpose, a device comprising a digital microfluidic system and a patterned superhydrophobic/superhydrophilic silicon nanowire interface was developed. The digital microfluidic system serves for the displacement of the droplets containing analytes, via an electrowetting actuation, inside the superhydrophilic patterns. The nanostructured silicon interface acts as an inorganic target for matrix-free laser desorption-ionization mass spectrometry analysis of the dried analytes. The proposed device can be easily used to realize several basic operations of a Lab-on-Chip such as analyte displacement and rinsing prior to MS analysis. We have demonstrated that the analysis of low molecular weight compounds (700 m/z) can be achieved with a very high sensitivity (down to 10 fmol µL(-1)).

Chips from chips: application to the study of antibody responses to methylated proteins.

Peptide microarrays are useful tools for the characterization of humoral responses against peptide antigens. The study of post-translational modifications requires the printing of appropriately modified peptides, whose synthesis can be time-consuming and expensive. We describe here a method named "chips from chips", which allows probing the presence of antibodies directed toward modified peptide antigens starting from unmodified peptide microarrays. The chip from chip concept is based on the modification of peptide microspots by simple chemical reactions. The starting peptide chip (parent chip) is covered by the reagent solution, thereby allowing the modification of specific residues to occur, resulting in the production of a modified peptide chip (daughter chip). Both parent and daughter chips can then be used for interaction studies. The method is illustrated using reductive methylation for converting lysines into dimethyllysines. The rate of methylation was studied using specific antibodies and fluorescence detection, or surface-assisted laser desorption ionization mass spectrometry. This later technique showed unambiguously the efficient methylation of the peptide probes. The method was then used to study the humoral response against the Mycobacterium tuberculosis heparin-binding hemagglutinin, a methylated surface-associated virulence factor and powerful diagnostic and protective antigen.

In situ chemical modification of peptide microarrays: characterization by desorption/ionization on silicon nanowires.

Peptide microarrays are useful devices for the high throughput study of biomolecular or peptide-cell interactions. Whereas the synthesis of unmodified peptide libraries is an easy task and can be performed at reasonable cost, the synthesis of libraries of modified peptides remains expensive and time consuming. This bottleneck led us to examine the possibility to produce modified peptide microspots by in situ chemical modification of unmodified peptide microspots. The great advantage would be the preparation of a series of complex microarrays (daughter microarrays) starting from an easy-to-make and cost-effective unmodified peptide microarray (parent microarray). One step toward this goal has been presented in the accompanying chapter dealing with the in situ methylation methodology for studying the specificity of antibodies directed toward methylated epitopes. Here we describe the development of a novel desorption/ionization on silicon nanowires mass spectrometry (DIOSiNWs-MS) technique for characterizing the in situ chemical modification of peptides.

Matrix-free laser desorption/ionization mass spectrometry on silicon nanowire arrays prepared by chemical etching of crystalline silicon.

This paper reports on the use of silicon nanowires (SiNWs), easily prepared in a single step by chemical etching of crystalline silicon in HF/AgNO(3) aqueous solution, as a highly sensitive substrate for laser desorption/ionization mass spectrometry (LDI-MS) analysis. The SiNWs' diameter and length depend on the etchant concentration and dissolution time. Optimized LDI substrate consists of nanowires with an average diameter in the range of 20-100 nm and 2.5 mum in length. The optimized SiNWs' surface morphology coupled to a controlled surface chemistry allowed a significant LDI-MS performance through measurements of a broad range of analytes, including small molecules, peptides, and a bovine serum albumin (BSA) digest. A signal-to-noise ratio of 250 was ascertained for a 10 fmol bradykinin pick, in reflector mode acquisition. Likewise, the sutent, a small tyrosine kinase inhibitor, could be observed down to 10 fmol, as compared to 500 fmol limit detection using the classical matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We have further investigated the optical properties of the nanowires, and our results suggest that they have a small or no effect on the desorption/ionization (D/I) process. On the contrary, the surface morphology and thermal properties of the silicon nanostructures are found to be the essential features contributing to the D/I performance.

Selective adhesion of Bacillus cereus spores on heterogeneously wetted silicon nanowires.

The article reports on the selective adhesion of Bacillus cereus spores on patterned and heterogeneously wetted superhydrophobic silicon nanowires surfaces. Superhydrophilic patterns on superhydrophobic silicon nanowire (SiNW) surfaces were prepared by a standard optical lithography technique. Exposure of the patterned surface to a suspension of B. cereus spores in water led to their specific adsorption in superhydrophobic areas. Comparable results were obtained on a patterned hydrophobic/hydrophilic flat silicon (Si) surface even though a higher concentration of spores was observed on the hydrophobic areas, as compared to the superhydrophobic regions of the SiNW substrate. The surfaces were characterized using scanning electron microscopy (SEM), fluorescence spectroscopy, and contact angle measurements.

Biomolecule and nanoparticle transfer on patterned and heterogeneously wetted superhydrophobic silicon nanowire surfaces.

We report on the use of patterned superhydrophobic silicon nanowire surfaces for the efficient, selective transfer of biological molecules and nanoparticles. Superhydrophilic patterns are prepared on superhydrophobic silicon nanowire surfaces using standard optical lithography. The resulting water-repellent surface allows material transfer and physisorption to the superhydrophilic islands upon exposure to an aqueous solution containing peptides, proteins, or nanoparticles.