The application of 3D micropatterning of agarose substrate for cell culture and in situ comet assays.

We report the fabrication of a 3D micropatterned agarose substrate that enables the culture of single or multiple cells. Patterning was performed on dried agarose using deep UV irradiation leading to 6-microm-deep micropatterns of 25-70 microm in diameter. Cell adhesion was facilitated by the specific grafting of ECM (extra cellular matrix) proteins such as fibronectin into the micropatterns. We show that the pattern size induced the adhesion of one or more cells, thus allowing precise control of the cell number used in the assay, and that cells proliferated similarly as in standard culture conditions. Moreover, cell polarity appeared well preserved on this substrate, so polarized cells like hepatoma HepaRG cells might maintain their differentiation status and act as primary human hepatocytes for hepatotoxicity testing. These 3D patterned culture slides have been successfully used for in situ comet assays and there is evidence that the genotoxic effects of sub-cytotoxic concentrations of drugs could be analyzed in a large number of single HeLa cells. Coupled with the parallel-based design of the 3D micropatterning, which allows automated image analysis, these results strongly indicate that this new cell array system is suitable for high-throughput cytotoxicity and genotoxicity screening applications.

Polypyrrole oligosaccharide array and surface plasmon resonance imaging for the measurement of glycosaminoglycan binding interactions.

In order to construct tools able to screen oligosaccharide-protein interactions, we have developed a polypyrrole-based oligosaccharide chip constructed via a copolymerization process of pyrrole and pyrrole-modified oligosaccharide. For our study, GAG (glycosaminoglycans) or GAG fragments, which are involved in many fundamental biological processes, were modified by the pyrrole moiety on their reducing end and then immobilized on the chip. The parallel binding events on the upperside of the surface can be simultaneously monitored and quantified in real time and without labeling by surface plasmon resonance imaging (SPRi). We show that electrocopolymerization of the oligosaccharide-pyrrole above a gold surface enables the covalent immobilization of multiple probes and the subsequent monitoring of their binding capacities using surface plasmon resonance imaging. Moreover, a biological application was made involving different GAG fragments and different proteins, including stromal cell-derived factor-1alpha (SDF-1alpha), interferon-gamma (IFN-gamma), and monoclonal antibody showing different affinity pattern.

Surface plasmon resonance imaging on polypyrrole protein chips: application to streptavidin immobilization and immunodetection.

Initially developed for the construction of DNA chips, the polypyrrole approach has been extended to other biochemical compounds (mainly proteins and oligosaccharides). This method allows one to copolymerize a pyrrole monomer with a biomolecule bearing a pyrrole group; this reaction is based on an electrochemical process allowing a very fast coupling of the biomolecule (probe) to a gold layer used as a working electrode. Fluorescence-based detection processes are classically used for evidence biorecognition on biochips; in order to avoid the labeling of the targets, we propose an alternative method--surface plasmon resonance imaging (SPRi). Surface plasmon resonance (SPR) is a typical label-free method for real-time detection of the binding of biological molecules onto functionalized surfaces. This surface-sensitive optical method is based upon evanescent wave sensing on a thin metal layer. The SPR approach described herein is performed in an imaging geometry that allows simultaneous monitoring of biorecognition reactions occurring on an array of immobilized probes (chip). In a SPRi experiment, local changes in reflectivity are recorded with a charge-coupled device (CCD) camera and are exploited to monitor up to 100 different biological reactions occurring on the molecules linked to the polypyrrole matrix. This method will be applied to protein recognition.

Analogies and surprising differences between recombinant nitric oxide synthase-like proteins from Staphylococcus aureus and Bacillus anthracis in their interactions with l-arginine analogs and iron ligands.

Genome sequencing has recently shown the presence of genes coding for NO-synthase (NOS)-like proteins in bacteria. The roles of these proteins remain unclear. The interactions of a series of l-arginine (l-arg) analogs and iron ligands with two recombinant NOS-like proteins from Staphylococcus aureus (saNOS) and Bacillus anthracis (baNOS) have been studied by UV-visible spectroscopy. SaNOS and baNOS in their ferric native state, as well as their complexes with l-arg analogs and with various ligands, exhibit spectral characteristics highly similar to the corresponding complexes of heme-thiolate proteins such as cytochromes P450 and NOSs. However, saNOS greatly differs from baNOS at the level of three main properties: (i) native saNOS mainly exists under an hexacoordinated low-spin ferric state whereas native baNOS is mainly high-spin, (ii) the addition of tetrahydrobiopterin (H4B) or H4B analogs leads to an increase of the affinity of l-arg for saNOS but not for baNOS, and (iii) saNOS Fe(II), contrary to baNOS, binds relatively bulky ligands such as nitrosoalkanes and tert-butylisocyanide. Thus, saNOS exhibits properties very similar to those of the oxygenase domain of inducible NOS (iNOS(oxy)) not containing H4B, as expected for a NOSoxy-like protein that does not contain H4B. By contrast, the properties of baNOS which look like those of H4B-containing iNOS(oxy) are unexpected for a NOS-like protein not containing H4B. The origin of these surprising properties of baNOS remains to be determined.

A polypyrrole protein microarray for antibody-antigen interaction studies using a label-free detection process.

Protein microarray is a promising technology that should combine rapidity and easy use with high throughput and versatility. This article describes a method in which an electrocopolymerization process is employed to graft biological molecules on to a chip so that surface plasmon resonance imaging may be used to detect molecular interactions. Copolymerization of pyrrole-modified protein and pyrrole is an efficient grafting process which immobilizes molecules at defined positions on a gold surface. Surface plasmon resonance imaging is an optical technique that allows real-time simultaneous detection of molecular interactions on a large number of spots without labeling. This method was successfully used to analyze antibody-antigen interactions. This illustrates its high specificity and good sensitivity and demonstrates its suitability for biological studies.