Molecular arrangement between multivalent glycocluster and Pseudomonas aeruginosa LecA (PA-IL) by atomic force microscopy: influence of the glycocluster concentration.

New therapeutics strategy against cystic fibrosis seeks to prevent the adhesion of the bacterium Pseudomonas aeruginosa (PA) on the epithelial cells in the lungs. One of the factors that induces the adhesion is the interaction between natural glycocluster present on the cells and lectins such as the PA lectin LecA (PA-IL) present on the bacterium. By introducing synthetic glycoclusters with a great affinity with the lectin PA-IL, the adhesion can be prevented. In this study, we characterized, by atomic force microscopy, the interaction between a tetra-galactosylated glycocluster and the PA-IL lectin for high concentration of lectins (2.5 µM).We showed that the strong lectin/lectin interaction is reduced even for low concentration of glycoclusters (1 for 20 000 lectins). We assumed that it is due to the tensioactive behavior of the glycoclusters. It was shown that the arrangement of the created complexes induced different structures evolving from one-dimensional elongated aggregates to two-dimensional compact islands when increasing the glycocluster concentration. This evolution can be interpreted as the predominance of the glycocluster/lectin interaction.

Assessment of porous silicon substrate for well-characterised sensitive DNA chip implement.

A biochip approach based on porous silicon as substrate is presented. The goal is to enhance the sensitivity of the biochip by increasing the specific surface area on the support. The elaboration of porous silicon layers has been optimized to guarantee good accessibility for large bio-molecule targets. Oligonucleotide probes are synthesised directly on the surface using phosphoramidite chemistry. The high specific surface area of porous silicon allows the direct characterisation, by infrared spectroscopy, of the porous layer formation and the functionalisation steps. The monolayer grafting and derivatisation protocol is additionally characterized by wettability and fluorescence microscopy. The surface modification of porous layers (i.e. thermal oxidation and chemical derivatisation) ensures the stability of the structure against strong chemical reagents used during the direct oligonucleotide synthesis. Finally the protocol is successfully transferred to a flat Si/SiO(2) substrate, and validated by biological target specific recognition during hybridisation tests. In particular, radioactive measurements show a 10-fold enhancement of the oligonucleotide surface density on the porous silicon substrate compared to the flat thermal silica.

Optimisation of a silicon/silicon dioxide substrate for a fluorescence DNA microarray.

This paper presents a comprehensive theory and experimental characterisation of the modulation of the fluorescence intensity by the construction of optical interferences on oxidised silicon substrates used for DNA microarrays. The model predicts a 90-fold variation of the fluorescence signal depending on the oxide thickness. For a Cy3 dye, the signal is maximal for a 90 nm oxide thickness corresponding to a 7.5-fold enhancement with respect to a standard glass substrate. For experimental validation of the model, we have prepared Si/SiO2 substrates with different parallel steps of decreasing oxide thicknesses on the same sample using a buffered oxide etch (BOE) etching process after thermal oxidation. The SiO2 surface has been functionalized by a silane monolayer before in situ synthesis of L185 oligonucleotide probes. After hybridisation with complementary targets, the variations of the fluorescence intensity versus oxide thickness are in very good accordance with the theoretical model. The experimental comparison against a glass substrate shows a 10-fold enhancement of the detection sensitivity. Our results demonstrate that a Si/SiO2 substrate is an attractive alternative to standard glass slides for the realisation of fluorescence DNA microarrays whenever detection sensitivity is an important issue.

Immobilization of homooligonucleotide probe layers onto Si/SiO(2) substrates: characterization by electrochemical impedance measurements and radiolabelling.

Radiolabelling and electrochemical impedance measurements were used to characterize the immobilization of single stranded homooligonucleotides onto silica surfaces and their subsequent hybridization with complementary strands. The immobilization procedure consists of grafting an epoxysilane onto microelectronic grade Si/SiO(2) substrates, and coupling oligonucleotides bearing a hexylamine linker onto the epoxy moiety. Radiolabelling was used as a reference method to quantify the amount of immobilized and hybridized oligonucleotides. These results show that the Si/SiO(2) substrates modified with an epoxysilane yield a surface concentration of approximately 10(11) strands/cm(2) for the immobilized oligonucleotides, after vigorous washings, and that approximately 36% of these undergo hybridization with complementary strands. The impedance measurements, which provide a direct means of detecting variations in electrical charge accumulation across the semiconductor/oxide/electrolyte structure when the oxide surface is chemically modified, show that the semiconductor's flat band potential undergoes reproducible shifts of -150 and -100 mV following the immobilization and the hybridization step, respectively. These results demonstrate that electrochemical impedance measurements using chemically modified semiconductor/oxide/electrolyte structures of this type offer a viable alternative for the direct detection of complementary DNA strands upon hybridization.

Comparison between electrochemical and optoelectrochemical impedance measurements for detection of DNA hybridization.

The principles of the electrochemical and optoelectrochemical impedance measurements on bare electrolyte/dielectric/semiconductor structures are described. The analysis of the experimental curves allows access to several indications concerning the electrical behavior of such structures. The application of these techniques to follow the electrical behavior of structures modified with two biological systems was investigated. The antibody/antigen recognition did not change the surface charge and, therefore, did not affect the impedance curves with respect to the applied potential. By contrast, the hybridization of two complementary DNA strands on the surface of the structure induced a variation of flat band potential of the semiconductor leading to a shift of impedance curves along the potential axis. This means that it is possible to detect directly the DNA hybridization without the use of labeled probes. The use of light allows the surface to be probed locally. In the future, the application of this technique for direct detection of hybridization on DNA chips should be possible.

Monitoring of melamine contamination in fat watery milk by the photoluminescence analysis.

A photoluminescence method to detect the toxic melamine contamination in fat watery milk has been proposed. Despite the quite different luminescence origins of milk and melamine patterns, their wide emission spectra under UV excitation are similar and in the range of 2.2-3.5 eV. The complex milk photoluminescence spectrum composed of riboflavin, furosine, lactulose, Vitamin E and tryptophan emitting species can be modified if milk pattern is undergone by acid treatment (for example, in vinegar). At the same time the melamine emission is not subjected to any modification in vinegar. It allows quantitatively discriminating the melamine contamination in milk in linear range, at least, 0.05-7 g/l from different photoluminescence spectra of milk (water) with and without melamine. Limit of melamine detection achieves 0.01 g/l.

New method for DNA microarrays development: applied to human platelet antigens polymorphisms.

DNA microarrays are a powerful experimental tool for the detection of specific genomic sequences and are invaluable to a broad array of applications: clinical diagnosis, personalized medicine, drug research and development, gene therapy, food control technologies, and environmental sciences. Alloimmunization to human platelet antigens (HPAs) is commonly responsible for neonatal alloimmune thrombocytopenia, post-transfusional purpura and platelet transfusion refractoriness. Using DNA microarrays, we developed a diagnosis to type the biallelic HPA-1 platelet group. The region for the human genomic DNA sequence that contains the polymorphism responsible for HPA-1 alleles was amplified by polymerase chain reaction (PCR). The expected DNA fragments were hybridized on DNA microarrays, and the data were analyzed using specially developed software. Our initial results show that the two HPA-1 antigens polymorphisms containing a single base difference were detected using DNA microarrays.

Implementation of DNA chips obtained by microprojection for diagnostic and personalized medicine.

It is expected that rapidly emergent new fields of application for DNA chips will be Diagnostic and Personalized Medicine. These new applications will require a limited number of probes, generally from 100 to 1000. So, after a brief review of the existing techniques to manufacture DNA chips, which are efficient for R&D applications and which often require a higher number of probes, we shall first report some advances in the silanization of the substrates and the grafting of probes to improve the robustness and the reliability of the devices. Then we shall discuss two manufacturing processes working at the scale of a nanoliter of reactant: ex situ and in situ fabrication by microprojection. We shall see how these processes are complementary and may be used to design and produce chips, at a large scale, for these new applications.