DNA chips are potentially powerful technologies for genotyping and gene expression profiling that rely on comparative analyses of up to thousands of "spots of analysis" on a glass support. The spot quality throughout the support influences spot-to-spot variations within an array and the repeatability of data across experiments. For glass slide DNA microarrays, droplets of DNA solution are deposited on functionalized glass slides and left to react through complete evaporation of the droplet. On hydrophobic flat surfaces, different modes of droplet evaporation can be attained. Under atmospheric pressure, water droplets tend to evaporate under two main regimes. Initially, the droplet flattens with a constant contact area, and then the droplet shrinks at a constant contact angle. As a result, the diameter and morphology of thousands of spots on microarrays are not uniform. This leads to poor and unreliable data processing results. In this work, we report the evaporation of an aqueous solution under a constant contact area mode. Evaporation under reduced pressure and the effect of reagent additives to the solution have been investigated. Video microscopy and digital image analysis techniques were applied to monitor the evaporation of the droplets. A mixture of surfactants was developed to maintain a constant area regime during evaporation and to form homogeneous spots. The control of some physicochemical properties (wetting, evaporation rate) of the droplet allows the formation of well-controlled spots compatible with DNA grafting. The influence of surfactant molecules on the mechanisms of evaporation is also discussed.

Homo-oligomer DNA strands were immobilized onto silicon/silicon dioxide electrodes using 3-aminopropyltriethoxysilane. These modified substrates were used as working electrodes in a three-electrode electrochemical cell. In-phase and out-of-phase impedances were measured in the range -1 to +1 V with respect to an Ag/AgCl reference electrode, with a superimposed 10 mV ac signal at frequencies of 20 and 100 kHz. Ex situ hybridization with complementary oligomer strands, performed at the surface of modified electrodes, is clearly reflected by negative shifts of about 100 mV in the flat-band potential of the semiconductor. Consecutive hybridization-denaturation steps show that the shifts are reproducible and the process is reversible. The in situ hybridization of complementary strands has also been observed with impedance measurements at Si/SiO 2 substrates and with the use of a field effect device. The direct detection of hybridization with a field effect device was performed under constant drain current mode, and the corresponding variations observed for the gate potential during hybridization are in good agreement with the flat band potential shifts observed with the impedance experiments. Measurements made in the presence of noncomplementary strands demonstrate the selectivity of the device.

Carbohydrates and glycoconjugates play a major role in key biological events such as cell-cell recognition, pathogenesis, and inflammation. [1,2] As a consequence, there is a need to understand the structural parameters governing the recognition of carbohydrates by their receptors. This knowledge will be of use for both fundamental research and potential applications in diagnostics or therapeutics. However, research in this field is slowed by the wide diversity of carbohydrate structures and by the minute amounts of materials available for experimentation. The design of sensitive and highthroughput technologies for the characterization of oligosaccharide/protein interactions [3] is therefore emerging as an attractive tool for chemists and biochemists. Available techniques such as isothermal calorimetry, enzyme-linked lectin assay, and even crystallographic studies provide data on carbohydrate/protein interactions, but they are often limited by the amount of available material.Carbohydrate microarray technology [4][5][6][7][8][9][10][11][12][13][14][15][16] is a promising approach for probing carbohydrate/protein interactions, and it permits the simultaneous screening of a number of biological interactions with only minute amounts of material. A large family of carbohydrate derivatives has been designed for immobilization on surfaces by various means. [5][6][7][8][9][10][11][12][13][14] However, this technology has various limitations. Relative surface densities of bound ligands are often not assessed. A careful optimization of the orientation and the distance separating the carbohydrate probe from the surface is often required.The interactions of oligosaccharides with lectins are usually weak (mm range) and can be enhanced using the "cluster effect" with multivalent ligands. [17][18][19][20] In the latter case, the distance between the residues should be optimized for binding. [21][22][23] Finally, the syntheses of functionalized oligosaccharide ligands are labor intensive.We report herein an original approach for the surface immobilization of oligosaccharides using glycoconjugate molecules that present a DNA sequence for anchoring onto DNA chips through hybridization. This approach has been used in the field of protein microarrays, [24,25] but to our knowledge this is the first time that such a strategy has been reported in the field of glycoarrays.Several syntheses of glycoconjugated oligonucleotides have been reported, but none are suitable for introducing different carbohydrate moieties. [26][27][28] We designed a conjugate that incorporates carbohydrate residue(s) for interacting with a lectin, an oligonucleotide sequence for anchoring on the surface, and a fluorescent tag at the 5'-end for the determination of relative surface densities (Figure 1). These moieties were assembled through a combination of automated oligonucleotide synthesis, and amidative oxidation and 1,3-dipolar cycloaddition ("click" chemistry) performed on a solid support (Scheme 1).[29] We introduced either one or three saccharide residue...

The synthesis of propargylated pentaerythrityl phosphodiester oligomers (PePOs) was achieved using a DNA synthesizer with a bis-propargylated pentaerythritol-based phosphoramidite. An azido fucose derivative was reacted under "click" chemistry conditions activated by microwaves to construct a series of glycosylated PePOs bearing 4, 6, 8, and 10 L-fucose residues. Binding to the fucose-specific bacterial lectin (PA-IIL) was determined for the fucosylated PePOs through an enzyme-linked lectin amplification competition assay. The IC50 values measured are 10-20 times better than for monovalent l-fucose and denotate for a "macromolecular" effect rather than a "cluster" effect.

Carbohydrates and glycoconjugates play a major role in key biological events such as cell-cell recognition, pathogenesis, and inflammation. [1,2] As a consequence, there is a need to understand the structural parameters governing the recognition of carbohydrates by their receptors. This knowledge will be of use for both fundamental research and potential applications in diagnostics or therapeutics. However, research in this field is slowed by the wide diversity of carbohydrate structures and by the minute amounts of materials available for experimentation. The design of sensitive and highthroughput technologies for the characterization of oligosaccharide/protein interactions [3] is therefore emerging as an attractive tool for chemists and biochemists. Available techniques such as isothermal calorimetry, enzyme-linked lectin assay, and even crystallographic studies provide data on carbohydrate/protein interactions, but they are often limited by the amount of available material.Carbohydrate microarray technology [4][5][6][7][8][9][10][11][12][13][14][15][16] is a promising approach for probing carbohydrate/protein interactions, and it permits the simultaneous screening of a number of biological interactions with only minute amounts of material. A large family of carbohydrate derivatives has been designed for immobilization on surfaces by various means. [5][6][7][8][9][10][11][12][13][14] However, this technology has various limitations. Relative surface densities of bound ligands are often not assessed. A careful optimization of the orientation and the distance separating the carbohydrate probe from the surface is often required.The interactions of oligosaccharides with lectins are usually weak (mm range) and can be enhanced using the "cluster effect" with multivalent ligands. [17][18][19][20] In the latter case, the distance between the residues should be optimized for binding. [21][22][23] Finally, the syntheses of functionalized oligosaccharide ligands are labor intensive.We report herein an original approach for the surface immobilization of oligosaccharides using glycoconjugate molecules that present a DNA sequence for anchoring onto DNA chips through hybridization. This approach has been used in the field of protein microarrays, [24,25] but to our knowledge this is the first time that such a strategy has been reported in the field of glycoarrays.Several syntheses of glycoconjugated oligonucleotides have been reported, but none are suitable for introducing different carbohydrate moieties. [26][27][28] We designed a conjugate that incorporates carbohydrate residue(s) for interacting with a lectin, an oligonucleotide sequence for anchoring on the surface, and a fluorescent tag at the 5'-end for the determination of relative surface densities (Figure 1). These moieties were assembled through a combination of automated oligonucleotide synthesis, and amidative oxidation and 1,3-dipolar cycloaddition ("click" chemistry) performed on a solid support (Scheme 1).[29] We introduced either one or three saccharide residue...

Atomic force microscopy reveals that Pseudomonas aeruginosa LecA (PA-IL) and a tetra-galactosylated 1,3-alternate calix[4]arene-based glycocluster self-assemble according to an aggregative chelate binding mode to create monodimensional filaments. Lectin oligomers are identified along the filaments and defects in chelate binding generate branches and bifurcations. A molecular model with alternate 90° orientation of LecA tetramers is proposed to describe the organisation of lectins and glycoclusters in the filaments.

Pseudomonas aeruginosa (PA) is a major public health issue due to its impact on nosocomial infections as well as its impact on cystic fibrosis patient mortality. One of the main concerns is its ability to develop antibiotic resistance. Therefore, inhibition of PA virulence has been proposed as an alternative strategy to tackle PA based infections. LecA (or PA-IL), a galactose binding lectin from PA, is involved in its virulence. Herein, we aimed at designing high affinity synthetic ligands toward LecA for its inhibition and at understanding the key parameters governing the binding of multivalent galactosylated clusters. Twenty-five glycoclusters were synthesized and their bindings were studied on a carbohydrate microarray. Monosaccharide centered clusters and linear comb-like clusters were synthesized with different linkers separating the core and the galactosyl residues. Their length, flexibility, and aromaticity were varied. Our results showed that the binding profile of LecA to galactosylated clusters was dependent on both the core and the linker and also that the optimal linker was different for each core. Nevertheless, an aryl group in the linker structure drastically improved the binding to LecA. Our results also suggest that optimal distances are preferred between the core and the aromatic group and the core and the galactose.

Pseudomonas aeruginosa (PA) is a Gram negative opportunistic pathogen and is the major pathogen encounter in the cystic fibrosis (CF) lung airways. It often leads to chronic respiratory infection despite aggressive antibiotic therapy due to the emergence of resistant strains and to the formation of biofilm. The lectin PA-IIL (LecB) is a fucose-specific lectin from PA suspected to be involved in host recognition/adhesion and in biofilm formation. Thus, it can be foreseen as a potential therapeutic target. Herein, 16 fucosylated glycoclusters with antenna-like, linear, or crown-like spatial arrangements were synthesized using a combination of DNA solid-phase synthesis and alkyne azide 1,3-dipolar cycloaddition (CuAAC). Their binding properties toward PA-IIL were then evaluated based on DNA directed immobilization (DDI) carbohydrate microarray. Our results suggested that the antenna-like scaffold was preferred to linear or crown-like glycoclusters. Among the crown-like carbohydrate centered fucosylated glycoclusters, mannose-based core was better than glucose- and galactose-based ones. The influence of the linker arm was also evaluated, and long linkers between fucoses and the core led to a slight better binding than the short ones.