André Roget

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.

Polypyrrole based DNA hybridization assays: study of label free detection processes versus fluorescence on microchips.

In this paper, we present different ways to detect DNA hybridization on a solid support. The grafting chemistry is based on the electro-controlled copolymerization of a pyrrole-modified oligonucleotide and pyrrole. This process allows an easy functionalization of conducting materials. Three kind of devices were studied: silicon chips bearing an array of addressable 50 or 4 microm microelectrodes, quartz crystal microbalance (QCM) and a non patterned gold/glass slide bearing 500 microm spots. Each device is compatible with a specific detection process: a classical indirect fluorescence detection for the microchips, a microgravimetric measurement for the QCM and a surface plasmon resonance imaging process (SPRi) for the gold slides. Both QCM and SPRi are a label-free real time detection process whereas the fluorescence methodology gives end-point data but only the fluorescence and the SPRi give multiparametric results. Although the hybridization experiments show that the detection limit for an oligonucleotide is better for the fluorescence (1-10 pM) than that found for SPRi (10 nM) and QCM (250 nM), the information content of real time measurement techniques such as SPRi is of interest for many biological studies.

Reversible oligonucleotide immobilisation based on biotinylated polypyrrole film

Abstract In this paper, we describe the design of a new versatile and reversible DNA sensor. The system is based on biotin grafting-units, covalently linked to a polypyrrole matrix, able to anchor large biomolecules, thanks to biotin/avidin affinity. These grafting-units can be regenerated after the “denaturation” of the biotin/avidin link allowing to reuse the matrix for the immobilisation of an other assembly and then the possibility to generate a new sensor.

Biosensing effects in functionalized electroconducting conjugated polymer layers: addressable DNA matrix for the detection of gene mutations

Abstract A new methodology for the preparation of addressed DNA matrices is described. The process is based on a one-step electrodeposition of a polypyrrole film functionalized by a covalently linked oligonucleotide. Even in the bulk of the film the single-stranded DNA fragment can be hybridized or denatured. A support bearing four oligonucleotide spots was obtained by successive copolymerization on a four electrode matrix. This device allows detection of three ras mutations on a synthetic DNA fragment without cross contamination.

Conducting polymers as a link between biomolecules and microelectronics

Abstract Biosensors based on electronic conducting polymers (ECPs) appear particularly well suited to the requirements of modern biological analysis: multiparametric assays, high information density and miniaturization. We describe a new methodology for the preparation of addressed DNA matrices. The process includes an electrochemically directed copolymerization of pyrrole and oligonucleotides bearing on their 5′ end a pyrrole moiety. The resulting polymer film deposited on the addressed electrode consists of pyrrole chains bearing covalently linked oligonucleotides. An oligonucleotide array was constructed on a silicon device bearing a matrix of 48 addressable 50 μm x 50 μm gold microelectrodes. This technology was successfully applied to the genotyping of Hepatitis C Virus in blood samples. Fluorescence detection results show good sensivity and a high degree of dimensional resolution. The need for versatile processes for the immobilization of biological species on surface led us to extend our methodology. A biotinylated surface was obtained by co-electropolymerization of pyrrole and biotin-pyrrole monomers. The efficiency for recognition (and consequently immobilization) of R-phycoerythrin-avidin was demonstrated by fluorescence detection. Copolymerization of decreasing ratios of pyrrole-biotin over pyrrole allowed us to obtain a decreasing scale of fluorescence.

Versatile analysis of multiple macromolecular interactions by SPR imaging: application to p53 and DNA interaction

The greatest challenge in the postgenomic era is the description of proteome interactions, such as protein–protein or protein–DNA interactions. Surface plasmon resonance (SPR) is an optical technique in which binding of an analyte to the surface changes the refractive index at the surface/solution interface. Molecular interactions are analysed in real time without a labeling step. Currently, the limit to SPR imaging is the small number of reactions that can be simultaneously analysed. Using a novel grafting technology and a new imaging system, we increased the throughput of SPR imaging. The interaction between p53 and DNA was chosen as a paradigm for validation of this assay. Using a tagged DNA methodology, we simultaneously targeted multiple DNA sequences on a single chip. The interaction between p53 and these DNA sequences was monitored by SPR imaging. Qualitative and quantitative analysis provides results similar to those obtained with conventional technologies.

Polypyrrole electrospotting for the construction of oligonucleotide arrays compatible with a surface plasmon resonance hybridization detection

We describe in this paper a new electrospotting process allowing a straightforward functionalization of homogeneous gold surfaces. It is based on a direct electro-copolymerization of pyrrole and pyrrole linked to an oligonucleotide on a gold substrate via the use of a moving micro electrochemical cell. The polypyrrole spots obtained by this way were characterized in terms of thickness and DNA hybridization efficiency. Results show that either fluorescence or surface plasmon resonance detection of DNA hybridization can be carried out on these substrates.

Electropolymerization as a versatile route for immobilizing biological species onto surfaces

Biosensors based on electronic conducting polymers appear particularly well suited to the requirements of modern biological analysis—multiparametric assays, high information density, and miniaturization. We describe a new methodology for the preparation of addressed DNA matrices. The process includes an electrochemically directed copolymerization of pyrrole and oligonucleotides bearing on their 5′ end a pyrrole moiety. The resulting polymer film deposited on the addressed electrode consists of pyrrole chains bearing covalently linked oligonucleotides (ODN). An oligonucleotide array was constructed on a silicon device bearing a matrix of 48 addressable 50 × 50 µm gold microelectrodes. This technology was successfully applied to the genotyping of hepatitis C virus in blood samples. Fluorescence detection results show good sensitivity and a high degree of spatial resolution. In addition, gravimetric studies carried out by the quartz crystal microbalance technique provide quantitative data on the amount of surface-immobilized species. In the case of ODN, it allows discrimination between hybridization and nonspecific adsorption. The need for versatile processes for the immobilization of biological species on surfaces led us to extend our methodology. A biotinylated surface was obtained by coelectropolymerization of pyrrole and biotin-pyrrole monomers. The efficiency for recognition (and consequently immobilization) of R-phycoerythrin-avidin was demonstrated by fluorescence detection. Copolymerization of decreasing ratios of pyrrole-biotin over pyrrole allowed us to obtain a decreasing scale of fluorescence.

Binding of chondroitin 4-sulfate to cathepsin S regulates its enzymatic activity.

Human cysteine cathepsin S (catS) participates in distinct physiological and pathophysiological cellular processes and is considered as a valuable therapeutic target in autoimmune diseases, cancer, atherosclerosis, and asthma. We evaluated the capacity of negatively charged glycosaminoglycans (heparin, heparan sulfate, chondroitin 4/6-sulfates, dermatan sulfate, and hyaluronic acid) to modulate the activity of catS. Chondroitin 4-sulfate (C4-S) impaired the collagenolytic activity (type IV collagen) and inhibited the peptidase activity (Z-Phe-Arg-AMC) of catS at pH 5.5, obeying a mixed-type mechanism (estimated Ki = 16.5 ± 6 μM). Addition of NaCl restored catS activity, supporting the idea that electrostatic interactions are primarly involved. Furthermore, C4-S delayed in a dose-dependent manner the maturation of procatS at pH 4.0 by interfering with the intermolecular processing pathway. Binding of C4-S to catS was demonstrated by gel-filtration chromatography, and its affinity was measured by surface plasmon resonance (equilibrium dissociation constant Kd = 210 ± 40 nM). Moreover, C4-S induced subtle conformational changes in mature catS as observed by intrinsic fluorescence spectroscopy analysis. Molecular docking predicted three specific binding sites on catS for C4-S that are different from those found in the crystal structure of the cathepsin K-C4-S complex. Overall, these results describe a novel glycosaminoglycan-mediated mechanism of catS inhibition and suggest that C4-S may modulate the collagenase activity of catS in vivo.

Carbohydrates as new probes for the identification of closely related Escherichia coli strains using surface plasmon resonance imaging.

Prevention of foodborne diseases depends highly on our ability to control rapidly and accurately a possible contamination of food. So far, standard procedures for bacterial detection require time-consuming bacterial cultures on plates before the pathogens can be detected and identified. We present here an innovative biochip, based on direct differential carbohydrate recognitions of five closely related Escherichia coli strains, including the enterohemorragic E. coli O157:H7. Our device relies on efficient grafting of simple carbohydrates on a gold surface and on the monitoring of their interactions with bacteria during their culture using surface plasmon resonance imaging. We show that each of the bacteria interacts in a different way with the carbohydrate chip. This allows the detection and discrimination of the tested bacterial strains in less than 10 h from an initial bacterial concentration of 10(2) CFU·mL(-1). This is an improvement over previously described systems in terms of cost, easiness to use, and stability. Easily conceived and easily regenerated, this tool is promising for the future of food safety.