Thierry Livache

Electroconducting polymers for the construction of DNA or peptide arrays on silicon chips

We wish to show in this paper new developments and new applications of the pyrrole copolymerization process allowing the addressing of pyrrole-modified biomolecules on microelectrode arrays. Two main developments are described: the first one concerns the development of multiplexed silicon chips bearing 128 microelectrodes instead of 48 for the passive chips. The second one deals with new applications of this grafting process concerning not only DNA chips but peptide chips too. In this way, copolymerization of pyrrole peptides on the chip (leading to peptide chip) and their immunological detection is illustrated. This technology shows a high dimensional resolution and a real versatility.

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.

Salt Concentration Effects on Equilibrium Melting Curves from DNA Microarrays

DNA microarrays find applications in an increasing number of domains where more quantitative results are required. DNA being a charged polymer, the repulsive interactions between the surface of the microarray and the targets in solution are increasing upon hybridization. Such electrostatic penalty is generally reduced by increasing the salt concentration. In this article, we present equilibrium-melting curves obtained from dedicated physicochemical experiments on DNA microarrays in order to get a better understanding of the electrostatic penalty incurred during the hybridization reaction at the surface. Various salt concentrations have been considered and deviations from the commonly used Langmuir adsorption model are experimentally quantified for the first time in agreement with theoretical predictions.

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.

Point Mutation Detection by Surface Plasmon Resonance Imaging Coupled with a Temperature Scan Method in a Model System

The detection of point mutations in genes presents clear biological and medical interest. Various methods have been considered. In this paper, we take advantage of surface plasmon resonance imaging, a technique allowing detection of unlabeled DNA hybridization. Coupled with a temperature scan, this approach allows us to determine the presence of single-point mutations in oligonucleotide samples from the analysis of DNAs melting curves in either the homozygous or heterozygous case. Moreover, these experimental data are confirmed in good agreement with numerical calculations.

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.

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.

Conducting polymers on microelectronic devices as tools for biological analyses

In the field of biological analysis, the need for multiparametric analysis has prompted the development of supports bearing a series of biomolecules linked to a support in a precise location (addressed). To reach a high information density, miniaturization of this kind of support has to be carried out. We describe in this paper an approach involving the use of electro-conducting polymers such as polypyrrole. This technology is based on an electro-directed copolymerization of pyrrole and oligodeoxynucleotides (ODN) linked to a pyrrole residue. The process allows the grafting of the selected ODN at the surface of the successively addressed microelectrodes. In this way, the syntheses are carried out on 50 microm electrodes on passive chips or on active (multiplexed) chips bearing 48 or 128 gold microelectrodes, respectively. The detection of biological targets recognized by the biochip is carried out by using fluorescent tracers. This technology, involving prepurified materials precisely addressed, allows better reproducibility of the biochip preparation and, then, an easy interpretation of the fluorescence results. The versatility of this technology is illustrated by ODN or peptide copolymerizations leading to DNA chips or peptide chips, respectively. This would open the field for other biological interaction studies.

Effects of formamide on the thermal stability of DNA duplexes on biochips

In molecular biology, formamide (FA) is a commonly used denaturing agent for DNA. Although its influence on DNA duplex stability in solution is well established, little is known about immobilized DNA on microarrays. We measured thermal denaturation curves for oligonucleotides immobilized by two standard protocols: thiol self-assembling and pyrrole electrospotting. A decrease of the DNA denaturation temperature with increasing FA fraction of the solvent was observed on sequences with mutations for both surface chemistries. The average dissociation temperature decrease was found to be -0.58+/-0.05 degrees C/% FA (v/v) independently of grafting chemistry and probe sequence.