Yves Levy

One hundred spots parallel monitoring of DNA interactions by SPR imaging of polymer-functionalized surfaces applied to the detection of cystic fibrosis mutations

Abstract In the present paper, we report the detection of mutations implicated in human cystic fibrosis (CF). Nine different oligonucleotides are studied, including three possible mutations related to this specific genetic disease: a deletion of three bases, ΔF508, and two single-nucleotide polymorphisms 1540A/G and 1716G/A. We monitor, in real time and in parallel, hybridizations of a solution of unlabeled oligonucleotide targets to a matrix of 100 spots of oligonucleotide probes using surface plasmon resonance (SPR) imaging of a bio-functionalized gold slide. In order to functionalize our gold slide with the DNA probes, we have developed a self-assembled multilayer (SAM) based on electrostatic interactions and formed with 11-mercaptoundecanoic acid (MUA), poly(ethylenimine) (PEI) and ExtrAvidin layers. Probes are then linked to this SAM by the usual strong binding affinity of the avidin–biotin duplex. The 100 spots array deposited by a robot can be addressed either several times, sequentially, with the various oligonucleotide targets, or once, in parallel, with a mixture of some oligonucleotides. The specific response of our system is established along with the possibility of discriminating between a totally complementary sequence and its mutant form, even for a single base mismatch thus demonstrating the capacity of parallel diagnostic using patient like material.

Electrooptic properties of a ferroelectric polymer studied by attenuated total reflection

Abstract The attenuated total reflection method is used for measuring the electrooptic properties of a thin film of a polarized ferroelectric polymer. The piezoelectric character of the sample is shown to be of major importance. A very simple theory is presented which explains an unexpected change of sign of the electrooptic signal. A method is given for extracting the molecular electrooptic coefficients.

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.

Second-order optical nonlinearities of azo chromophores covalently attached to a sol-gel matrix

Optically nonlinear disperse red 1 (DR1) was covalently bound into a silica gel network by the coupling of DR1 and 3-isocyanatopropyltriethoxysilane. Organic-inorganic hybrid materials present a large resonant second-order susceptibility (d33 = 55 pm/V for a fundamental wavelength of 1.06 μm) with an excellent room-temperature stability.

Effect of chromophore–chromophore electrostatic interactions in the NLO response of functionalized organic–inorganic sol–gel materials ☆

Abstract In the last years, important non-linear optical (NLO) results on sol–gel and polymeric materials have been reported, with values comparable to those found in crystals. These new materials contain push–pull chromophores either incorporated as guest in a high T g polymeric matrix (doped polymers) or grafted onto the polymeric matrix. These systems present several advantages, however they require significant improvement at the molecular level—by designing optimized chromophores with very large molecular figure of merit, specific to each application targeted. Besides, it was recently stated in polymers that the chromophore–chromophore electrostatic interactions, which are dependent of chromophore concentration, have a strong effect into their NLO properties. This has not been explored at all in sol–gel systems. In this work, the sol–gel route was used to prepare hybrid organic–inorganic thin films with different NLO chromophores grafted into the skeleton matrix. Combining a molecular engineering strategy for getting a larger molecular figure of merit and by controlling the intermolecular dipole–dipole interactions through both: the tuning of the push–pull chromophore concentration and the control of tetraethoxysilane concentration, we have obtained a r 33 coefficient around 15 pm/V at 633 nm for the classical DR1 azo-chromophore and a r 33 around 50 pm/V at 831 nm for a new optimized chromophore structure.

Stable photorefractive memory effect in sol-gel materials

We report on the synthesis and the optical characterization of a photorefractive sol-gel material which contains nonlinear azo chromophore and carbazole charge transporting molecules. Both of these functional groups are covalently attached to the silica based backbone. Thin sol-gel films exhibit stable optically nonlinear properties: the electro-optic coefficient r13 measured at 633 nm is found to be 17 pm/V one day after corona poling and 15 pm/V after three weeks and over a period of several months. Photorefractivity is demonstrated by two beam coupling experiments without external applied electric field. Sol-gel films present a stable photorefractive memory effect with a net internal gain of 200 cm−1.

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.

Novel photorefractive sol-gel materials

We have developed new photorefractive media based on hybrid organic-inorganic materials containing a charge transporting (CT) molecule either as side-chain or main-chain substituents on the silica backbone. Second order nonlinear optical (NLO) chromophores were introduced either as side chain or as guest units. These materials were prepared by the sol-gel process in the form of thin films of a few μm-thick. NLO and photorefractive properties have been evaluated using electro-optic measurements, two beam coupling experiments and photoconductivity measurements.

The reversible binding of hydrophobically end-capped poly(ethylene glycol)s to poly-β-cyclodextrin-coated gold surfaces: a surface plasmon resonance investigation

Abstract Polymers of β-cyclodextrin (β-CD) bearing amino groups (poly-β-CD-NH2) were grafted to gold surfaces by two different procedures. Hydrophobically end-capped model-polymers were adsorbed onto resulting (poly-β-CD-NH2)-coated sensor chips by formation of inclusion complexes. Their adsorption onto the surface was followed in real time by surface plasmon resonance (SPR). Multilayered structures obtained by inclusion of adamantyl-modified poly(ethylene glycol)s (Ad-PEG) into immobilized β-cyclodextrin cavities were highly stable in aqueous media. Conditions leading to the regeneration of initial (poly-β-CD-NH2)-coated surfaces were studied by SPR. Regeneration by competitors such as polymers of β-cyclodextrin was possible. However, it was found to be a complex mechanism involving two opposite phenomena depending on the structure and composition of the (poly-β-CD-NH2) layer. Complete desorption of the bound Ad-PEG layer was also observed after exposure of the multilayered structures to organic solvents or sodium dodecylsulfate (SDS).

Temperature dependence of second-order nonlinear relaxation of a poled chromophore-doped sol–gel material

Second-order optical nonlinearity relaxations of hybrid inorganic/organic sol–gel films doped with poled Disperse Red 1 chromophores were measured by second harmonic generation at several temperatures. The decay curves were fitted to biexponentials and Kohlrausch, Williams, and Watts (KWW) stretched exponentials. It can be concluded that the KWW model well describes chromophore thermal reorientation in hybrid sol–gel materials, as is known for all-organic polymers. Using the Arrhenius law, the activation energy of the reorientation process has been determined to be about 50 or 70 kcal mol−1. A stability of roughly a decade has been extrapolated at room temperature. Another experiment at room temperature gave an estimation of the stability that was found to be in qualitative agreement. It is shown that poled chromophore-doped sol–gel materials have the potential to be stable enough for applications in telecommunication devices.