Dextran based polymers derived surfaces : characterization by attenuated total reflection infrared (ATR-IR) spectroscopy and investigation of protein-carbonhydrate interactions
Thèse de doctorat : Université de Neuchâtel, 2007 ; Th 2027.
Sensitive surfaces imply responsive elements lodged onto a carrier which eventually allows for reaction, recognition and detention. Interfaces require fine tuning for response performance optimization. Observation needs to be focused on the two media common boundary. Currently, dextran based polymers are still a major class of tunable materials for surface modifications. To simply and... PlusAjouter à la liste personnelle
- Sensitive surfaces imply responsive elements lodged onto a carrier which eventually allows for reaction, recognition and detention. Interfaces require fine tuning for response performance optimization. Observation needs to be focused on the two media common boundary. Currently, dextran based polymers are still a major class of tunable materials for surface modifications. To simply and simultaneously work as biocompatible spacer, antifouling agent, passivation means, and flexible surface chemistry tailor, they require covalent binding to the support. Besides the well defined chemical methods, photochemical procedures provide straightforward and non invasive protocols to generate reactive intermediates. The latter quickly and indistinctively interact with both the surface and the target, establishing definitive ties. Among photochemical methods, photobonding technology (arrayon Biotechnology, Switzerland) still remains an outstanding procedure for sample immobilization on inert material surfaces by light-induced processes. Actinic dextrans (like OptoDex ®) are used as photolinker polymers. The generated intermediate is a carbene, a highly reactive organic molecule with a divalent carbon atom which basically interposes itself into an existing bond. The insertion generally does not affect the 3D structure holding the “docking” point. OptoDex ® functionalized platforms are a specific product issue of this technology, intended for bioarray applications. They are plain glass platforms that become appropriate for biomolecule patterning (micropipetting, nanodispensing) after OptoDex ® conditioning. The technology proved successful for a large protein portfolio immobilization and motivated the progress of investigation in two directions. First, a deeper characterization of the photolinker behavior at interface was judged worthy of a physico-chemical characterization. One should note that the chemical functionalities were already well-established by the supplier quality control procedures (arrayon Biotechnology proprietary). A spectroscopic method (molecular-level, label free) could uphold routine analytical methods and functional screening using indirect detection (like fluorescence labeling). Second, the search for a new cutting-edge application which might also fulfill a new market spot, led to the extension of such technology to the domain of glycomics (carbohydrates decorated biomolecules interaction screening and monitoring via middle-high density throughput layout). Attenuated Total Reflection Infrared (ATR-IR) spectroscopy is a powerful method to investigate liquid-solid interfaces. Simplifying, it can be considered an adaptation of the classical IR spectroscopy in transmission (appropriate for solutions) to investigate reactions at the solid-liquid interfaces. The molecular fingerprint is retained, the sensitivity is higher, and quantification is accessible. The characteristics of this surface-sensitive spectroscopy are useful for examining the interface properties of dextran based photolinker polymers. These characteristics are: a penetration depth of evanescent wavelength beam up to 1 µm, and the chemical detectable entities of interest C-H bonds and –OH groups. The effects of the photoreaction at the interface and the physico-chemical characteristics of the adlayer have been explored and defined on a molecular basis (orientation, kinetic evaluations, film thickness determination). Particular attention was paid to physisorbtion of dextran polymers structurally similar to OptoDex ®. Results differ if chemical immobilization is performed allowing previous spontaneous physisorption. This study defined the role of the physisorption intermediate step and elucidated the role of charge as well. To further explore the potential of the photoinduced immobilization, 2 nm monodispersed gold nanoparticles were nestled onto the dextran adlayer and covalently grafted to the surface by OptoDex ®. ATR-IR equipment proved a powerful tool to detect organic shell capping gold cores down to a small scale. The application of the photobonding technology in the field of glycomics resulted in the design and optimization of carbohydrate micro arrays. OptoDex ® A mediated immobilization of low and high molecular weight sugar moieties (glycoproteins and exopolisaccharides, respectively) was achieved while evidencing retained bioactivity by fluorescence labeled probes (lectins, namely specific sugar tether binding proteins). Flexibility of the method was demonstrated permuting the probe/target pair: lectins were microprinted in array format, immobilized onto the surface, and probed by fluorescent labeled sugar moieties (glycoproteins). Such a portfolio of detectable interactions and binding tests in miniaturized format allowed monitoring of the glycoexpression related to the evolution of two different cell lines. Carbohydrate microarrays were manufactured and the relative protocols for glycoprofiling established.