A VERSTAILE POLYMERIC COATING FOR MICROARRAYS USING COPPER-MEDIATED CLICK CHEMISTRY

In this thesis we introduce a method to functionalize the surface of glassy materials with alkynes using a polymer that produces a coating by a facile 'dip and rinse' method. The alkyne groups on the tri-dimensional surface can be subsequently linked to azide-containing carbohydrates using Cu-catalyzed azide/alkyne cycloaddition (CuAAC, click chemistry)1,2. The research is aimed at developing a new strategy to generate a polymer coating enabling the attachment of complex sugars via click chemistry by a method that does not require skilled personnel and chemistry laboratories. The proposed approach combines the advantages of high sensitivity and superior signal-to-noise ratio of a Si-SiO2 substrate with the quality of a 3D coating. The Si/SiO2 surface was used as the substrate to take advantage from the superior optical properties of this material. A novel polymer named poly (DMA-PMA-MAPS), obtained from the polymerization of N,N-dimethylacrylamide (DMA), 3-trimethylsilanyl-prop-2-yn methacrylate (PMA) and 3(trimethoxysilyl)-propylmethacrylate (MAPS) was synthesized and characterized. It consists of: 1) a segment of polydimethylacrylamide that interacts with the surface by weak, non covalent interactions such as hydrogen bonding, Van der Waals or hydrophobic forces, 2) a pending silane hydrolysable monomers that promote condensation of the polymer with surface silanols or between contiguous chains and 3) chemically active monomers whose reactivity is selected on the basis of the reactivity of the molecules that have to be immobilized. The polymer reported herein is similar to another polymer developed in 2004 by Pirri et al.3 to form a coating on glass slides by a combination of physi- and chemi-sorption. The novelty of this work consists in the presence of an alkyne functional monomer that replaces the succinimide active ester. By exploiting the presence of a stable coating that allows regio-specific and bio-orthogonal immobilization, a glycan microarray was built and its performance was deeply investigated. First, a qualitative assay (fluorescence analysis) was carried out to obtain a fast screening of the affinity of the interaction of nine glycomimetics with Concanavlin A. Second, thanks to the high-sensitivity of the Si/SiO2 platform used, a study of the influence of the multivalency presentation of glycans during lectin interaction was made. The high-performing substrate used allows a dramatic decrease of glycan surface densities (from 1,96•1014 down to 5,07•1012 molecules/cm2), offering the possibility to calculate and compare the avidity in different conditions by providing density dependent surface dissociation constants (KD,surf). In a second different application, the new poly(DMA-PMA-MAPS) copolymer coating was used to functionalize microarray slides with orientated antibodies taking advantage from the regio-specific reaction between alkyne on the surface and azido groups on the biomolecule. Inspired by the work of Zeglis et al.4 an enzymatic procedure was devised to obtain site-specific modified antibodies using an unnatural UDP-6-azidogalactose and two commercially available enzymes: β-(1,4)-galactosidase and β-(1,4)-galactosyltransferase. As the 6-azidogalactose is sterically less hindered, it is expected to display a higher reactivity in the surface immobilization process. The strategy adopted was, in part, mutated from the procedure reported by Bosco et al.5. To validate the methodology and highlight its advantages, a sandwich microray test for the detection of interleukin-6 (IL-6) was developed. Cytokines, a set of proteins implicated in the onset and development of almost every major life-threatening disease are amongst the most intensively studied biomarkers. They play a prominent role in cancer, neurodegenerative diseases, cardiovascular diseases, sepsis and many other pathologies6. Although enzyme-linked immunosorbent assay (ELISA) is the gold standard for the measurement of a single cytokine concentration, the key to successful identification of biomarkers is the simultaneous detection of multiple cytokines with high sensitivity. IL-6 was chosen as a model of a typical inflammatory biomarker, to demonstrate the senistivity provided by an oriented immobilization of the capturing antibody in a microarray based immunoassay. Our site-specifically modified antibody was compared to both a randomly azido-pegylated antibody and a site-specifically modified antibody derivatized by a commercial Kit (Site-Click Antibody Labelling purchase from Life Technology), which makes use of an unnatural UDP-2-azidogalactose instead of UDP-6-azidogalactose, and of a mutant GalT (Y289L) instead of a commercially available GalT. Furthermore, through the use of the label-free sensing platform IRIS, we have correlated the efficiency of the Ab-antigen interaction given by the fluorescence signal with the mass of antibody immobilized per surface unit (ng/mm2). The fluorescence per mass unit allows to assess the importance the antibody orientation on its capturing ability. In particular its was demonstrated that a higher amount of immobilized probe does not necessary lead to a higher antibody-antigen interaction. (1) H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem. Int. Ed., 2001, 40, 2004. (2) C. W. Tornøe, C. Christensen and M. Meldal, J. Org. Chem., 2002, 67, 3057. (3) G. Pirri, F. Damin, M. Chiari, E. Bontempi, L. E. Depero, Anal. Chem., 2004, 76, 1352. (4) B. M. Zeglis, C. B. Davis, R. Aggeler, H. C. Kang, A. Chen, B. J. Anew and J. S. Lewis, Bioconjugate Chem., 2013, 24, 1057. (5) M. Bosco, S. Le Gall, C. Rihouey, S. Couve-Bonnaire, M. Bardol, P. Lerouge, X. Pannecoucke, Tethraedron Lett., 2008, 49, 2294. (6) R.P. Huang, B. Burkholder, V.S. Jones, W.D. Jiang, Y.Q. Mao, Q.L. Chen, Z. Shi, Current Proteomics, 2012, 9, 55.