Thesis presented December 08, 2006
Abstract: Microarrays are orderly arrangement of chemical, biological or cellular entities. This research field is rapidly growing because of high expectations in fundamental and applied sciences. The microarray technology is supported by three main methods: surface chemistry for slides functionalization, accurate technologies for manufacturing, and detection. Among possible applications, studying enzymatic activity is of high interest as it allows for monitoring the behaviour of enzymes in parallel, in the presence of a variety of substrates or inhibitors. Proteases play a key role in all living organisms and are therapeutic targets of choice, which make them promising enzymological models for microarrays.
Fast creation of microarrays made of hundreds of separated microdroplets on surface of a few cm
2 is achieved with the technology developed in our lab. Each droplet behaves as an individual microreactor. The aim is to use for the first time this technology for chemical synthesis and proteolysis monitoring. These two approaches are then to be combined in an original way to carry out a screening experiment.
These microdroplets arrays were applied successfully to chemical synthesis of a library of inhibitors as well as for the screening of fluorescent products. Enzymology on chip was performed with papaïn, a metalloprotease and HCV protease NS3, using three different kinds of fluorogenic substrates. Finally, a sequential method based on the library assembly by chemical synthesis on microarray, followed by NS3 assay on the same microarray was tried for the first the screening of NS3 inhibitors. Promising hits displaying were discovered.
Keywords: Microarray, chip, technology, combinatorial chemical synthesis, bioactive molecules, fluorogenic substrates, proteases, NS3, VHC, antiviral agent
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