Thesis presented November 14, 2005

Abstract:
This work deals with: (i) the electrochemical detection of nucleosides on a new carbon electrode material, Boron Doped Diamond (BDD), and (ii) the use of a recent apparatus, scanning electrochemical microscopy (SECM) to micropattern surfaces with oligonucleotides and to detect the hybridization event. On the one hand, we illustrate the capacity of scanning electrochemical microscopy to micropattern and characterize surfaces. By direct mode of SECM, using the microelectrode as a counter electrode, polypyrrole-oligonucleotide spots with micrometric dimensions are deposited on a gold surface. This patterning method is optimized studying the spots hybridization by fluorescence microscopy. By the difference in surface conductivity between gold and polypyrrole-oligonucleotide, SECM can detect the spots on the gold surface using the feedback mode. Then, the hybridization event is detected using the feedback mode by the precipitation of an insulating product on the surface generated by the reaction catalyzed by the peroxidase which is coupled by a biological assembly to the complementary oligonucleotide. Finally, this electrochemical method is coupled to an optical one, Surface Plasmon Resonance imaging (SPRi), to visualize in real time the patterning step of the gold surface with oligonucleotide spots or surface oligonucleotide gradients. On the other hand, the oxidation reactions of both nucleosides, 2'-deoxyguanosine and 2'-deoxyadenosine, are performed on an oxygenated boron doped diamond electrode. This points out the interest of this new material thanks to its enlarged potential window in aqueous media and its little and stable background current. Then, we show the formation of a continuous film of oxidized products on the BDD surface by a macroscopic study analyzing cyclic voltametries of a redox mediator, and in a microscopic way with the use of scanning electrochemical microscopy.

Keywords:
Scanning electrochemical microscopy (SECM), DNA biosensor, nucleoside oxidation, Boron Doped Diamond (BDD), Surface Plasmon Resonance imaging (SPRi), surface gradients, hybridization

On-line thesis.