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The recently proposed bioassay procedure that is based on the substrate induced quenching (SIQ) of an indicator fluorescence for the measurement of analyte concentrations is evaluated. In this type of assay a enzynatic reaction and a fluorescence quenching interaction are coupled together. Typically, an appropriate dehydrogenase enzyme reduces or oxidises the nicotinamide adenine dinucleotide cofactor. The change in the concentration of NADH results in variations in the excited fluorophore population as observed through fluorescence intensity. This latter aspect is used to monitor substrate (analyte) concentrations. Results on the investigation of the substrate induced quenching bioassay method and possibilities of using it as the basis of (i) a novel enzyme bioassay technique and (ii) a novel bioprobe format are presented. Ethanol was chosen as the model analyte, and a new assay procedure for its measurement was developed. A generic theoretical relation is discussed for the observed assay kinetics of substrate induced quenching (SIQ) and a model is described that includes the effects due to dynamic/static quenching of the fluorophore by either the enzyme substrate or product. The validity of the derived model is shown by comparison with experimental results for a SIQ based ethanol assay. The option of running the dehydrogenase reaction so as to consume NADH rather than generate it is also investigated. In order to demonstrate this approach acetaldehyde was chosen as the model analyte, and a assay procedure for its measurement was developed. The potential of the SIQ technique for incorporation into biosensor based upon a 'reservoir' format was demonstrated through the development of custom optical instrumentation and resevoir flowcell. Applicability of the SIQ technique to other biosensor formats such as flow-injection analysis and 'dry reagent' technology is discussed. The overall applicability of the SIQ technique is assessed through the generation of a number of SIQ assays on the following substrates: ethanol, glucose, glucose-6- phosphate, L-glutamic acid, isocitric acid, acetaldehyde, pyruvic acid, ot-ketoglutaric acid, and oxalacetic acid.

Les travaux présentés dans ce manuscrit décrivent le développement de trois supports innovants basés sur des nanofibres de matériaux hybrides obtenus par électrofilage pour la réalisation de biocapteur électrochimiques. La performance des biocapteurs est fortement améliorée du fait de l'utilisation de matériaux nanostructurés qui leurs confèrent des propriétés uniques. Les fibres obtenues par électrofilage trouvent des applications dans de nombreux domaines, mais leur utilisation pour l'élaboration de biocapteurs, bien que très prometteuse, est encore très peu abordée. Dans ce travail, différentes nanofibres polymériques contenant des nanotubes de carbone ou recouvertes de nanoparticules d'or ou d'un film de copolymère polypyrolle / poly(pyrolle-3-carboxylique acide) ont été utilisées comme support pour le développement de biocapteurs. La glucose oxydase a été utilisée comme enzyme modèle pour valider les performances des biocapteurs réalisés. Cette enzyme a été incorporée directement dans les nanofibres ou fixée de façon covalente à leur surface. Les biocapteurs ainsi obtenus, caractérisés par différentes techniques microscopiques et électrochimiques, ont permis la détection du glucose avec succès, en utilisant la voltammétrie cyclique et la spectroscopie d'impédance électrochimique, tout en montrant des performances (sensibilité, reproductibilité, stabilité) supérieures à celles des biocapteurs conventionnels / The work detailed within this manuscript describes the development of three novel efficient electroactive platforms based on electrospun nanofibrous hybrid materials for further application to electrochemical biosensors elaboration. The performance of biosensors is enhanced by their coupling with nanoscale materials, due to the unique properties that the latter exhibit. Although electroctrospun fibers find applications in various fields, their exploitation for biosensing is still in an early but promising stage. Herein, different polymeric nanofibers incorporating carbon nanotubes, decorated with gold nanoparticles or coated with conducting polypyrrole/poly(pyrrole-3-carboxylic acid) films were used as platforms for the development of biosensors. Glucose oxidase was used as a model enzyme to validate the performance of the developed biosensors. The enzyme was either incorporated into the nanofibers or covalently immobilized onto their surface. These innovative biosensors, characterized by different microscopic and electrochemical techniques, enabled successful detection of glucose by employing cyclic voltammetry and electrochemical impedance spectroscopy, whilst demonstrating enhanced performances over conventional biosensors in terms of sensitivity, reproducibility and stability