Development of oxidoreductase based electrochemical biosensors

Dissertação para obtenção do Grau de Mestre em Biotecnologia

This thesis is divided in 2 sections, each describing the development of an oxidoreductase based biosensor. In the first part human Cytochrome P450 1A2 (CYP1A2) electrochemistry was studied, while the second is focused on the optimization of immobilization platforms and operation methods for amperometric biosensors, using cytochrome c nitrite reductase (ccNiR), (Desulfovibrio desulfuricans ATCC 27774) as a model enzyme. The direct electrochemistry of P450s immobilized in water-based sol-gel thin films was described for the first time. The optimization of the film showed that only the combination of the inorganic matrix and the PEG400 enabled the direct electron transfer reaction and electrocatalytic activity towards oxygen. The amount of dissolved oxygen in solution revealed itself a significant feature in CYP’s electrochemistry – in anaerobic conditions, when small amounts of oxygen are added the PFeIII=II signal’s intensity increased, while in aerobic conditions it disappeared; probably PFeIII is not being regenerated. However, this was not observed with the CYPOR complex, indicating that the reductase has an essential role in the CYP’s catalytic cycle completion; this was also sustained by the fact that only in its presence organic substrates catalysis (caffeine) occurs. The hybrid sol-gel developed for CYP, was optimized for a nitrite biosensor. ccNiR was successfully incorporated while promptly displaying catalytic currents. Although the bioelectrode’s response decreases after day one, it was able to maintain a reasonable catalytic activity over a time span of 2 weeks. Another electrode modification strategy, studied with ccNiR, was based on the electrophoretic deposition of macroporous assemblies of single-walled carbon nanotubes. The macroporous structure was created as a result of the presence of polystyrene beads co-deposited with the carbon nanotubes. An increase in the amount of material was correlated with a higher enzyme activity. Finally, an oxygen scavenger system consisting of glucose oxidase, glucose, and catalase was employed for oxygen removal in an open electrochemical cell. The system completely removed oxygen for over 1 h and was successfully applied to a ccNiR based nitrite sensor.