New methods of immobilization of oxidases at electrodes

Renata Bilewicz ,  Ewa Nazaruk ,  Kazimierz Chmurski ,  Jerzy Rogalski ,  Maciej Klis 

Warsaw University, Faculty of Chemistry, Pasteura 1, Warszawa 02-093, Poland


Retaining enzymes in functionally-active forms on the electrode surface is a challenging and difficult task. Ideally, the immobilization of protein should be performed under conditions that provide membrane like environment in which all the normal interactions of the proteins are preserved. Providing electronic contact of the protein molecules with the conducting substrate by means of a biocompatible medium is even more difficult. In the present talk several ways of contacting oxidases with electrodes will be discussed.Three step sequential self-assembly procedure was applied to prepare gold electrodes functionalized in a stable and controlled way with a β-cyclodextrin (β-CD). When thiolated cyclodextrins are used as active sites in the organothiol monolayers modifying electrodes, the electron transfer from solution species to the electrode is facilitated in the presence of appropriate mediators in the cyclodextrin cavity. Methylene blue in the cavities provided electrical contact between the electrode and solution resident enzyme - laccase catalyzing reduction of oxygen to water. Covalent binding of laccase to thiol monolayers using 11-mercaptoundecanoic acid as the organic junction was found to provide direct electrical contact with the enzyme together with its stable immobilization at the electrode. Direct electrochemistry of the enzyme can be observed and its catalytic activity demonstrated. Immobilization in thin films of lyotropic liquid crystal - lipidic matrix with cubic symmetry is discussed as a way of immobilizing enzymes by means of hydrophobic-hydrophilic interactions with lipid molecules. The electrochemical behaviour and method of immobilizing the fungal laccases in diamond type monoolein cubic phase will be discussed. The cubic phase prevents the enzymes from denaturation on the electrode surface, and provides contact of enzyme with the electrode. Thin layer of the cubic phase embedding glucose and pyranose oxidase activated glucose oxidation. The potentials and ratio of catalytic to diffusion currents are modified by choosing the appropriate electroactive probes as mediators. Ferrocenecarboxylic acid and Ru(NH3)62+ provided good contact between the electrode and the enzyme. The system based on Ru(NH3)62+ was found most suitable in terms of catalytic efficiency, low potential.

Acknowledgement: This work was financially supported by Ministry of Scientific Research and Information Technology - Project No. PBZ 18-KBN-098/T09/2003.

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Presentation: Tutorial lecture at SMCBS'2005 Workshop, by Renata Bilewicz
See On-line Journal of SMCBS'2005 Workshop

Submitted: 2005-09-19 17:34
Revised:   2009-06-07 00:44
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