Electrochemistry of immobilized redox enzymes: kinetic characteristics of NADH oxidation catalysis at diaphorase monolayers affinity immobilized on electrodes |
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Authors: | Limoges Benoît Marchal Damien Mavré François Savéant Jean-Michel |
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Affiliation: | Laboratoire d'Electrochimie Moléculaire, Université de Paris 7-Denis Diderot, 2 place Jussieu,75251 Paris Cedex 05, France. limoges@paris7.jussieu.fr |
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Abstract: | In the class of NADH:acceptor oxidoreductases, the diaphorase from Bacillus stearothermophilusis a particularly promising enzyme for sensing NADH, and indirectly a great number of analytes, when coupled with a NAD-dependent dehydrogenase as well as for the design of mono- and multienzyme affinity sensors. The design and rational optimization of such systems require devising immobilization procedures that prevent dramatic losses of the enzymatic activity and a full kinetic characterization of the immobilized enzyme system. Two immobilization procedures are described, which involve recognition of the biotinylated diaphorase by a monolayer of neutravidin adsorbed on the electrode surface either directly or through the intermediacy of a monolayer of biotinylated rabbit immunoglobulin. Thorough kinetic characterization of the two systems is derived from cyclic voltammetric responses. A precise estimate of the enzyme coverages is obtained after comparing the enzyme kinetics of the immobilized and the homogeneous system. |
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