Utility of wiring nitrate reductase by alkylpyrroleviologen-based redox polymers for electrochemical biosensor and bioreactor applications

The purpose of this work was to see if the alkylpyrroleviologen redox polymer technology previously developed for a reagentless nitrate biosensor based on nitrate reductase (NaR) from Escherichia coli (Cosnier, S.; Innocent, C.; Jouanneau, Y. Anal. Chem. 1994, 66, 3198-3201) could be applied to the isozyme from Aspergillus niger. In particular, the enzyme viability after immobilization was of great interest, as Cosnier et al. reported a residual activity of only 0.33% of the amount initially applied. The present work showed that A. niger NaR lost 99.2% of soluble activity on vacuum-drying in the presence of 2.5 nM N-methyl-N'-(12-[pyrrol-1-yl]dodecyl)-4,4'-bipyridinium ditetrafluoroborate monomer (C12V2+) and that most of this loss was due to monomer inhibition (91%). The loss due to dehydration was only 8%. In the biosensor configuration, the enzyme gave a residual activity of 0.18% of the amount originally applied and a specific response of 1.7 mA M-1 cm-2, but all activity was lost after 4 d storage at 4 degrees C in phosphate buffer. It was concluded that for practical biosensors and bioreactors, modification of the redox polymer format was needed, for example by covalent immobilization, to effect higher loading of viable NaR and improved enzyme stability.