Utility of Wiring Nitrate Reductase by Alkylpyrroleviologen-Based Redox Polymers for Electrochemical Biosensor and Bioreactor Applications

Abstract

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.

Electroimmobilization of nitrate reductase and nicotinamide adenine dinucleotide into polypyrrole films for potentiometric detection of nitrate

“…The enzyme (NaR) is usually immobilized onto a suitable support matrix to minimise the use of enzyme and achieve compatibility of the biosensor. The range of materials that have been used for the immobilization of NaR for construction of nitrate biosensor include acrylamide copolymer, polythiophene-bipyridinium [24], polyvinyl alcohol [27], polystyrene-polybutadiene [30], viologen-acrylamide [31], sol-gel matrix [15], decanethiol [32], alkylpyrroleviologen [29,33], and polyviologen [26]. The detection of nitrate with the immobilized NaR is often achieved in the presence of NAD(P)H or other suitable co-factor by amperometry [19,22,25,27,34,35], cyclic voltammetry [19,29], luminescence [16,36,37], optical [15,38] and field effect transistor [39] detection.…”

Biosensors based on electropolymerized films: new trends

“…In particular, no significant loss of enzymatic activity was observed after 1 year for phenol biosensors based on polyphenol oxidase [40]. Ramsay et al have exploited this concept to study the immobilization and electrical wiring of a nitrate reductase from Aspergillus niger by a poly(amphiphilic pyrrole-viologen) film [41].…”

Electrochemical detection of nitrate in PM2.5 with a copper-modified carbon fiber micro-disk electrode

“…It is characterized by low sensitivity and irreproducibility and associated with surface passivation effects [29]. Therefore, a large number of strategies have been developed to modify electrode with some sensitive materials, such as copper [30,31], silver [32,33], cadmium [34,35], lead [36,37], palladium [38,39], polypyrrol materials [40], boron-doped diamond electrodes [41,42], and more recently, biocatalysts [43,44]. Among these materials, metallic copper is the most popular one because of its great advantages of low cost, good electrocatalysis and easy eletrodeposition [45][46][47].…”