Electrochemical nitrate biosensor based on poly(pyrrole-viologen) film-nitrate reductase-clay composite

The immobilization of nitrate reductase (NR) was performed by entrapment in a laponite clay gel and cross-linking by glutaraldehyde. In presence of nitrate and methyl viologen, a catalytic current appeared at -0.60 V illustrating the enzymatic reduction of nitrate into nitrite via the reduced form of the freely diffusing methyl viologen. The electropolymerization of a water-soluble pyrrole viologen derivative within the interlamellar spaces and channels of the host clay matrix successfully carried out the electrical wiring of the entrapped NR. Rotating disk measurements led to the determination of kinetic constants, namely k(2)=10.7 s(-1) and K(M)=7 microM. These parameters reflect the efficiency of the electro-enzymatic reduction of nitrate and the substrate affinity for the immobilized enzyme.