Copper, zinc superoxide dismutase and nitrate reductase coimmobilized bienzymatic biosensor for the simultaneous determination of nitrite and nitrate

This work presents a novel bienzymatic biosensor for the simultaneous determination of nitrite (NO2-) and nitrate (NO3-) ions using copper, zinc superoxide dismutase (SOD1) and nitrate reductase (NaR) coimmobilized on carbon nanotubes (CNT)-polypyrrole (PPy) nanocomposite modified platinum electrode. Morphological changes of the PPy and CNT modified electrodes were investigated using scanning electron microscopy. The electrochemical behavior of the bienzymatic electrode (NaR-SOD1-CNT-PPy-Pt) was characterized by cyclic voltammetry exhibiting quasi-reversible redox peak at +0.06 V and reversible redox peaks at -0.76 and -0.62 V vs. Ag/AgCl, for the immobilized SOD1 and NaR respectively. The electrocatalytic activity of SOD1 towards NO2- oxidation observed at +0.8 V was linear from 100 nM to 1 mM with a detection limit of 50 nM and sensitivity of 98.5 +/- 1.7 nA mu M-1 cm(-2). Similarly, the coimmobilized NaR showed its electrocatalytic activity towards NO3- reduction at -0.76 V exhibiting linear response from 500 nM to 10 mM NO3- with a detection limit of 200 nM and sensitivity of 84.5 +/- 1.56 nA mu M-1 cm(-2). Further, the present bienzymatic biosensor coated with cellulose acetate membrane for the removal of non-specific proteins was used for the sensitive and selective determinations of NO2- and NO3- present in human plasma, whole blood and saliva samples. (C) 2013 Elsevier B.V. All rights reserved.