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

• A novel bienzymatic biosensor for the simultaneous determination of nitrite and nitrate has been developed.
• SOD1 and NaR are the more specific enzymes for NO2− oxidation and NO3− reduction respectively.
• Using CNT–PPy nanocomposite, SOD1 and NaR were effectively coimmobilized and attained higher sensitivity.
• It has been applied to measure NO2− and NO3− in human plasma, blood, and saliva samples.

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 µM−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 µM−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.