Development of Electrochemical Assay Kit for the Measurement of Nitrite/Nitrate Using Microelectrode Immobilized with Carbon Nanotubes and Copper, Zinc Superoxide Dismutase

Determination of nitric oxide (NO), nitrite (NO₂ ^-) and nitrate (NO₃ ^-) is progressively becoming more important during hypoxia, nutrition and therapeutics. Hence, we have developed a novel electrochemical assay for the measurement of NO, NO₂ ^- and NO₃ ^- in volume miniaturized sample using cost effective screen printed microelectrode. It is based on the electrochemical oxidation and reduction reactions of NO and NO₂ ^- on the biosensor electrode immobilized with carbon nanotubes and copper, zinc superoxide dismutase (Cu,ZnSOD). The electrochemical characterization of Cu,ZnSOD (SOD1) immobilized microelectrode exhibited the characteristic quasi-reversible redox peak at the potential, +0.06 V vs. Ag/AgCl, confirming the immobilization of SOD1. Further, the carbon nanotubes modified electrode showed 2-fold increase in current response perhaps by enhancing the immobilization of SOD1 and also direct electron transfer between the deeply buried active site and the electrode surface. In order to measure these analytes selectively among the other co-existing biological substrates, nafion membrane for NO and cellulose acetate membrane for NO₂ ^- and NO₃ ^- were used. It showed a wide linear range of response over the concentration 100 nM to 1 mM for NO and 200 nM to 5 mM for NO₂ ^- with a detection limit of 50 nM NO₂ ^-. Moreover, it exhibited very higher sensitivity 96.4 nA μM^-1 for NO and 88.4 nA μM^-1 for NO₂ ^-. Using this microelectrode, the electrochemical assay kit for the determination of the NO₂ ^- and NO₃ ^- levels were estimated in human serum samples.