We have developed here a novel, highly sensitive and selective nitrate (NO– 3) biosensor by covalent immobilization of nitrate reductase (NaR) in self-assembled monolayer (SAM) of cysteine on gold nanoparticles (GNP)-polypyrrole (PPy) modified platinum electrode. Incorporation of GNP in highly microporous PPy matrix was confirmed by morphological scanning electron microscope (SEM) images. The electrochemical behavior of the NaR modified electrode exhibited the characteristic reversible redox peaks at the potential, –0.76 and –0.62 V versus Ag/AgCl. Further, the GNP-PPy nanocomposite enhanced the current response by 2-fold perhaps by enhancing the immobilization of NaR and also direct electron transfer between the deeply buried active site and the electrode surface. The common biological interferences like ascorbic acid, uric acid were not interfering with the NO– 3 measurement at low concentration levels. This biosensor showed a wide linear range of response over the concentration of NO– 3 from 1 μM to 1 mM, with higher sensitivity of 84.5 nA μM–1 and a detection limit of 0.5 μM. Moreover, the NO– 3 level present in the nitrate-rich beetroot juice and the NO– 3 release from the lipopolysaccharide treated human breast cancer cells were estimated
