Polypyrrole-based bilayer nitrate amperometric biosensor with an integrated permselective poly-ortho-phenylenediamine layer for exclusion of inorganic interferences

A bilayer amperometric nitrate biosensor with an integrated permselective layer has been developed for exclusion of inorganic anion and cation interferences. The inner PPy(polypyrrole)–NaR–NADH layer of the biosensor is formed by galvanostatic polymerization of pyrrole (Py) in presence of nitrate reductase (NaR) and nicotinamide adenine dinucleotide (NADH), followed by formation of the outer permselective poly-ortho-phenylenediamine (P-o-PDA) layer by potentiodynamic polymerization of ortho-phenylenediamine (o-PDA). The exclusion efficiency (Eeff) of the outer layer in rejecting inorganic cation and anion interferences is evaluated by a new proposed relationship. 73–87% and 47–84% of anion and cation interferences, respectively, were efficiently rejected with the permselective layer. Further improvement in the exclusion efficiency for cations was accomplished by combining the use of the outer layer with the addition of 1 mM EDTA into the measurement solution. The addition of EDTA improved the Eeff achieved for cation rejection by 10–40% to give net Eeff of 89–94%. The inclusion of the outer layer also aided the retention of NaR and NADH in the inner PPy–NaR–NADH layer and, hence, enabled improved amperometric detection of nitrate, achieving a detection limit of 0.20 μM and a linear concentration range of 10–500 μM with a 3.4% rsd (n = 10).