Amperometric determination of ammonium with bienzyme/poly(carbamoyl) sulfonate hydrogel-based biosensor

A bienzyme sensor for the amperometric determination of ammonium (NH4+) was constructed by immobilizing glutamate oxidase (GXD, E.C. 1.4.3.11) and glutamate dehydrogenase (GlDH, E.C. 1.4.1.2) on a Clark-type oxygen electrode. The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. GlDH consumes ammonium for specific amination of 2-oxoglutarate in the presence of NADH. GXD consumes dissolved oxygen for the oxidative deamination of glutamate produced by GlDH. Dissolved oxygen acts as an essential material for GXD during its enzymatic reactions. Therefore, a signal detected as the maximum rate of the consumption of dissolved oxygen by GXD, was observed in the measurement of ammonium. A Teflon membrane was used to fabricate the biosensor in order to avoid interferences from real samples. The biosensor has a fast response (2 s) and short recovery time (2 min). The total test time for a measurement by using this ammonium biosensor (4 min) was faster than using a commercial ammonium testing kit (up to 20 min). The biosensor has a linear range between 10 and 300 μM ammonium, with a detection limit of 2.06 μM. A good agreement (R2 = 0.9984) with a commercial ammonium testing kit was obtained in the measurement of wastewater sample.