Development of a Disposable Amperometric NH Biosensor Based on a Chemically Modified Screen-Printed Carbon Electrode Coated with Glutamate Dehydrogenase, 2-Oxoglutarate, and NADH

Abstract

A screen-printed carbon electrode (SPCE), impregnated with the electrocatalyst Meldola's Blue (MB), has been investigated as the base transducer in a disposable amperometric NH biosensor. The MB-SPCE detects the cofactor NADH when it is polarized at a potential of only +0.05 V (vs. Ag/AgCl); electrocatalytic oxidation of the cofactor readily occurs at this potential. The device was converted into an NHA biosensor by coating the surface of the MB-SPCE with glutamate dehydrogenase, 2-oxoglutarate and NADH. When ammonium ions are present in the sample solution, a decrease in the anodic current occurs as a result of the enzymatic conversion of 2-oxoglutarate to glutamate which requires NADH. Chronoamperometry was performed on 40 μL aliquots of solutions containing various concentrations of NH4+. A 30 s incubation period was used, then the potential was stepped from open circuit to +0.05 V (vs. Ag/AgCl); response currents were measured from the resulting chronoamperograms at a time of 120 s (t120s). The detection limit was found to be about 2 μM with biosensors containing 4.6 U of enzyme. The stability of these biosensors was examined after storage at 4°C in a desiccator containing silica gel; the response was found to be constant for a period of about 29 days. The proposed biosensors were evaluated on samples of unspiked, and spiked, river water; the recovery and precision data indicated that the devices could be expected to give reliable results for the low levels of NHA normaly expected in rivers.