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Determination of Ammonium Ion Using a Reagentless Amperometric Biosensor Based on Immobilized Alanine Dehydrogenase
School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor 43600, Malaysia
Faculty of Science and Technology, Universiti Sains Islam Malaysia, Bandar Baru Nilai, Nilai, Negeri Sembilan D.K. 71800, Malaysia
* Author to whom correspondence should be addressed.
Received: 14 July 2011; in revised form: 14 August 2011 / Accepted: 5 September 2011 / Published: 29 September 2011
Abstract: The use of the enzyme alanine dehydrogenase (AlaDH) for the determination of ammonium ion (NH4+) usually requires the addition of pyruvate substrate and reduced nicotinamide adenine dinucleotide (NADH) simultaneously to effect the reaction. This addition of reagents is inconvenient when an enzyme biosensor based on AlaDH is used. To resolve the problem, a novel reagentless amperometric biosensor using a stacked methacrylic membrane system coated onto a screen-printed carbon paste electrode (SPE) for NH4+ ion determination is described. A mixture of pyruvate and NADH was immobilized in low molecular weight poly(2-hydroxyethyl methacrylate) (pHEMA) membrane, which was then deposited over a photocured pHEMA membrane (photoHEMA) containing alanine dehydrogenase (AlaDH) enzyme. Due to the enzymatic reaction of AlaDH and the pyruvate substrate, NH4+ was consumed in the process and thus the signal from the electrocatalytic oxidation of NADH at an applied potential of +0.55 V was proportional to the NH4+ ion concentration under optimal conditions. The stacked methacrylate membranes responded rapidly and linearly to changes in NH4+ ion concentrations between 10–100 mM, with a detection limit of 0.18 mM NH4+ ion. The reproducibility of the amperometrical NH4+ biosensor yielded low relative standard deviations between 1.4–4.9%. The stacked membrane biosensor has been successfully applied to the determination of NH4+ ion in spiked river water samples without pretreatment. A good correlation was found between the analytical results for NH4+ obtained from the biosensor and the Nessler spectrophotometric method.
Keywords: biosensor; ammonium ion; reduced nicotinamide adenine dinucleotide; poly(2-hydroxyethyl methacrylate); alanine dehydrogenase
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MDPI and ACS Style
Tan, L.L.; Musa, A.; Lee, Y.H. Determination of Ammonium Ion Using a Reagentless Amperometric Biosensor Based on Immobilized Alanine Dehydrogenase. Sensors 2011, 11, 9344-9360.
Tan LL, Musa A, Lee YH. Determination of Ammonium Ion Using a Reagentless Amperometric Biosensor Based on Immobilized Alanine Dehydrogenase. Sensors. 2011; 11(10):9344-9360.
Tan, Ling Ling; Musa, Ahmad; Lee, Yook Heng. 2011. "Determination of Ammonium Ion Using a Reagentless Amperometric Biosensor Based on Immobilized Alanine Dehydrogenase." Sensors 11, no. 10: 9344-9360.