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.
References
[1]
Kuo, CT; Wang, PY; Wu, CH. Fluorometric determination of ammonium ion by ion chromatography using postcolumn derivatization with o-phthaldialdehyde. J. Chromatogr. A 2005, 1085, 91–97, doi:10.1016/j.chroma.2005.05.042. 16106853
[2]
Martinez, YM; Hernandez, RH; Falco, PC. Improved detection limit for ammonium/ammonia achieved by Berhelot’s reaction by use of solid-phase extraction coupled to diffuse reflectance spectroscopy. Anal. Chim. Acta 2005, 534, 327–334, doi:10.1016/j.aca.2004.11.044.
[3]
Thomas, DH; Rey, M; Jackson, PE. Determination of inorganic cations and ammonium in environmental waters by ion chromatography with a high-capacity cation-exchange column. J. Chromatogr. A 2002, 956, 181–186, doi:10.1016/S0021-9673(02)00141-3. 12108649
[4]
Deyhimi, F; Choobar, BG. Potentiometric determination of activity coefficients for NH4Cl in the ternary NH4Cl/LiCl/H2O mixed electrolyte system. J. Electroanal. Chem 2005, 584, 141–146, doi:10.1016/j.jelechem.2005.07.012.
[5]
Hassan, SSM; Marei, SA; Badr, IH; Arida, HA. Novel solid-state ammonium ion potentiometric sensor based on zirconium titanium phosphate ion exchanger. Anal. Chim. Acta 2001, 427, 21–28, doi:10.1016/S0003-2670(00)01189-2.
[6]
Andrew, KN; Worsfold, PJ; Comber, M. On-line flow injection monitoring of ammonia in industrial liquid effluents. Anal. Chim. Acta 1995, 314, 33–43, doi:10.1016/0003-2670(95)00269-6.
[7]
Aminot, A; Kerouel, R; Birot, D. A flow injection-fluorometri method for the determination of ammonium in fresh and saline waters with a view to in situ analyses. Wat. Res 2001, 35, 1777–1785, doi:10.1016/S0043-1354(00)00429-2.
[8]
Haghighi, B; Kurd, SF. Sequential flow injection analysis of ammonium and nitrate using gas phase molecular absorption spectrometry. Talanta 2004, 64, 688–694, doi:10.1016/j.talanta.2004.03.037. 18969660
[9]
Kerouel, R; Aminot, A. Fluorometric determination of ammonia in sea and estuarine waters by direct segmented flow analysis. Mar. Chem 1997, 57, 265–275, doi:10.1016/S0304-4203(97)00040-6.
[10]
Oliveira, SM; Lopes, TIMS; Toth, IV; Rangel, AOSS. A multi-commuted flow injection system with a multi-channel propulsion unit placed before detection: Spectrophotometric determination of ammonium. Anal. Chim. Acta 2007, 600, 29–34, doi:10.1016/j.aca.2007.01.019. 17903461
[11]
Staden, JFV; Taljaard, RE. Determination of ammonia in water and industrial effluent streams with the indophenol blue method using sequential injection analysis. Anal. Chim. Acta 1997, 344, 281–289, doi:10.1016/S0003-2670(96)00523-5.
[12]
Mori, M; Tanaka, K; Helaleh, MIH; Xu, Q; Ikedo, M; Ogura, Y; Sato, S; Hu, W; Hasebe, K. Selective determination of ammonium ios by high-speed ion-exclusion chromatography on a weakly baic anion-exchange resin column. J. Chromatogr. A 2003, 997, 191–197, doi:10.1016/S0021-9673(03)00625-3. 12830892
[13]
Kazanskaya, N; Kukhtin, A; Manenkova, M. FET-based sensors with robust photosensitive polymer membranes for determination of ammonium ion and urea. Biosens. Bioelectron 1996, 11, 253–261, doi:10.1016/0956-5663(96)88412-0.
[14]
Senillou, A; Renault, NJ; Griffe, CMF. A miniaturized ammonium sensor based on the integration of both ammonium and reference FETs in a sungle chip. Mater. Sci. Eng. C 1998, 6, 59–63, doi:10.1016/S0928-4931(98)00037-X.
[15]
Dubas, ST; Pimpan, V. Green synthesis of silver nanoparticles for ammonia sensing. Talanta 2008, 76, 29–33, doi:10.1016/j.talanta.2008.01.062. 18585235
[16]
Li, PQ; Zhang, JZ; Millero, FJ; Hansell, DA. Continuous colorimetric determination of trace ammonium in seawater with a long-path liquid waveguide capillary cell. Mar. Chem 2005, 107, 73–85.
[17]
Abass, AK; Hart, JP; Cowell, DC; Chappell, A. Development of an amperometric assay for NH4+ based on a chemically modified screen-printed NADH sensor. Anal. Chim. Acta 1998, 373, 1–8, doi:10.1016/S0003-2670(98)00368-7.
[18]
Bertocchi, P; Compagnone, D. Amperometric ammonium ion and urea determination with enzyme-based probes. Biosens. Bioelectron 1996, 11, 1–10, doi:10.1016/0956-5663(96)83708-0. 8600914
[19]
Kwan, RCH; Hon, PYT; Renneberg, R. Amperometric determination of ammonium with bienzyme/poly(carbamoyl) sulfonate hydrogel-based biosensor. Sens. Actuat. B 2005, 107, 616–622, doi:10.1016/j.snb.2004.11.028.
[20]
Agren, D; Stehr, M; Berthold, CL; Kapoor, S; Oehlmann, W; Singh, M; Schneider, G. Three-dimensional structures of apo- and hoho-L-alanine dehydrogenase from mycobacterium tuberculosis reveal conformational changes upon coenzyme binding. J. Mol. Biol 2008, 377, 1161–1173, doi:10.1016/j.jmb.2008.01.091. 18304579
[21]
Hashimoto, SI; Katsumata, R. l-alanine fermentation by an alanine racemase-deficient mutant of the dl-alanine hyperproducing bacterium Arthrobacter oxydans HAP-1. J. Ferment. Bioeng 1998, 86, 385–390, doi:10.1016/S0922-338X(99)89009-6.
[22]
Kato, SI; Ohshima, T; Galkin, A; Kulakova, L; Yoshimura, T; Esaki, N. Purification and characterization of alanine dehydrogenase from a marine bacterium, vibrio proteolyticus. J. Mol. Catal. Part B 2003, 23, 373–378, doi:10.1016/S1381-1177(03)00101-2.
[23]
Ohashima, T; Soda, K. Purification and properties of alanine dehydrogenase from Bacillus spharicus. Eur. J. Biochem 1979, 100, 29–39, doi:10.1111/j.1432-1033.1979.tb02030.x. 488097
[24]
Nassef, HM; Radi, AE; Sullivan, CKO. Electrocatalytic sensing of NADH on a glassy carbon electrode modified with electrografted o-aminophenol film. Electrochem. Commun 2006, 8, 1719–1725, doi:10.1016/j.elecom.2006.07.045.
[25]
Behera, S; Retna, CR. Mediatorless voltammetric oxidation of NADH and sensing of ethanol. Biosens. Bioelectron 2007, 21, 949–956.
[26]
Retna, CR; Behera, S. Self-assembled monolayers of thio-substituted nucleobases on gold electrode for the electroanalysis of NADH, ethanol and uric acid. Sens. Actuat. B 2005, 128, 31–38.
[27]
Rao, TN; Yagi, I; Miwa, T; Tryk, DA; Fujishima, A. Electrochemical oxidation of NADH at highly boron-doped diamond electrodes. Anal. Chem 1999, 71, 2506–2511, doi:10.1021/ac981376m. 21662795
[28]
Schroder, I; Vadas, A; Johnson, E; Lim, S; Monbouquette, HG. A novel archeal alanine dehydrogenase homologous to ornithine cyclodeaminase and μ-crystalin. J. Bacteriol 2004, 186, 7680–7689, doi:10.1128/JB.186.22.7680-7689.2004. 15516582
[29]
Carr, PW; Bowers, LD. Immobilised Enzymes in Analytical and Clinical Chemistry; John Wiley & Sons: New York, NY, USA, 1980.
[30]
Kwan, RCH; Hon, PYT; Renneberg, R. Amperometric biosensor for rapid determination of alanine. Anal. Chim. Acta 2004, 523, 81–88, doi:10.1016/j.aca.2004.07.019.
[31]
Renkema, JMS; Gruppen, H; Vliet, TV. Influence of pH and ionic strength on heat-induced formation and rheological properties of soy protein gels in relation to denaturation and their protein compositions. J. Agric. Food Chem 2002, 50, 6064–6071, doi:10.1021/jf020061b. 12358481
[32]
Carville, M; Robinson, H. Leachate treatment. Available online: http://www.leachate-treatment.com (accessed on 14 July 2011).
[33]
Nur Ellina, A; Jaafar, A; Musa, A; Heng, LY; Hamidah, S; Nadarajah, K. Biosensor based on glutamate dehydrogenase immobilized in chitosan for the determination of ammonium in water samples. Anal. Biochem 2009, 388, 28–32, doi:10.1016/j.ab.2009.02.005. 19454217
