An Optical Based Biosensor for the Determination of Ammonium in Aqueous Environment


A simple and rapid optical biosensor for the determination of ammonium was developed by immobilization of gluta-mate dehydrogenase (GLDH) and diaphorase (Dph) in chitosan film coated on a glass slide employing thiazolyl blue tetrazolium bromide (MTT) as a color indicator. The developed biosensor displays a purple color formation of formazan attributed to the unreacted NADH in the reaction system in the presence of ammonium. The color intensity was found to decrease proportionally with the increase of ammonium concentrations after 10 min exposure. The linearity of the biosensor towards ammonium was in the range of 16.8 – 70 μM (R2 = 0.9955) with detection limit of 11 μM. A good agreement (R2 = 0.9984) with indothymol method was obtained in the measurement of fish pond water samples. The effect of potential interferences such as metals ion has also been evaluated.

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N. Azmi, J. Abdullah, M. Ahmad, H. Sidek, L. Heng and S. Rahman, "An Optical Based Biosensor for the Determination of Ammonium in Aqueous Environment," American Journal of Analytical Chemistry, Vol. 3 No. 5, 2012, pp. 364-370. doi: 10.4236/ajac.2012.35048.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] F. Valentini, V. Biagiotti, C. Lete, G. Palleschi and J. Wang, “The Electrochemical Detection of Ammonia in Drinking Water Based on Multi-Walled Carbon Nano-tube/Copper Nanoparticle Composite Paste Electrodes,” Sensors and Actuators B: Chemical, Vol. 128, No. 1, 2007, pp. 326-333. doi:10.1016/j.snb.2007.06.010
[2] R. C. H. Kwan, P. Y. T. Hon and R. Renneberg, “Ampe-rometric Determination of Ammonium with Bienzyme/Poly (Carbamoyl) Sulfonate Hydrogel-Based Biosensor,” Sen- sors and Actuators B: Chemical, Vol. 107, No. 2, 2005, pp. 616-622. doi:10.1016/j.snb.2004.11.028
[3] B. Strehlitz, B. Grundig and H. Kopinke, “Sensor for Amperometric Determination of Ammonia and Ammonia- Forming Enzyme Reactions,” Analytica Chimica Acta, Vol. 403, No. 1-2, 2000, pp. 11-23. doi:10.1016/S0003-2670(99)00594-2
[4] K. Fukushi, H. Ito, K. Kimura, K. Yokota, K. Saito, K. Chayama, S. Takeda and S. Wakida, “Determination of Ammonium in River Water and Sewage Samples by Ca- pillary Zone Electrophoresis with Direct UV Detection,” Journal of Chromatography A, Vol. 1106, No. 1-2, 2006, pp. 61-66. doi:10.1016/j.chroma.2005.10.054
[5] Q. P. Li, J. Z. Zhang, F. J. Millero and D. A. Hansell, “Continuous Colorimetric Determination of Trace Ammo-nium in Seawater with a Long-Path Liquid Waveguide Capillary Cell,” Marine Chemistry, Vol. 107, No. 1-2, 2005, pp. 73-85. doi:10.1016/j.marchem.2004.12.001
[6] K. T. Lau, S. Edwards and D. Diamond, “Solid-State Ammonia Sensor Based on Berthelot’s Reaction,” Sensors and Actuators B: Chemical, Vol. 98, No. 1, 2004, pp. 12-17. doi:10.1016/j.snb.2003.08.004
[7] A. K. Abbas, J. P. Hart, D. C. Cowell and A. Chappell, “Development of an Amperometric Assay for NH4+ Based on a Chemically Modified Screen-Printed NADH,” Analytica Chimica Acta, Vol. 373, No. 1, 1998, pp. 1-8. doi:10.1016/S0003-2670(98)00368-7
[8] P. Bertocchi and D. Compagnone, “Amperometric Ammo- nium Ion and Urea Determination with Enzyme-Based Probes,” Biosensors and Bioelectronics, Vol. 11, No. 1-2, 1996, pp. 1-10. doi:10.1016/0956-5663(96)83708-0
[9] R. Quiles, J. M. F. Romero, E. Fernandez and M. D. L. Castro, “Continuous Flow Assay of Ammonia in Plasma Using Immobilized Enzymes,” Analytica Chimica Acta, Vol. 294, No. 1, 1996, pp. 43-47. doi:10.1016/0003-2670(94)85044-5
[10] S. Hashimoto, H. Y. Sun, A. Otsuki, S. Kawakama and M. Yokomizo, “Enzymatic Determination of Ammonia in Lake Water Using a Semi-Automatic Analyser,” Inter- national Journal of Environmental Analytical Chemistry, Vol. 48, No. 3-4, 1992, pp. 155-161. doi:10.1080/03067319208027396
[11] H. Jaegfelt, A. B. C. Torstensson, L. Gorton and G. Johansson, “Catalytic Oxidation of Reduced Nicotina-mide Adenine Dinucleotide by Graphite Electrodes Modi- fied with Adsorbed Aromatics Containing Catechol Functionalities,” Analytical Chemistry, Vol. 53, No. 13, 1981, pp. 1979-1982. doi:10.1021/ac00236a007
[12] J. Moiroux and P. J. Elving, “Adsorption Phenomena in the NAD+/NADH System at Glassy Carbon Electrodes,” Journal of Electroanalytical Chemistry, Vol. 102, No. 1, 1979, pp. 93-108. doi:10.1016/S0022-0728(79)80033-9
[13] E. Wang, L. Zhu, L. Ma and H. Patel, “Optical Sensors for Sodium, Potassium and Ammonium Ions Based on Lipophilic Fluorescein Anionic Dye and Neutral Carriers,” Analytica Chimica Acta, Vol. 357, No. 1-2, 1997, pp. 85-90. doi:10.1016/S0003-2670(97)00532-1
[14] Y. M. Martinez, R. H. Hernandez and P. C. Falco, “Improved Detection Limit for Ammonium/Ammonia Achieved by Berhelot’s Reaction by Use of Solid-Phase Extraction Coupled to Diffuse Reflectance Spectroscopy,” Analytica Chimica Acta, Vol. 534, No. 2, 2005, pp. 327- 334. doi:10.1016/j.aca.2004.11.044
[15] A. K. Sarker, H. Ukeda, D. Kawana and M. Sawamura, “Enzymatic Assay of Dehydrogenase Substrate Based on the Detection of Superoxide Anion,” Food Research International, Vol. 34, No. 5, 2001, pp. 393-399. doi:10.1016/S0963-9969(00)00183-6
[16] N. E. Azmi, J. Ab-dullah, M. Ahmad, L. Y. Heng, H. Sidek and K. Nadarajah, “Biosensor Based on Glutamate Dehydrogenase Immobilized in Chitosan for the Deter- mination of Ammonium in Water Samples,” Analytical Biochemistry, Vol. 388, No. 1, 2009, pp. 28-32. doi:10.1016/j.ab.2009.02.005
[17] P. Odman, W. B. Wellborn and A. S. Bommarius, “An Enzymatic Process to α-Ketoglutarate from L-Glutamate: the Coupled System L-Glutamate Dehydrogenase/NADH Oxidase,” Tetrahedron: Assymetry, Vol. 15, No. 18, 2004, pp. 2933-2937. doi:10.1016/j.tetasy.2004.07.055
[18] B. Kuswandi, Nuriman, H. H. Dam, D. N. Reinhoud and W. Verboom, “Development of a Disposable Mercury Ion-Selective Optode Based on Tri-tyl-Picolinamide as Ionophore,” Analytica Chimica Acta, Vol. 591, No. 2, 2007, pp. 208-213. doi:10.1016/j.aca.2007.03.064
[19] A. Bollmann and N. P. Revsbech, “An NH4+ Biosensor Based on Ammonia-Oxidizing Bacteria for Use under Anoxic Conditions,” Sensor and Actua-tors B, Vol. 105, No. 2, 2005, pp. 412-418. doi:10.1016/j.snb.2004.06.030
[20] W. D. Boer, P. A. K. Gunnewiek and H. J. Laanbroek, “Ammonium-Oxidation at Low pH by a Chemolithotrophic Bacterium Belonging to the Genus Nitrosospira,” Soil Biology Biochemistry, Vol. 27, No. 2, 1995, pp. 127-132. doi:10.1016/0038-0717(94)00157-V
[21] J. N. Miller and J. C. Miller, “Statistics and Chemometrics for Analytical Chemistry,” 4th Edition, Pearson Education Limited, Essex, 2000, pp. 42-51.

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