Abstract: Urease is the enzyme catalyzing the hydrolysis of urea into carbon dioxide andammonia. This enzyme is substrate-specific, which means that the enzyme catalyzes thehydrolysis of urea only. This feature is a basic diagnostic criterion used in thedetermination of many bacteria species. Most of the methods utilized for detection ofurease are based on analysis of its enzyme activity – the hydrolysis of urea. The aim of thiswork was to detect urease indirectly by spectrometric method and directly by voltammetricmethods. As spectrometric method we used is called indophenol assay. The sensitivity ofdetection itself is not sufficient to analyse the samples without pre-concentration steps.Therefore we utilized adsorptive transfer stripping technique coupled with differential pulse voltammetry to detect urease. The influence of accumulation time, pH of supporting electrolyte and concentration of urease on the enzyme peak height was investigated. Under the optimized experimental conditions (0.2 M acetate buffer pH 4.6 and accumulation time of 120 s) the detection limit of urease evaluated as 3 S/N was 200 ng/ml. The activity of urease enzyme depends on the presence of nickel. Thus the influence of nickel(II) ions on electrochemical response of the enzyme was studied. Based on the results obtained the interaction of nickel(II) ions and urease can be determined using electrochemical methods. Therefore we prepared Ni nanoelectrodes to measure urease. The Ni nanoelectrodes was analysed after the template dissolution by scanning electron microscopy. The results shown vertically aligned Ni nanopillars almost covered the electrode surface, whereas the defect places are minor and insignificant in comparison with total electrode surface. We were able to not only detect urease itself but also to distinguish its native and denatured form.
Keywords: urease; electrochemical methods; nanotechnology; nanotube; nickel electrode; hanging mercury drop electrode; spectrometry
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Hubalek, J.; Hradecky, J.; Adam, V.; Krystofova, O.; Huska, D.; Masarik, M.; Trnkova, L.; Horna, A.; Klosova, K.; Adamek, M.; Zehnalek, J.; Kizek, R. Spectrometric and Voltammetric Analysis of Urease – Nickel Nanoelectrode as an Electrochemical Sensor. Sensors 2007, 7, 1238-1255.
Hubalek J, Hradecky J, Adam V, Krystofova O, Huska D, Masarik M, Trnkova L, Horna A, Klosova K, Adamek M, Zehnalek J, Kizek R. Spectrometric and Voltammetric Analysis of Urease – Nickel Nanoelectrode as an Electrochemical Sensor. Sensors. 2007; 7(7):1238-1255.
Hubalek, Jaromir; Hradecky, Jan; Adam, Vojtech; Krystofova, Olga; Huska, Dalibor; Masarik, Michal; Trnkova, Libuse; Horna, Ales; Klosova, Katerina; Adamek, Martin; Zehnalek, Josef; Kizek, Rene. 2007. "Spectrometric and Voltammetric Analysis of Urease – Nickel Nanoelectrode as an Electrochemical Sensor." Sensors 7, no. 7: 1238-1255.