Urease is the enzyme catalyzing the hydrolysis of urea into carbon dioxide and ammonia. This enzyme is substrate-specific, which means that the enzyme catalyzes the hydrolysis of urea only. This feature is a basic diagnostic criterion used in the determination of many bacteria species. Most of the methods utilized for detection of urease are based on analysis of its enzyme activity – the hydrolysis of urea. The aim of this work was to detect urease indirectly by spectrometric method and directly by voltammetric methods. As spectrometric method we used is called indophenol assay. The sensitivity of detection 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.
Drábová L., Pulkrabová J., Kalachová K., Hradecký J., Suchanová M., Tomaniová M., Kocourek V., Hajšlová J. (2011): Novel approaches to determination of PAhs and halogenated PoPs in canned fish. Czech J. Food Sci., 29: 498-507. A simple method is described for simultaneous isolation of 7 indicator polychlorinated biphenyls (PCBs), 10 polybrominated diphenyl ethers (PBDEs), 22 organochlorine pesticides (OCPs), and 16 polycyclic aromatic hydrocarbons (16 EU PAHs). The sample preparation procedure, including a pressurised liquid extraction (PLE) followed by gel permeation chromatography (GPC) for the selective isolation of the target compounds, was optimised and validated. For the final identification/quantitation of the target PCBs, PBDEs, OCPs, and PAHs, gas chromatography (GC) coupled to a high speed time-of-flight mass spectrometer (TOF MS) was used. The performance characteristics of the procedure were assessed including the recoveries (86-118% for PCBs, 73-113% for PBDEs, 71-113% for OCPs, and 85-111% for PAHs), repeatabilities (3-12% PCBs, 3-9% PBDEs, 1-11% OCPs and 3-10% PAHs), and limits of quantitation (LOQs -0.5 µg/kg PCBs, 0.1-0.3 µg/kg PBDEs, 0.1-0.5 µg/kg OCPs, and 0.03-0.1 µg/kg PAHs). Within the follow-up study, this method will be used for the monitoring of contamination of canned fish and sea food products available at the Czech market.
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