title: Development of urease and glutamic dehydrogenase amperometric assay for heavy metals screening in polluted samples.
abstract: An amperometric assay based on urease inactivation has been developed for the screening of heavy metals in environmental samples. The enzyme urease catalyses the hydrolysis of urea and the formation of NH(4)(+) is determined using a NADH-glutamate dehydrogenase coupled reaction system. NADH consumption is monitored amperometrically using screen-printed three electrode configuration and its oxidation current is then correlated to urease activity. The presence of heavy metals in the samples inhibits the urease activity, resulting in a lower NH(4)(+) production and therefore a decrease in NADH oxidation. The use of metallised carbon electrodes gave a decrease in NADH oxidation potential from +300 mV versus Ag/AgCl compared with > +600 mV for bare carbon electrodes, and thus minimised interferences from oxidizable species present in the samples. Electrodes fouling and possible contamination after reuse and cleaning was also eliminated by using screen-printed disposable electrodes. The linear range obtained for Hg(II) and Cu(II) was 10-100 microgl(-1) with a detection limit of 7.2 microgl(-1) and 8.5 microgl(-1), respectively. Cd(II) and Zn(II) produced enzyme inhibition in the range 1-30 mgl(-1), with limits of detection of 0.3 mgl(-1) for Cd(II) and 0.2 mgl(-1) for Zn(II). Pb(II) did not inactivate the urease enzyme significantly at the studied range (up to 50 mgl(-1)). Coefficients of variation (CV) values were 6-9% in all cases. Application of the assay system to leachate samples gave reliable and accurate toxicity assessments when compared to atomic absorption spectrometry (AAS) and inductively coupled plasma atomic emission spectroscopy (ICP-MS) analysis. This approach provides to be a simple and rapid (15 min, including enzyme inhibition time) method for metal ions detection.
authors.affiliation: Cranfield Biotechnology Centre, Institute of Bioscience, Cranfield University, Silsoe, Bedforshire MK45 4DT, UK. b.bello-rodriguez.1999@cranfield.ac.uk
authors.forename: Belen Bello
authors.initials: BB
authors.lastname: Rodriguez
authors.forename: John A
authors.initials: JA
authors.lastname: Bolbot
authors.forename: Ibtisam E
authors.initials: IE
authors.lastname: Tothill
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D003300:Copper / Q000032:analysis; D004566:Electrodes; D004581:Electronics / Q000379:methods; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D008628:Mercury / Q000032:analysis; D019216:Metals, Heavy / Q000032:analysis*; D009243:NAD; D012989:Soil Pollutants / Q000032:analysis; D014510:Urease / Q000378:metabolism*; D014873:Water Pollutants / Q000032:analysis
chemical_list: D019216:Metals, Heavy; D012989:Soil Pollutants; D014873:Water Pollutants; D009243:NAD; D003300:Copper; D005969:Glutamate Dehydrogenase; D014510:Urease; D008628:Mercury