A Microtiter-Plate Urease Inhibition Assay-Sensitive, Rapid and Cost-Effective Screening for Heavy Metals in Water

Abstract

The described microtiter-plate urease inhibition assay is a cost-effective tool to detect mercury and other heavy metals in surface water, ground water and waste water samples with highly specific sensitivity. The advantages of this test procedure are biological effect-based detection and quantifying of contamination, sensitivity, low costs and rapidity, thus meeting essential requirements for screening methods in environmental research and control. The test system is based on the incubation of samples with the isolated enzyme urea-amidohydrolase (urease) and the subsequent determination of the released ammonia with an indophenol reaction along with parallel determination of the ammonia background in the water sample. The assay indicates very sensitively mercury and other heavy metals. Highest sensitivity was found for mercury(II)chloride with a detection limit of 0.05 µg l−1 for Hg++. This substance was chosen as reference for the urease inhibition assay. Inhibitory effects of unknown samples were transformed to mercury (II) equivalents. The relative sensitivity of the test system to inorganic metal ions was in the order Hg>Co>Cr>Zn=Cd>Pb. For Fe(III) a concentration range exists with medium to weak inhibitory effects and an atypical biphasic dose-response relation. Tests with organic solvents and potent genotoxic substances like 2-aminoanthracene and 4-nitroquinolin-N-oxid showed moderate to weak inhibitory potential. Furthermore organic alkylants were tested, but inhibition effects mostly occur only at high concentrations (>10 mg l−1). Basic test conditions are described and discussed.