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Abstract
We used a luminescence-based biosensor to determine mercury in urine samples of subjects with or without dental amalgam restorations. The system utilizes Escherichia coli as a host organism and firefly luciferase luc gene as a reporter under the control of the mercury-inducible promoter from the mer operon from transposon Tn21. The presence of mercury induces the specific gene sequence with consequent synthesis of the luciferase enzyme, and luciferin/luciferase-mediated light output occurs. The method showed a linear relationship between the light signal intensity and the Hg2+ concentration in the range of 1.67 × 10−13–1.67 × 10−7 M with a detection limit of 1.67 × 10−13 M, corresponding to 4.0 × 10−18 mol/tube. The system proved precise, accurate, and highly selective for mercury and methylmercury, with no light emission induced by other heavy metals; the matrix effect caused by urine components was minimal. The analysis of urine specimens showed that urinary mercury levels in subjects with amalgam fillings were slightly significantly higher than those in subjects without amalgam fillings (p < 0.1) and the statistically significant difference improved when creatinine-normalized values were considered (p < 0.05). In summary, from the analytical point of view this luminescent microbial biosensor represents an easy, rapid, and sensitive tool to analyse Hg2+ in biological and environmental samples. Along with the possibility of using 384-well microtitre plates requiring very low reagent volumes, these characterisitcs make the system suitable for the high-throughput screening of Hg2+ on large numbers of specimens. This method could help better clarify the relationship between dental amalgam and urinary mercury excretion by permitting large-scale analysis of many selected groups of subjects, with strict control of all the relevant parameters affecting Hg2+ concentration in urine, such as diet and environmental and occupational exposure.
ACKNOWLEDGMENTS
This work was partly supported by the Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST). The authors thank Dr. M. Virta (University of Turku, Finland) for supplying E. coli MC1061 cells transformed by the plasmid pTOO11. Mr. Robin M.T. Cooke is also thanked for helping revise the manuscript.