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A High Sensitivity Micro Format Chemiluminescence Enzyme Inhibition Assay for Determination of Hg(II)
Chemistry Group, Birla Institute of Technology and Science, Pilani-Goa Campus, Zuarinagar Goa, PIN 403726, India
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Received: 26 March 2010; in revised form: 17 May 2010 / Accepted: 11 June 2010 / Published: 28 June 2010
Abstract: A highly sensitive and specific enzyme inhibition assay based on alcohol oxidase (AlOx) and horseradish peroxidase (HRP) for determination of mercury Hg(II) in water samples has been presented. This article describes the optimization and miniaturization of an enzymatic assay using a chemiluminescence reaction. The analytical performance and detection limit for determination of Hg(II) was optimized in 96 well plates and further extended to 384 well plates with a 10-fold reduction in assay volume. Inhibition of the enzyme activity by dissolved Hg(II) was found to be linear in the range 5–500 pg.mL−1 with 3% CVin inter-batch assay. Due to miniaturization of assay in 384 well plates, Hg(II) was measurable as low as 1 pg.mL−1 within15 min. About 10-fold more specificity of the developed assay for Hg(II) analysis was confirmed by challenging with interfering divalent metal ions such as cadmium Cd(II) and lead Pb(II). Using the proposed assay we could successfully demonstrate that in a composite mixture of Hg(II), Cd(II) and Pb(II), inhibition by each metal ion is significantly enhanced in the presence of the others. Applicability of the proposed assay for the determination of the Hg(II) in spiked drinking and sea water resulted in recoveries ranging from 100–110.52%.
Keywords: high throughput assay; miniaturization; Hg(II); alcohol oxidase; peroxidase; enzyme inhibition; chemiluminescence; water
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Deshpande, K.; Mishra, R.K.; Bhand, S. A High Sensitivity Micro Format Chemiluminescence Enzyme Inhibition Assay for Determination of Hg(II). Sensors 2010, 10, 6377-6394.
Deshpande K, Mishra RK, Bhand S. A High Sensitivity Micro Format Chemiluminescence Enzyme Inhibition Assay for Determination of Hg(II). Sensors. 2010; 10(7):6377-6394.
Deshpande, Kanchanmala; Mishra, Rupesh K.; Bhand, Sunil. 2010. "A High Sensitivity Micro Format Chemiluminescence Enzyme Inhibition Assay for Determination of Hg(II)." Sensors 10, no. 7: 6377-6394.