A High Sensitivity Micro Format Chemiluminescence Enzyme Inhibition Assay for Determination of Hg(II)

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% CV in inter-batch assay. Due to miniaturization of assay in 384 well plates, Hg(II) was measurable as low as 1 pg·mL−1 within 15 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%.

Modern nanobiotechnologies for efficient detection and remediation of mercury

“…The inhibition of carbonic anhydrase by heavy metalsspecifically Pb, Cu and Hg has been reported (Ek _ Inc _ et al, 2007). The biosensors for Hg 21 detection developed using the inhibition of alcohol oxidase (Deshpande et al, 2010) showed great amplification of the chemiluminescent signal. The specificity was also tested with Pb and Cd and the developed enzyme biosensor revealed a LOD of 1 pg mL À1 and good specificity for Hg 21 .…”

Bio-Recognition in Spectroscopy-Based Biosensors for *Heavy Metals-Water and Waterborne Contamination Analysis

“…The enzymatic inhibition results in a CL suppression that is analyte concentration dependent. Though, in a previous work, Deshpande et al [41], exploiting a two enzyme based (i.e., alcohol oxidase (AlOx) and HRP) inhibition assay for single HM ion determination, showed a lower LOD (1 pg/mL) for Hg 2+ ions.…”

Detection

“…Many of these tests and devices are being developed to address needs other than security (e.g., water quality issues such as arsenic in well water); however, these tests may also serve in enhanced water security. Examples include microfluidic sensors that can detect low concentrations of contaminants such as lead (Chang et al, 2005), a biosensor used to detect arsenic in drinking water (de Mora et al, 2011), micro-chemiluminescence enzyme tests for mercury in drinking water (Deshpande et al, 2010), a self-powered enzyme biofuel cell on a microchip for cyanide detection (Deng et al, 2010), and a biosensor for arsenic detection that is inexpensive and field deployable (Joshi et al, 2009). Advancements have also been made in testing for lead in drinking water (Wang et al, 2001).…”