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

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%.

[1]  Julia Xiaojun Zhao,et al.  Sensing Mercury for Biomedical and Environmental Monitoring , 2009, Sensors.

[2]  I. J. van der Klei,et al.  Alcohol oxidase: a complex peroxisomal, oligomeric flavoprotein. , 2005, FEMS yeast research.

[3]  M. Karve,et al.  Invertase inhibition based electrochemical sensor for the detection of heavy metal ions in aqueous system: Application of ultra-microelectrode to enhance sucrose biosensor's sensitivity. , 2008, Biosensors & bioelectronics.

[4]  Danila Moscone,et al.  Reversible Enzyme Inhibition–Based Biosensors: Applications and Analytical Improvement Through Diagnostic Inhibition , 2009 .

[5]  Phil Monbet,et al.  Measurements of trace concentrations of mercury in sea water by stripping chronopotentiometry with gold disk electrode: influence of copper , 2000 .

[6]  Mark R. Geier,et al.  A Case-Control Study of Mercury Burden in Children with Autistic Spectrum Disorders , 2003 .

[7]  Aziz Amine,et al.  Mercury–enzyme inhibition assays with an amperometric sucrose biosensor based on a trienzymatic-clay matrix , 2005 .

[8]  D. Barceló,et al.  Biosensors for environmental applications: Future development trends , 2004 .

[9]  G. Ozolins,et al.  WHO guidelines for drinking-water quality. , 1984, WHO chronicle.

[10]  K. Tsukagoshi,et al.  Metal ion analysis using microchip CE with chemiluminescence detection based on 1,10-phenanthroline-hydrogen peroxide reaction. , 2009, Journal of separation science.

[11]  Filiz Kuralay,et al.  Inhibitive determination of Hg2+ ion by an amperometric urea biosensor using poly(vinylferrocenium) film , 2007 .

[12]  G. Palleschi,et al.  Enzyme inhibition-based biosensors for food safety and environmental monitoring. , 2006, Biosensors & bioelectronics.

[13]  Jia Cao,et al.  An assay for inorganic mercury(II) based on its post-catalytic enhancement effect on the potassium permanganate-luminol system. , 2006, Luminescence : the journal of biological and chemical luminescence.

[14]  T. N. Shekhovtsova,et al.  Determination of Mercury at the Picogram per Milliliter Level Using Immobilized Horseradish Peroxidase , 1994 .

[15]  M. Soylak,et al.  Mercury(II) and methyl mercury determinations in water and fish samples by using solid phase extraction and cold vapour atomic absorption spectrometry combination. , 2009, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[16]  I. Tothill,et al.  Urease–glutamic dehydrogenase biosensor for screening heavy metals in water and soil samples , 2004, Analytical and Bioanalytical Chemistry.

[17]  T. N. Shekhovtsova,et al.  Enzymatic Method for Determination of Organomercury in Sea Water , 1998 .

[18]  R. Blomberg,et al.  Design of a highly specific and noninvasive biosensor suitable for real‐time in vivo imaging of mercury (II) uptake , 2008, Protein science : a publication of the Protein Society.

[19]  Claudia Preininger,et al.  The enzymatic determination of mercury and copper using acid urease. The effects of buffers , 1999 .

[20]  G. Palleschi,et al.  Limitations in the analytical use of invertase inhibition for the screening of trace mercury content in environmental samples. , 2004, Analytical sciences : the international journal of the Japan Society for Analytical Chemistry.

[21]  T. Rao,et al.  Overview of Analytical Methodologies for Sea Water Analysis: Part I—Metals , 2005 .

[22]  A. Mahmod.  Investigation of the Reversible Inhibition of Butrylcholinesterase by Mercury Chloride , 2001 .

[23]  A. Ishaque,et al.  Differential Toxicological Interaction among Arsenic, Cadmium, Lead, and Mercury on MCF 7 Cell Line , 2009 .

[24]  T. Ninomiya,et al.  Expansion of methylmercury poisoning outside of Minamata: an epidemiological study on chronic methylmercury poisoning outside of Minamata. , 1995, Environmental research.

[25]  H. Obata,et al.  Automated determination of iron in seawater by chelating resin concentration and chemiluminescence detection , 1993 .

[26]  M. Trojanowicz,et al.  Inhibitive determination of mercury and other metal ions by potentiometric urea biosensor. , 2000, Biosensors & bioelectronics.

[27]  S. Han,et al.  A methylene blue-mediated enzyme electrode for the determination of trace mercury(II), mercury(I), methylmercury, and mercury-glutathione complex. , 2001, Biosensors & bioelectronics.

[28]  H. S. Wolff,et al.  iRun: Horizontal and Vertical Shape of a Region-Based Graph Compression , 2022, Sensors.

[29]  Ruey-an Doong,et al.  Simultaneous determination of pH, urea, acetylcholine and heavy metals using array-based enzymatic optical biosensor. , 2005, Biosensors & bioelectronics.

[30]  Jonathan J. Burbaum,et al.  Miniaturization technologies in HTS: how fast, how small, how soon? , 1998 .

[31]  Antonio Turco,et al.  Inhibitive determination of metal ions by an amperometric glucose oxidase biosensor: Study of the effect of hydrogen peroxide decomposition , 2008 .