Du-D; Wang-J; Wang-L; Lu-D; Smith-JN; Timchalk-C; Lin-Y
Anal Chem 2011 May; 83(10):3770-3777
We report a new approach for electrochemical quantification of enzymatic inhibition and phosphorylation for biomonitoring of exposure to organophosphorus (OP) pesticides and nerve agents based on a magnetic bead (MB) immunosensing platform. The principle of this approach is based on the combination of MB immunocapture-based enzyme activity assay and competitive immunoassay of the total amount of enzyme for simultaneous detection of enzyme inhibition and phosphorylation in biological fluids. Butyrylcholinesterase (BChE) was chosen as a model enzyme. In competitive immunoassay, the target BChE in a sample competes with the BChE immobilized on the MBs to bind to the limited sites of anti-BChE antibody labeled with quantum dots (QD-anti- BChE), followed by stripping voltammetric analysis of the bound QD conjugate on the MBs. This assay shows a linear response over the total BChE concentration range of 0.1-20 nM. Simultaneous real time BChE activity was measured on an electrochemical carbon nanotube-based sensor coupled with a microflow injection system after immunocapture by the MB-anti-BChE conjugate. Therefore, the formed phosphorylated BChE adduct (OP-BChE) can be estimated by the difference values of the total amount of BChE (including active and OP-inhibited) and active BChE from established calibration curves. This approach not only eliminates the difficulty in screening of low-dose OP exposure (less than 20% inhibition of BChE) because of individual variation of BChE values but also avoids the drawback of the scarce availability of OP-BChE antibody. It is sensitive enough to detect 0.5 nM OP-BChE, which is less than 2% BChE inhibition. This method offers a new method for rapid, accurate, selective, and inexpensive quantification of OP-BChE and enzyme inhibition for biomonitoring of OP and nerve agent exposures.
Agricultural-chemicals; Analytical-chemistry; Biochemical-analysis; Biochemistry; Biodynamics; Biological-effects; Biological-monitoring; Biostatistics; Biomarkers; Cell-biology; Cellular-reactions; Exposure-assessment; Exposure-levels; Exposure-methods; Health-hazards; Immunochemistry; Immunotoxins; insecticide-poisoning; Insecticides; Occupational-exposure; Organo-phosphorus-compounds; Pesticides; Pesticides-and-agricultural-chemicals; Quantitative-analysis; Risk-analysis; Risk-factors; Statistical-analysis; Toxic-effects; Work-environment; Worker-health; Work-operations; Workplace-studies
Jun Wang, Pacific Northwest National Laboratory, Richland, WA 99352, USA
Agriculture, Forestry and Fishing
Battelle Pacific Northwest Laboratories