Magnetic Electrochemical Sensing Platform for Biomonitoring of Exposure to Organophosphorus Pesticides and Nerve Agents Based on Simultaneous Measurement of Total Enzyme Amount and Enzyme Activity

作者：Du, Dan; Wang, Jun^*; Wang, Limin; Lu, Donglai; Smith, Jordan N.; Timchalk, Charles; Lin, Yuehe

来源：Analytical Chemistry, 2011, 83(10): 3770-3777.

DOI：10.1021/ac200217d

• PHOSPHORYLATED ACETYLCHOLINESTERASE
• HUMAN SERUM
• BUTYRYLCHOLINESTERASE
• ASSAY
• IMMOBILIZATION
• BIOMARKER
• CHOLINESTERASES
• AMPLIFICATION
• IMMUNOSENSOR
• IMMUNOASSAYS

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 ME 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 Alas 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.

• 出版日期2011-5-15
• 单位华中师范大学