title: 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.
abstract: 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.
authors.affiliation: Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Du
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.forename: Limin
authors.initials: L
authors.lastname: Wang
authors.forename: Donglai
authors.initials: D
authors.lastname: Lu
authors.forename: Jordan N
authors.initials: JN
authors.lastname: Smith
authors.forename: Charles
authors.initials: C
authors.lastname: Timchalk
authors.forename: Yuehe
authors.initials: Y
authors.lastname: Lin
mesh_terms: D000906:Antibodies / Q000276:immunology; D002091:Butyrylcholinesterase / Q000737:chemistry / Q000378:metabolism*; D002619:Chemical Warfare Agents / Q000032:analysis* / Q000737:chemistry; D055664:Electrochemical Techniques / Q000379:methods*; D007118:Immunoassay / Q000379:methods; D008280:Magnetics; D037742:Nanotubes, Carbon / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000032:analysis* / Q000737:chemistry; D010575:Pesticides / Q000032:analysis* / Q000737:chemistry; D010766:Phosphorylation; D045663:Quantum Dots
chemical_list: D000906:Antibodies; D002619:Chemical Warfare Agents; D037742:Nanotubes, Carbon; D009943:Organophosphorus Compounds; D010575:Pesticides; D002091:Butyrylcholinesterase