An evanescent wave sensor for the detection of organophosphorus compounds based on the inhibition of cholinesterase

Abstract

261-265<span style="font-size:11.0pt;line-height: 115%;font-family:Calibri;mso-fareast-font-family:" times="" new="" roman";mso-bidi-font-family:="" "times="" roman";mso-ansi-language:en-us;mso-fareast-language:en-us;="" mso-bidi-language:ar-sa"="" lang="EN-US">A flow injection analysis (FIA) system coupled with an evanescent wave (EW) sensor employing total internal reflection of fluorescence radiation (TIRF) for the detection of the organophosphorus compounds is reported. The detection is based on the measurement of acetylcholinesterase (AChE) inhibition in the presence of organophosphorus compounds. AChE was immobilized on controlled pore glass (CPG) particles and packed into a teflon column which was then coupled to the evanescent wave sensor. A constant concentration of acetylthiocholine in Tris-HCI buffer (10 mM, pH 7.5) was pumped through the enzyme column. The thiocholine formed as a result of the AChE catalyzed reaction in the column was monitored by the injection of 7-diethylamino-3-( 4' -maleimidylphenyl)-4-methylcoumarin (CPM) which forms a highly fluorescent compound with thiocholine. The CPM-thiocholine complex was excited with the evanescent wave and the resulting fluorescence was detected by a Himamastsu S-2387/66R detector. Varying concentrations of paraoxon were injected through the enzyme column and the extent of enzyme inhibition was recorded by the injection of the optimum concentration of the CPM. The difference of the two responses in the presence and absence of the paraoxon determines the extent of inhibition. The acetycholinesterase activity was then reactivated by the injection of pyridine 2-aldoxime methiodide (2-PAM) which results in the recovery of the initial response.</span