AN EVANESCENT-WAVE SENSOR FOR THE DETECTION OF ORGANOPHOSPHORUS COMPOUNDS BASED ON THE INHIBITION OF CHOLINESTERASE

A flow injection analysis (FIA) system coupled with an evanescent wave (EW) sensor employing total internal reflection of fluorescence radia tion (TIRP) for the detection of the organophosphorus compounds is rep orted. The detection is based on the measurement of acetylcholinestera se (AChE) inhibition in the presence of organophosphorus compounds. AC hE was immobilized on controlled pore glass (CPG) particles and packed into a teflon column which was then coupled to the evanescent wave se nsor. A constant concentration of acetylthiocholine in Tris-HCl 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 m onitored by the injection of thylamino-3-(4'-maleimidylphenyl)-4-methy lcoumarin (CPM) which forms a highly fluorescent compound with thiocho line. The CPM-thiocholine complex was excited with the evanescent wave and the resulting fluorescence was detected by a Himamastsu S-2387/66 R detector. Varying concentrations of paraoxon were injected through t he enzyme column and the extent of enzyme inhibition was recorded by t he injection of the optimum concentration of the CPM. The difference o f the two responses in the presence and absence of the paraoxon determ ines the extent of inhibition. The acetycholinesterase activity was th en reactivated by the injection of pyridine 2-aldoxime methiodide (2-P AM) which results in the recovery of the initial response.