Acetylcholinesterase fiber-optic biosensor for detection of anticholinesterases.
An optical sensor for anticholinesterases (AntiChEs) was constructed by immobilizing fluorescein isothiocyanate (FITC)-tagged eel electric organ acetylcholinesterase (AChE) on quartz fibers and monitoring enzyme activity. The pH-dependent fluorescent signal generated by FITC-AChE, present in the evanescent zone on the fiber surface, was quenched by the protons produced during acetylcholine (ACh) hydrolysis. Analysis of the fluorescence response showed Michaelis-Menten kinetics with a Kapp value of 420 microM for ACh hydrolysis. The reversible inhibitor edrophonium (0.1 mM) inhibited AChE and consequently reduced fluorescence quenching. The biosensor response immediately recovered upon its removal. The carbamate neostigmine (0.1 mM) also inhibited the biosensor response but recovery was much slower. In the presence of ACh, the organophosphate (OP) diisopropylfluorophosphate (DFP) at 0.1 mM did not interfere with the ACh-dependent fluorescent signal quenching, but preexposure of the biosensor to DFP in absence of ACh inhibited totally and irreversibly the biosensor response. However, the DFP-treated AChE biosensor recovered fully after a 10-min perfusion with pralidoxime (2-PAM). Echothiophate, a quaternary ammonium OP, inhibited the ACh-induced fluorescence quenching in the presence of ACh and the phosphorylated biosensor was reactivated with 2-PAM. These effects reflected the mechanism of action of the inhibitors with AChE and the inhibition constants obtained were comparable to those from colorimetric methods. The biosensor detected concentrations of the carbamate insecticides bendiocarb and methomyl and the OPs echothiophate and paraoxon in the nanomolar to micromolar range. Malathion, parathion, and dicrotophos were not detected even at millimolar concentrations; however, longer exposure or prior modification of these compounds (i.e., to malaoxon, paraoxon) may increase the biosensor detection limits. This AChE biosensor is fast, sensitive, reusable, and relatively easy to operate. Since the instrument is portable and can be self-contained, it shows potential adaptability to field use.
Rogers
K R
KR
Department of Pharmacology and Experimental Therapeutics, University of Maryland, School of Medicine, Baltimore 21201.
Cao
C J
CJ
Valdes
J J
JJ
Eldefrawi
A T
AT
Eldefrawi
M E
ME
D000110:Acetylcholinesterase; D000818:Animals; D015374:Biosensing Techniques; D002800:Cholinesterase Inhibitors / Q000032:analysis*; D004524:Eels; D004800:Enzymes, Immobilized; D005336:Fiber Optic Technology; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009388:Neostigmine / Q000494:pharmacology; D055100:Optical Fibers; D013861:Thiocyanates
D002800:Cholinesterase Inhibitors; D004800:Enzymes, Immobilized; D005452:Fluoresceins; D013861:Thiocyanates; D009388:Neostigmine; D000110:Acetylcholinesterase; D016650:Fluorescein-5-isothiocyanate