Biosensors for pesticide detection based on alkaline phosphatase-catalyzed chemiluminescence

An enzyme-based detection methodology for sensing and quantitation of organophosphorus pesticides is described. The enzyme, alkaline phosphatase, is used in soluble form for reactions in bulk solutions, and in immobilized form for reactions on optical fibers or in glass capillaries. The immobilization strategy involves building a molecular assembly of the enzyme and a conjugated copolymer, poly (3-undecylthiophene-co-3-thiophenecarboxyldehyde-biotin-LC-hydrozone) on a glass surface. Hydrophobic or specific biotin-streptavidin interactions are used to immobilize the biotinylated copolymer on a silanized glass surface and attach a streptavidin conjugate of alkaline phosphatase to the copolymer. Alkaline phosphatase catalyzes the dephosphorylation of a macrocyclic compound, chloro-3-(4-methoxy spiro[1,2 dioxetane-3-2′-tricyclo-“3.3.1.1”-decan]-4-yl) phenyl phosphate and releases light; the chemiluminescence signal is detected with a simple photomultiplier tube. The enzyme activity, and proportionately the chemiluminescence signal strength, are inhibited in the presence of organophosphorus pesticides such as paraoxon. Quantitation of pesticides is thus possible from the inverse correlation between the enzyme activity or signal strength and pesticide concentration. Detection of paraoxon at a concentration of about 50 ppb is achieved with this system. Reaction kinetics are examined for the enzyme in both states. The immobilized enzyme can be reused a number of times, and there is no significant loss of enzyme activity over a period of eight weeks. The design of a flow cell for continuous analysis of the pesticides is described.