Methodology is described for the creation of a molecular assembly consisting of the enzyme alkaline phosphatase immobilized onto a glass surface using a biotinylated conjugated copolymer, poly(3-undecylthiophene-co-3-thiophenecarboxaldehyde) 6-biotinamidohexanohydrazone. The biotinylated polymer is attached to the inside walls of a silanized glass capillary via hydrophobic interactions, and a streptavidin-conjugated alkaline phosphatase is interfaced with the polymer through the classical biotin-streptavidin interaction. Utilizing a simple optical setup, we can detect the activity of as little as approximately 0.1 fmol of alkaline phosphatase with this molecular assembly. The assembly is mechanically robust and retains the majority of bound enzyme activity for up to 30 days. We have utilized this molecular assembly for the detection of organophosphorus-based pesticides. Both paraoxon and methyl parathion inhibit the enzyme-mediated generation of chemiluminescence signal. We are able to detect paraoxon and methyl parathion concentrations down to 500-700 ppb.
The use and application of the enzyme alkaline phosphatase in a chemiluminescence assay are discussed. The enzyme catalyzes the hydrolysis of a macrocyclic phosphate compound generating a chemiluminescence signal. On the basis of inhibition of this signal, a methodology for the detection and quantitation of organophosphorus-based pesticides has been developed. The methodology is studied with alkaline phosphatase in the bulk aqueous phase, and detection of the signal is accomplished by a simple optical setup. Parts per billion level detection of paraoxon and methyl parathion in bulk solutions is achieved. The technique is rapid and sensitive and is applicable to the detection of most organophosphorus-based pesticides. The results from kinetic studies indicate a mixed type of inhibition of the enzyme by paraoxon and methyl parathion. The detection methodology forms an integral part of a biosensor under development and is adaptable to incorporating optical fibers for remote detection of pesticides.
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