Development of a bifunctional sensor using haptenized acetylcholinesterase and application for the detection of cocaine and organophosphates.

We developed a dual piezoelectric/amperometric sensor for the detection of two unrelated analytes in one experiment that uses propidium to anchor acetylcholinesterases (AChE) at the surface. This mass-sensitive sensor does not only allow the examination of the interaction between AChE and the modified surface but also the detection of in situ inhibition of the surface-bound AChE. Here we describe the application of the propidium-based sensor in combination with a modified AChE. For this reason the cocaine derivative benzoylecgonine (BZE) was coupled via a 10A long hydrophilic linker - 1,8-diamino-3,4-dioxaoctane - to carboxylic groups of the AChE after EDC/NHS activation. Thus the modified AChE (BZE-AChE) possesses an additional recognition element besides the inhibitor binding site. After the deposition of BZE-AChE on the sensor surface the binding of an anti-BZE-antibody to the BZE-AChE can be monitored. This makes it possible to determine two analytes - cocaine and organophosphate - in one experiment by measuring antibody binding and decrease in enzymatic activity, respectively. Furthermore it was also shown that other cocaine-binding enzymes, e.g., butyrylcholinesterase, can bind to the modified BZE-AChE. The competitive immunoassay allowed the detection of cocaine with a dynamic range from 10(-9) to 10(-7)M. The organophosphate chlorpyrifos-oxon could be detected in concentrations from 10(-6) down to 10(-8)M after 20 min of injection time (equals to 500 microL sample volume.