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 10 Å 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 1 0 − 9 to 1 0 − 7 M. The organophosphate chlorpyrifos-oxon could be detected in concentrations from 1 0 − 6 down to 1 0 − 8 M after 20 min of injection time (equals to 500 μ L sample volume.