Development of highly sensitive sensor based on bioengineered acetylcholinesterase immobilized by affinity method

A new method for the immobilization of a bioengineered His-tagged acetylcholinesterase onto a chemically modified graphite is described for the fabrication of screen-printed electrodes. The choice of the enzyme, the functionalization of the graphite and the analytical characteristics of the electrodes were studied. The nonspecific binding and the effect of the pH were also evaluated. A sensitivity of 4 mA L mol(-1) was obtained between 1-60 mumol L-1 acetylthiocholine, one order of magnitude higher than in the case of entrapment in PVA-SbQ or Sol-Gel. The acetylcholinesterase sensors remained fully stable after 80 days storage at 4degreesC under vacuum, with a response of 327nA, with 1 mM substrate. In the absence of vacuum the electrodes lose more than 30% of the initial value after 15 days and they have completely lost their activity after 30 days. The sensors can be reused by elution of inhibited enzyme either with a solution of imidazole or with ethylenediaminetetraacetic acid (EDTANa(2)H(2)). The developed system was applied for the detection of an acetylcholinesterase inhibitor, paraoxon, a detection limit of 2 nmol L-1 being obtained following a 10 min incubation.