Acetylcholinesterase using acetylthiocholine

Another class of enzymes that has found wide application in the biosensor field in the last decades is that of the cholinesterases which have been mainly used for the detection of pesticides. For the amperometric detection of cholinesterase activity, both the substrates acetylcholine and acetylthiocholine have been extensively used [6-9], the latter being preferred because this avoids the use of another enzyme, choline oxidase, which is usually coupled with acetylcholinesterase. However, the amperometric measurement of thiocholine, produced by... 

Fig. 24.5. (a) Calibration curve for paraoxon inhibition of acetylcholinesterase-modified CoPC electrodes for paraoxon concentrations between 10-8 and 10 M using a concentration of 2 mM acetylthiocholine chloride (n — 3, S.D. < +11%). Insets show a typical current transient response for an AChE-modified electrode to 2 mM acetylthiocholine chloride, before (b) and after (c) the addition of 1 x 10-17 M paraoxon. 

In another report, several acetylcholinesterase (AChE) inhibitors, including tacrine, edrophonium, tetramethyl- and tetraethyl-ammonium chloride, carbofu-ran, and eserine were assayed on a chip [1045]. AChE converted the substrate, acetylthiocholine, to thiocholine. This product reacted with coumarinylphenyl-maleimide (CPM) to form thiocholine-CPM (a thioether) for LIF detection. Since the acetonitrile solvent used to dissolve CPM inhibited AChE activity, the CPM solution was added after the enzymatic reaction [1045]. Another enzyme inhibition assay using a peptide substrate was performed on a PMMA chip [1046]. 

Acetylcholinesterase (AChE) The lyophilized enzyme from electric eel (Sigma) was dissolved in 0.05M phosphate buffer (pH-7.4) at a concentration of 20 pg/mL. Acetylthiocholine iodide was used as substrate at a final concentration of 5x10 4M in buffer. 5,5 -Dithiobis-(2-nitrobenzoic acid) (DTNB) at a final concentration of 3.8xlO 2M was used to monitor the released thiocholine according to a published procedure (41) with slight modification. Acetone was used as a solvent for the inhibitors. 

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