Amperometric biosensors for pesticide detection based on acetylcholinesterase employ the enzymatically generated thiocholine as the electrochemical probe. The mechanism is generally assumed to be: 2 thiocholine -> 2H(+) + 2e(-) + dithio-bis-choline (dimer). However, this mechanism has never been confirmed experimentally, the detection of the dimer species. In the current work, anodic oxidation of synthetic thiocholine at a pyrolytic carbon electrode, modified with multiwalled carbon nanotubes, was studied for the first time using electrochemical, spectrophotometric and spectroelectrochemical techniques. Cyclic voltammetry was used to determine the linear relationship between I-p and thiocholine concentration. A detection limit of 5.96 (+/-0.22)mu M (S/N=3) and a detection sensitivity of 3.16 x 10(-2) (+/-2.65 x 10(-3)) mu A mu M-1 were observed with a linear range from 5.0 to 100.0 mu M and correlation coefficient of 0.9954. Using controlled potential electrolysis coupled to a spectrophotometric probe in situ, the number of electrons involved in the electrochemical oxidation was confirmed to be 2. By the reaction with dithiothreitol through Cleland's method, in which the disulfide compound generated presents an absorption peak at 283 nm, the dimeric product of the assumed oxidation reaction was also confirmed. (C) 2013 Elsevier Ltd. All rights reserved.