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We demonstrate that electrolyte-gated organic field-effect transistors (EGOFETs) can detect extremely minute modifications in the composition of the electrochemical double layers forming at the channel/electrolyte and gate/electrolyte interfaces, thus permitting real-time monitoring of alkylthiols self-assembling on a gold surface. We used this property to assay the enzymatic activity of acetylcholinesterase which, in the presence of its substrate (acetylthiocholine), produces thiocholine.