Thiamine biosensor based on oxidative trapping of enzyme-substrate intermediate

Abstract

In the present work, we describe a new thiamine amperometric biosensor based on thiamine pyrophosphate (ThDP)-dependent transketolase (TK)-catalyzed reaction, followed by the oxidative trapping of TK intermediate α,β-dihydroxyethylthiamine diphosphate (DHEThDP) within the enzymatic active site. For the biosensor design purpose, TK from Escherichia coli (TKec) was immobilized in Mg₂Al-NO₃ Layered Double Hydroxides (LDH) and the electrochemical detection was achieved with the TKec/LDH modified glassy carbon electrode (GCE). The transduction process was based on the ability of Fe(CN)₆^3- to oxidize DHEThDP to glycolic acid along with ThDP regeneration. The released Fe(CN)₆^4- was re-oxidized at +0.5V vs Ag-AgCl and the reaction was followed by chronoamperometry. The TKec/LDH/GCE biosensor was optimized using the best TK donor substrates, namely l-erythrulose and d-fructose-6-phosphate. ThDP was assayed with great sensitivity (3831mAM^-1cm^-2) over 20-400nM linear range.

Keywords: Biosensor; Hexacyanoferrate; Layered double hydroxides; Thiamine; Transketolase.