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Merck
  • Thiamine biosensor based on oxidative trapping of enzyme-substrate intermediate.

Thiamine biosensor based on oxidative trapping of enzyme-substrate intermediate.

Biosensors & bioelectronics (2016-09-23)
Matilte Halma, Bastien Doumèche, Laurence Hecquet, Vanessa Prévot, Christine Mousty, Franck Charmantray
摘要

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)63- to oxidize DHEThDP to glycolic acid along with ThDP regeneration. The released Fe(CN)64- 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.

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Sigma-Aldrich
L -焦谷氨酸 2-萘酰胺, ≥99% (TLC)
Sigma-Aldrich
L-(+)-赤藓酮糖, ≥75% (HPLC)