title: 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-NO3Layered 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.
authors.affiliation: Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, F-63000 Clermont-Ferrand, France; CNRS, UMR 6296, ICCF, F-63171 Aubière, France.
authors.forename: Matilte
authors.initials: M
authors.lastname: Halma
authors.affiliation: ICBMS (Institut de Chimie et Biochimie Moléculaires et Supramoléculaires) UMR 5246 CNRS, Université Lyon 1, France.
authors.forename: Bastien
authors.initials: B
authors.lastname: Doumèche
authors.affiliation: Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, F-63000 Clermont-Ferrand, France; CNRS, UMR 6296, ICCF, F-63171 Aubière, France.
authors.forename: Laurence
authors.initials: L
authors.lastname: Hecquet
authors.affiliation: Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, F-63000 Clermont-Ferrand, France; CNRS, UMR 6296, ICCF, F-63171 Aubière, France.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Prévot
authors.affiliation: Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, F-63000 Clermont-Ferrand, France; CNRS, UMR 6296, ICCF, F-63171 Aubière, France. Electronic address: Christine.MOUSTY@univ-bpclermont.fr.
authors.forename: Christine
authors.initials: C
authors.lastname: Mousty
authors.affiliation: Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, F-63000 Clermont-Ferrand, France; CNRS, UMR 6296, ICCF, F-63171 Aubière, France. Electronic address: Franck.CHARMANTRAY@univ-bpclermont.fr.
authors.forename: Franck
authors.initials: F
authors.lastname: Charmantray
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D055664:Electrochemical Techniques / Q000379:methods; D004800:Enzymes, Immobilized / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005295:Ferrocyanides / Q000378:metabolism; D006878:Hydroxides / Q000378:metabolism; D010084:Oxidation-Reduction; D013831:Thiamine / Q000032:analysis* / Q000378:metabolism; D013835:Thiamine Pyrophosphate / Q000032:analysis* / Q000378:metabolism; D014174:Transketolase / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D005295:Ferrocyanides; D006878:Hydroxides; D014174:Transketolase; C020354:hexacyanoferrate II; D013835:Thiamine Pyrophosphate; D013831:Thiamine
keywords: Biosensor; Hexacyanoferrate; Layered double hydroxides; Thiamine; Transketolase