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

Area of Science:

• Biochemistry
• Electrochemistry
• Biosensor Technology

Background:

• Thiamine pyrophosphate (ThDP) is a crucial coenzyme involved in various metabolic pathways.
• Accurate quantification of ThDP is essential for diagnosing ThDP-deficiency related disorders.
• Existing methods for ThDP detection can be complex and time-consuming.

Purpose of the Study:

• To develop a novel amperometric biosensor for sensitive and selective detection of thiamine pyrophosphate (ThDP).
• To utilize the transketolase (TK)-catalyzed reaction and electrochemical detection for ThDP quantification.
• To optimize the biosensor performance using specific TK donor substrates.

Main Methods:

• Immobilization of transketolase (TK) from Escherichia coli (TKec) in Mg2Al-NO3 Layered Double Hydroxides (LDH).
• Fabrication of a TKec/LDH modified glassy carbon electrode (GCE) for electrochemical detection.
• Employing the oxidative trapping of the TK intermediate α,β-dihydroxyethylthiamine diphosphate (DHEThDP) and subsequent electrochemical detection via chronoamperometry.
• Optimization of TK donor substrates (l-erythrulose and d-fructose-6-phosphate).

Main Results:

• The developed TKec/LDH/GCE biosensor demonstrated high sensitivity for ThDP detection (3831mAM-1cm-2).
• A linear detection range for ThDP was established between 20-400 nM.
• The biosensor effectively utilized the Fe(CN)63-/Fe(CN)64- redox couple for signal transduction, coupled with ThDP regeneration.

Conclusions:

• The novel amperometric biosensor provides a sensitive and efficient platform for ThDP quantification.
• The use of TK immobilized in LDH offers a promising approach for biosensor development.
• This biosensor has potential applications in clinical diagnostics and biochemical research requiring ThDP measurement.