Sensitive d-amino acid biosensor based on oxidase/peroxidase system mediated by pentacyanoferrate-bound polymer

• PVI[Fe(CN)5] selectively works as a mediator for POD in the DAAO/POD reaction.
• PVI[Fe(CN)5] shows the best performance in the DAAO/POD-based biosensor.
• The reductive detection of H2O2 drastically decreases noise.
• The use of Nafion and ascorbate oxidase eliminates most of the interference.
• This method can detect DAA as low as 2 μM in diluted urine.

A sensitive d-amino acid oxidase (DAAO)/peroxidase (POD) bienzyme biosensor is constructed, in which pentacyanoferrate-bound poly(1-vinylimidazole) polymer (PVI[Fe(CN)5]) is selected as a mediator. Reductive current of PVI[Fe(CN)5] related to the H2O2 concentration generated in the DAAO reaction was measured at −0.1 V vs. Ag|AgCl with DAAO/POD/PVI[Fe(CN)5]-modified electrode. The result revealed that PVI[Fe(CN)5] is suitable as a mediator for this bienzyme system due to its appropriate formal potential and its extremely low reactivity against DAAO. The stability of DAAO was improved by adding free flavin adenine dinucleotide and the electrode composition was optimized for the detection of d-alanine. Nafion and ascorbate oxidase-immobilized films worked successfully to prevent severe interference from uric acid and ascorbic acid. The low detection limits of d-alanine (2 μM) and d-serine (2 μM) imply its possibility for the determination of extremely low concentration of d-amino acids in physiological fluids. The proposed bienzyme biosensor is proved to be capable of detecting d-amino acids in urine.