For D-amino acid (DAA) electrochemical biosensors, it is necessary to achieve chiral recognition in racemic solutions or mixtures. However, common chiral recognition is only performed in a single isomer solution. Here, D-amino acid oxidase (DAAO) was used as a chiral selector, and carbon nanotubes (CNTs) as a signal amplifier to construct a non-mediator-style DAA biosensor. The biosensor showed high performance against enantiomer interference: in alanine (Ala) enantiomer mixtures, accurate quantification of D-Ala was achieved when the concentration ratio of L-Ala to D-Ala was 100. In Ala racemic solutions, the linear equation slope was almost consistent with that of standard D-Ala. This high performance was due to the combination of stereoselectivity (enzyme protein) and a catalytic reaction (redox center). The mechanism for the electrical signal change of the biosensor was explored and verified by cyclic voltammetry (CV). The results showed that (i) flavin adenine dinucleotide (FAD, redox center of DAAO) was a direct electroactive substance that produced a reduction peak current; in the presence of O2, the amount of FAD increased leading to an increase of the reduction peak current. (ii) In the presence of DAA, the chemical reaction FAD+DAA → imino acids+ FADH2 occurred and consumed FAD, which resulted in its decrease; thus, the reduction peak current also decreased. Under the same oxygen concentration, the linear decrease of the reduction peak current in the presence of DAA was due to FAD consumption. The biosensor was used for practical analyses in milk and urine samples with satisfactory results.
Keywords: Chiral recognition; D-amino acid oxidase; Electrochemical biosensor; Mechanism.
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