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.

中文翻译：

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基于D-氨基酸氧化酶的D-氨基酸电化学生物传感器：机理和对映体干扰的高性能。

对于D-氨基酸（DAA）电化学生物传感器，必须在外消旋溶液或混合物中实现手性识别。但是，常见的手性识别仅在单个异构体溶液中进行。在这里，D-氨基酸氧化酶（DAAO）被用作手性选择剂，而碳纳米管（CNT）被用作信号放大器，以构建非介体型的DAA生物传感器。该生物传感器显示出对映异构体干扰的高性能：在丙氨酸（Ala）对映异构体混合物中，当L-Ala与D-Ala的浓度比为100时，可以实现D-Ala的准确定量。在Ala外消旋溶液中，线性方程斜率是几乎与标准D-Ala一致。如此高的性能归因于立体选择性（酶蛋白）和催化反应（氧化还原中心）的结合。探索了生物传感器电信号变化的机制，并通过循环伏安法（CV）进行了验证。结果表明：（i）黄素腺嘌呤二核苷酸（FAD，DAAO的氧化还原中心）是直接的电活性物质，可产生降低的峰值电流；在氧气存在下，FAD的量增加，导致还原峰值电流增加。（ii）在DAA的存在下，发生了FAD + DAA→亚氨基酸+ FADH2的化学反应并消耗了FAD，导致其减少；因此，降低峰值电流也降低了。在相同的氧气浓度下，存在DAA时还原峰电流的线性下降是由于FAD消耗所致。该生物传感器用于牛奶和尿液样品的实际分析，结果令人满意。