A new L-phenylalanine dehydrogenase (PHD) and Toluidine Blue O (TBO) based amperometric biosensor for the determination of L-phenylalanine was reported in this study. An amperometric L-phenylalanine biosensor with immobilization of TBO (as a mediator,), PDH and nicotinamide adenine dinucleotide (NAD⁺) onto polypyrrole-polyvinylsulphonate (PPy-PVS) film was accomplished on the surface of a platinum electrode. Cross-linking with glutaraldehyde technique was used for immobilizing PDH on PPy-PVS film. Toluidine Blue O (TBO) was used as the mediator. The changes in the anodic current at −0.195 V vs Ag/AgCl produced by TBO was proportional to the L-phenylalanine concentration changes in the sample. The effects of substrate concentration, pH and temperature on the response of the L-phenylalanine biosensor were investigated. Optimum pH and temperature were found to be 7.0 and 60 °C, respectively. The linear working region of biosensor is ${5.0}\times {10}^{- {7}}$ - ${2.5}\times {10}^{- {5}}$ M (R² = 0.9816). The detection limit of the biosensor was ${1.0}\times {10}^{- {8}}$ M and response time of the biosensor was 200 s. The storage stability and application stability of the biosensor were also studied. Several possible interfering substances’ effects on the phenylalanine biosensor were investigated. The developed biosensor was tested in determinations of content in a synthetic blood sample.

中文翻译：

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一种用于苯丙酮尿症诊断的新型苯丙氨酸生物传感器的研制

本研究报道了一种新的 L-苯丙氨酸脱氢酶 (PHD) 和基于甲苯胺蓝 O (TBO) 的电流型生物传感器，用于测定 L-苯丙氨酸。一种固定化 TBO（作为介质）、PDH 和烟酰胺腺嘌呤二核苷酸 (NAD ⁺) 到聚吡咯-聚乙烯磺酸盐 (PPy-PVS) 膜上是在铂电极的表面上完成的。使用戊二醛交联技术将 PDH 固定在 PPy-PVS 薄膜上。甲苯胺蓝 O (TBO) 用作介质。TBO 产生的 -0.195 V 与 Ag/AgCl 的阳极电流变化与样品中的 L-苯丙氨酸浓度变化成正比。研究了底物浓度、pH 值和温度对 L-苯丙氨酸生物传感器响应的影响。发现最佳 pH 值和温度分别为 7.0 和 60 °C。生物传感器的线性工作区为 ${5.0}\times {10}^{- {7}}$ —— ${2.5}\times {10}^{- {5}}$ M (R ² = 0.9816)。生物传感器的检测限为 ${1.0}\times {10}^{- {8}}$ M 和生物传感器的响应时间为 200 s。还研究了生物传感器的储存稳定性和应用稳定性。研究了几种可能的干扰物质对苯丙氨酸生物传感器的影响。开发的生物传感器在合成血液样本中的含量测定中进行了测试。