Department of Biochemistry, M D University, Rohtak 124001, Haryana, India.
Bioprocess Biosyst Eng. 2013 Jan;36(1):81-9. doi: 10.1007/s00449-012-0763-8. Epub 2012 Jul 6.
A nickel hexacyanoferrate polypyrrole film was synthesized through an electrochemical two-step methodology leading to a very stable and homogenous robust hybrid film. A highly sensitive, specific and rapid amperometric D-amino acid biosensor was constructed by immobilizing D-amino acid oxidase on this film deposited over the surface of a glassy carbon electrode. The modified electrode was characterized by scanning electron microscopy, electrochemical impedance spectroscopy and Fourier transform infrared spectrophotometry. The biosensor showed optimum response within 1 s, when operated at 50 mV s(-1) in 0.01 M Tris HCl buffer, pH 7.0 at 30 °C. The biosensor exhibited excellent sensitivity with a detection limit of 1.5 µM (S/N = 3) for D-amino acids and wider linear range, 20-500 µM. Analytical recovery of added D-alanine (5 and 10 mM) in serum samples was 98.00 and 98.80 %, respectively. Within-batch and between-batch coefficients of variation in serum samples were 1.36 and 2.77 %, respectively. The enzyme electrode was used more than 50 times over 2 months, when stored at 4 °C. The proposed modified electrode exhibited sufficient mechanical and electrochemical stability and high sensitivity compared to earlier electrochemical D-amino acid biosensors. Interference by ascorbic acid and uric acid, the main interfering species in the biological samples, was negligible.
通过电化学两步法合成了镍六氰合铁酸聚苯胺薄膜，得到了非常稳定、均匀和坚固的混合薄膜。通过将 D-氨基酸氧化酶固定在沉积在玻碳电极表面的薄膜上，构建了一种高灵敏度、特异性和快速的安培型 D-氨基酸生物传感器。修饰电极的特征通过扫描电子显微镜、电化学阻抗谱和傅里叶变换红外光谱进行了表征。当在 30°C 下以 50 mV s(-1)的速度在 0.01 M Tris HCl 缓冲液中 pH 值为 7.0 时，该生物传感器在 1 s 内表现出最佳响应。该生物传感器具有出色的灵敏度，对 D-氨基酸的检测限为 1.5 µM（S/N = 3），线性范围较宽，为 20-500 µM。在血清样品中添加的 D-丙氨酸（5 和 10 mM）的分析回收率分别为 98.00%和 98.80%。在血清样品中，批内和批间变异系数分别为 1.36%和 2.77%。在 4°C 下储存时，该酶电极在 2 个月内使用超过 50 次。与早期的电化学 D-氨基酸生物传感器相比，该修饰电极具有足够的机械和电化学稳定性以及高灵敏度。生物样品中主要的干扰物质抗坏血酸和尿酸的干扰可以忽略不计。
