Almeida M Gabriela, Silveira Célia M, Moura José J G
REQUIMTE, Departamento de Química, Centro de Química Fina e Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Monte de Caparica, Portugal.
Biosens Bioelectron. 2007 May 15;22(11):2485-92. doi: 10.1016/j.bios.2006.09.027. Epub 2006 Nov 13.
This work describes the construction and voltammetric characterization of a nitrite biosensor based on a cytochrome c-type nitrite reductase (ccNiR) and the Nafion ionomeric matrix loaded with methyl viologen as redox mediator. Despite the potential electrostatic repulsions between the anionic substrate and the Nafion sulfonate groups, the resulting bioelectrode exhibited electrocatalytic activity toward nitrite. This phenomenon must be due to the nonuniformity of the enzyme/Nafion membrane, which allows the direct interaction between the substrate and numerous enzyme molecules. Nevertheless, the anionic nature of Nafion exerted a certain diffusion barrier to nitrite, as revealed by the unusually elevated limits of the linear dynamic range and k(m)(app). The irregularity of the composite membrane also contributed to slow down the rate of charge transfer throughout the Nafion polymer. The level of viologens incorporated within the Nafion membrane had a strong influence in the analytical parameters: as much mediator was present, lower was the sensitivity and wider was the linear range. For an optimized ratio enzyme/mediator the sensitivity was 445+/-8 mA M(-1)cm(-2), within the linear range 75-800 microM; the lowest detected nitrite concentration was 60 microM. The operational stability of the biosensor and the influence of some possible interferences were evaluated.
这项工作描述了一种基于细胞色素c型亚硝酸还原酶（ccNiR）和负载甲基紫精作为氧化还原介质的Nafion离子omeric基质的亚硝酸生物传感器的构建和伏安表征。尽管阴离子底物与Nafion磺酸基团之间存在潜在的静电排斥，但所得生物电极对亚硝酸盐表现出电催化活性。这种现象一定是由于酶/Nafion膜的不均匀性，这使得底物与众多酶分子之间能够直接相互作用。然而，Nafion的阴离子性质对亚硝酸盐施加了一定的扩散屏障，这通过线性动态范围和k(m)(app)的异常升高得以揭示。复合膜的不规则性也导致电荷在整个Nafion聚合物中的转移速率减慢。Nafion膜中所含紫精的水平对分析参数有很大影响：存在的介质越多，灵敏度越低，线性范围越宽。对于优化的酶/介质比例，灵敏度为445±8 mA M(-1)cm(-2)，线性范围为75 - 800 microM；检测到的最低亚硝酸盐浓度为60 microM。评估了生物传感器的操作稳定性以及一些可能干扰的影响。
