REQUIMTE-Dept. de Química, CQFB, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
Biosens Bioelectron. 2010 May 15;25(9):2026-32. doi: 10.1016/j.bios.2010.01.031. Epub 2010 Feb 6.
In this paper we propose the construction of a new non-mediated electrochemical biosensor for nitrite determination in complex samples. The device is based on the stable and selective cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans, which has both high turnover and heterogeneous electron transfer rates. In opposition to previous efforts making use of several redox mediators, in this work we exploited the capacity of ccNiR to display a direct electrochemical response when interacting with pyrolytic graphite (PG) surfaces. To enable the analytical application of such bioelectrode the protein was successfully incorporated within a porous silica glass made by the sol-gel process. In the presence of nitrite, the ccNiR/sol-gel/PG electrode promptly displays catalytic currents indicating that the entrapped ccNiR molecules are reduced via direct electron transfer. This result is noteworthy since the protein molecules are caged inside a non-conductive silica network, in the absence of any mediator species or electron relay. At optimal conditions, the minimum detectable concentration is 120 nM. The biosensor sensitivity is 430 mA M(-1) cm(-2) within a linear range of 0.25-50 microM, keeping a stable response up to two weeks. The analysis of nitrites in freshwaters using the method of standard addition was highly accurated.
在本文中，我们提出了一种新的非介导电化学生物传感器的构建，用于复杂样品中亚硝酸盐的测定。该装置基于来自脱硫弧菌的稳定且选择性的细胞色素 c 亚硝酸盐还原酶（ccNiR），其具有高周转率和非均相电子转移速率。与以前利用多种氧化还原介体的努力相反，在这项工作中，我们利用 ccNiR 与热解石墨（PG）表面相互作用时显示直接电化学响应的能力。为了使这种生物电极能够进行分析应用，成功地将蛋白质掺入了溶胶-凝胶过程制成的多孔硅玻璃中。在亚硝酸盐存在下，ccNiR/sol-gel/PG 电极迅速显示出催化电流，表明包埋的 ccNiR 分子通过直接电子转移被还原。这一结果值得注意，因为在没有任何介体或电子中继的情况下，蛋白质分子被笼封在非导电的硅网络中。在最佳条件下，最低检测浓度为 120 nM。在 0.25-50 microM 的线性范围内，生物传感器的灵敏度为 430 mA M(-1) cm(-2)，在两周内保持稳定的响应。使用标准添加法对淡水中亚硝酸盐的分析具有高度准确性。
