Development of the in vivo monitoring system of nitric oxide using a micro electrode.

通过对电极表面一氧化氮（NO）氧化反应机理的电化学分析，以及对电极本身及其表面涂层材料的考察，我们开发了三种用于检测生物组织中微量NO的电极；(1)覆盖有含铁卟啉的Nafion薄膜的玻碳电极，(2)覆盖有Nafion、α -环糊精和PDDA（阳离子聚合物）的铅笔芯电极，以及(3)覆盖有Nafion和PDDA的碳纤维电极。在电极表面包覆Nafion、PDDA和α -环糊精，可以有效消除组织中存在的氧化还原物质NO_2、抗坏血酸和儿茶酚胺对NO检测的干扰。在Nafion薄膜中加入铁卟啉，利用其催化性能提高了对No的敏感性。因此，电极可以检测溶液中的10^9MNO。采用包覆含铁卟啉的Nafion膜的玻碳电极，检测乙酰胆碱刺激离体大鼠胸主动脉释放NO，估计NO浓度在100nM左右。有证据表明，通过从血管中去除内皮细胞或添加L-NAME（一种NO合成酶抑制剂），安培信号会减弱，从而证实了这种特异性。此外，该电极还能检测体外培养的牛内皮细胞中NO的释放。我们正在尝试使用碳纤维电极的检测系统在体内的应用，结果是有希望的。

Following the electrochemical analysis of the oxidation reaction mechanism of nitric oxide(NO) on the electrode surface and examination of the materials for the electrode itself and its surface coating, we developed three types of electrode for the detection of trace amount of NO in biological tissues ; (1) a glassy carbon electrode covered with Nafion film containing iron porphyrins, (2) a pencil lead elecrode covered with Nafion, alpha-cyclodextrin and PDDA (cation polymer), and(3) a carbon fiber electrode covered with Nafion and PDDA.The interference in the detection of NO by several redox substances existed in tissues such as NO_2, ascorbic acid and catecholamines, could be effectively abolished by coating the electrode with Nafion, PDDA and alpha-cyclodextrin. The sensitivity for No was increased by adding iron porphyrins in Nafion film due to its catalytic properties. Thus, the electrodes could detect 10^9MNO in solution.Using the glassy carbon electrode coated with Nafion film containing iron porphyrins, we could detect NO release from the isolated rat thoracic aorta stimulated by acetyl choline, and the concentration of NO was estimated about 100nM.The specificity was confirmed by the evidence that the amperometric signal was diminished by removal of the endothel from the blood vessel or addition of L-NAME,a NO synthase inhibitor. Furthermore, the electrode could detect NO release from the cultured bovine endothelial cells. We are trying in vivo application of the detection system using the carbon fiber electrode, and the result is promising.