Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, P. R. China.
Anal Chem. 2020 Dec 15;92(24):15916-15926. doi: 10.1021/acs.analchem.0c03317. Epub 2020 Dec 2.
Endothelin-1 (ET-1) is a powerful endogenous vasoconstrictor and it is closely related to the pathogenesis of endothelial dysfunction that is commonly involved in the initiation of vascular inflammation and in the development of vascular diseases. A new method for the electrochemical immunoassay of ET-1 was put forward in this work. ET-1 antibodies (Ab), gold nanoparticles (GNPs), and copper ions were employed to synthesize nanoenzyme-labeled antibodies, Ab-GNPs-Cu(II) nanocomposites, and the latter was evaluated using transmission electron microscopy, dynamic light scattering, UV-vis absorption spectrophotometry, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. These nanocomposites could be captured on a glassy carbon electrode (GCE) modified with poly(thionine) (PTH) and ET-1, GCE/PTH/ET-1. The immobilized nanoenzymes, GNPs-Cu(II) nanoparticles, played a peroxidase mimic role. Hydroxyl radicals, OH, generated by a Fenton-type reaction, oxidized PTH and induced the considerable cathodic current on an assembled sandwich-type electrode. Owing to the competitive immunoreaction, ET-1 in the solution inhibited the capture of Ab-GNPs-Cu(II) nanocomposites. The deficiency of OH caused the decline of the electrochemical response. The cathodic current change was in proportion to the ET-1 concentration from 0.5 to 500 ng mL. Cell morphology and viability investigations show that human umbilical vein endothelial cells, HUVECs, suffered from dysfunction when they were incubated in the presence of high-concentration glucose. Analyses on the growth medium using the developed method reveal that ET-1 was secreted by the injured cells and the release level of ET-1 was associated positively with the glucose concentration in the growth medium.
内皮素-1（ET-1）是一种强大的内源性血管收缩剂，与内皮功能障碍的发病机制密切相关，内皮功能障碍通常涉及血管炎症的启动和血管疾病的发展。本工作提出了一种新的内皮素-1电化学免疫分析方法。利用 ET-1 抗体（Ab）、金纳米粒子（GNPs）和铜离子合成了纳米酶标记抗体 Ab-GNPs-Cu(II)纳米复合材料，并通过透射电子显微镜、动态光散射、紫外-可见吸收光谱、傅里叶变换红外光谱和 X 射线光电子能谱对其进行了评价。这些纳米复合材料可以被玻碳电极（GCE）上修饰的聚噻吩（PTH）和 ET-1 捕获，即 GCE/PTH/ET-1。固定化纳米酶 GNPs-Cu(II)纳米粒子具有过氧化物酶模拟作用。通过芬顿型反应生成的羟基自由基（OH）氧化 PTH 并在组装的三明治型电极上诱导出可观的阴极电流。由于竞争性免疫反应，溶液中的 ET-1 抑制了 Ab-GNPs-Cu(II)纳米复合材料的捕获。OH 的缺乏导致电化学响应的下降。阴极电流变化与 0.5 至 500ng mL 范围内的 ET-1 浓度成正比。细胞形态和活力研究表明，当人脐静脉内皮细胞（HUVEC）在高浓度葡萄糖存在下孵育时，它们会出现功能障碍。使用所开发的方法对生长培养基进行分析表明，受损细胞会分泌 ET-1，并且 ET-1 的释放水平与生长培养基中的葡萄糖浓度呈正相关。
