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活性氧(ROS)是细胞代谢的重要产物之一,其浓度与多种生理、病理现象密切相关;H2O2是ROS的重要代表,对细胞内H2O2及其释放过程的测定有助于理解相关疾病的形成机制,因而具有重要的意义.本文报道一种检测细胞内H2O2浓度、监测其生成和释放过程的电化学方法,该方法利用固定在天然矿物凹土(attapulgite,全称凹凸棒石黏土)表面的血红蛋白(Hb)对H2O2还原的电催化作用实现H2O2的定量测定.将Hb固定到凹土表面制备了Hb-凹土(Hb-Atta)纳米复合材料,用透射电子显微镜(TEM)、原子力显微镜(AFM)、红外光谱(FTIR)、紫外-可见光谱(UV-vis)等对其进行了表征;将该复合物修饰到玻碳(GC)电极表面制成了Hb-Atta/GC电极,用伏安法考察了Hb在凹土表面的直接电子转移性能,结果表明,Hb能进行有效的直接电子转移反应,其伏安曲线上呈现出一对良好的氧化还原峰,式量电位(E0′)约为-350mV(vs.SCE,50mV/s);进一步的结果表明,Hb-Atta对H2O2的电化学还原具有良好的催化性能,并能用于H2O2的定量测定,具有响应速率快、线性范围宽、检测限低等优点.将Hb-Atta/GC电极应用于嗜中性粒细胞内H2O2的测定,结果表明,该电极不仅能定量地检测细胞内的H2O2含量,还能监测细胞内H2O2生成和释放的过程,而且细胞内其他ROS和电活性物质如OCl-、NO.、ONOO-和抗坏血酸等不产生干扰.本文的结果为测量细胞内H2O2浓度、研究其生成及释放过程动力学以及进一步阐明由H2O2导致的相关疾病机制等提供了一个新的平台,可望在生物电化学、细胞生物学和病理生理学等领域得到应用.
Reactive oxygen species (ROS) is one of the important products of cell metabolism, its concentration is closely related to a variety of physiological and pathological phenomena; H2O2 is an important representative of ROS, the determination of intracellular H2O2 and its release process helps to understand the related diseases Forming mechanism, which is of great significance.In this paper, we report an electrochemical method to detect the concentration of H2O2 in cells and monitor the generation and release of H2O2 by using the method of immobilization on the surface of attapulgite (fully attapulgite clay) The electrocatalytic effect of hemoglobin (Hb) on the reduction of H2O2 to achieve the quantitative determination of H2O2 Hb-Atta nanocomposites were prepared by immobilizing Hb on the surface of the alluvial soil. Transmission electron microscopy (TEM), atomic force microscopy AFM, FTIR and UV-vis. The Hb-Atta / GC electrode was prepared by coating the surface of glassy carbon (GC) The results showed that Hb could conduct an efficient direct electron transfer reaction and showed a pair of good redox peaks on the voltammetric curve. About -350 mV (vs.SCE, 50 mV / s). Further results showed that Hb-Atta has good catalytic performance for the electrochemical reduction of H2O2, and can be used for the quantitative determination of H2O2, with the advantages of fast response rate, wide linear range, low detection limit, etc. The Hb-Atta The application of -Atta / GC electrode in the determination of neutrophil H2O2, the results showed that the electrode can not only quantitatively detect intracellular H2O2 content, but also monitor the process of intracellular H2O2 production and release, and the other intracellular ROS And electroactive substances such as OCl-, NO., ONOO- and ascorbic acid, etc. The results of this study provide a basis for measuring intracellular H2O2 concentration, studying its kinetics of production and release, and further elucidation of the underlying disease mechanisms induced by H2O2 A new platform is expected to be applied in bioelectrochemistry, cell biology and pathophysiology.