嗜中性粒细胞中过氧化氢及其释放过程的电化学检测

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.

Electrochemical approach for the measurement of intracellular hydrogen peroxide and its releasing process from neutrophil

Reactive oxygen species（ROS） are ubiquitous in life and death processes of cells.They are considered as the mediators of the biochemistry of cellular pathology and may be involved in a number of pathological events.Among the ROS,H2O2 is the most stable species.The quantitative determination of intracellular H2O2 could lead to a better understanding the clinical consequences of the enhancement in H2O2 concentration as well assisting in studies designed to elucidate the biological effect of H2O2 in cells.This work developed an electrochemical approach for measuring the concentration H2O2 in neutrophil and monitoring the releasing process of H2O2 from the cells.This approach is based on the electrocatalytic reduction of the released H2O2 by hemoglobin（Hb） immobilized on the surface of attapulgite,which is a natural mineral.The Hb-attapulgite（Hb-Atta） nanohybrids,fabricated based on the electrostatic interactions between the Hb and attapulgite,were characterized TEM,AFM,FTIR,and UV-vis techniques.The cyclic voltammetric results indicate that the immobilized Hb can undergo effective direct electron transfer at the surface of attapulgite and displays a well-defined redox peaks with the formal potential of-350 mV（vs SCE,at 50 mV/s）.The further results demonstrate the Hb-Atta nanohybrids exhibit a high electrocatalytic activity toward the reduction of H2O2 with a rapid response,a wide linear range,a high sensitivity,a low detection limit,as well as good stability due to using the natural mineral as the enzyme immobilization substrate.In addition,some common coexisting ROS and compounds in biological system such as hypochlorite（OCl-）,nitric oxide（NO.）,peroxynitrite（ONOO-）,and ascorbic acid（AA） etc.,did not cause any interference.Therefore,this work has demonstrated a simple and effective sensing platform for detection of cellular H2O2 releasing from cells such as neutrophil,which has potential utility to cellular biology and pathophysiology.