基于扫描电化学显微镜技术研究细胞实时释放ROS

Investigation on Real-time Release of Reactive Oxygen Species from Cells Based on Scanning Electrochemical Microscopy

本研究利用扫描电化学显微镜(SECM)技术考察了十四烷酰佛波醇乙酸酯(PMA)刺激肺癌细胞(A549)活性氧物种(ROS)的实时变化。以过氧化氢(H_2O_2)为目标检测物,获得了细胞氧化应激的电化学响应图像。以氯化六氨基合钌(Ru(NH_3)_6Cl_3)为体系介质,利用电流负反馈技术获得细胞形貌的清晰图像,同时优化获得探针与基底之间的最佳检测距离。将探针电位设定为过氧化氢的还原电位(-0.65 V)时,通过电流成像图可观察到ROS实时释放,实现了A549细胞被PMA即时刺激时ROS释放的实时检测。扩大扫描区域,获得了不同个体细胞ROS释放的SECM图像,实现了SECM对ROS信号的实时捕捉及再现性。结果显示:PMA可破坏细胞含氧物种的动态平衡,诱发细胞产生氧化应激响应。将探针放置在细胞ROS释放位点,检测其电流随时间变化,可观察到电流随时间呈现波动状态,推测ROS的胞外释放是一个动态的脉动过程。

An electrochemical method of scanning electrochemical microscopy (SECM )was developed for the detection of extracellular reactive oxygen species (ROS )based on H 2O 2.The release of ROS was investigated by SECM technique after stimulation of lung cancer cells (A549 )with tetradecanoylphorbol acetate (PMA ).The distance between the electrode and petri dish was determined as about 8 μm,and the cell morphology was shown clearly with hexamineammonium chloride (Ru (NH 3 ) 6Cl 3 )as the solution mediator in negative feedback mode.The release of ROS from A549 cells treated with PMA in real time were evaluated using SECM imaging when the potential of the probe was set at -0.65 V.Expanding the imaging range,SECM images of the release of ROS from different cells and the real-time capture of ROS signals were obtained.Overall,the PMA-induced ROS production was successfully determined.Moreover,at the corresponding ROS release site,the current variation with time was fluctuating.It was speculated that the release of ROS from cells was a dynamic pulsation process in this condition.