Analysis of mitochondrial oxygen consumption and hydrogen peroxide release from cardiac mitochondria using electrochemical multi-sensors
Analysis of mitochondrial oxygen consumption and hydrogen peroxide release from cardiac mitochondria using electrochemical multi-sensors
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DOI：
10.1016/j.snb.2022.131641
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发表时间：
2022-03-04
影响因子：
8.4
通讯作者：
Chen，Thomas W.
中科院分区：
文献类型：
--
作者：
Cheng，Ming-Hao;Chicco，Adam J.;Chen，Thomas W.
在大多数细胞类型中，线粒体是氧气消耗和能量代谢的主要部位，但也产生活性氧物种(ROS)，参与广泛的病理和生理过程。因此，同时监测细胞和线粒体制剂中线粒体ROS的释放和耗氧率(OCR)是生物学研究中一种有吸引力的研究方法，特别是在样本量稀少的情况下。本文介绍了一种灵敏的多传感器装置的研制，该装置能够在单个微室分析中测量生物样品中ROS的产生和OCR。对O2和过氧化氢(由线粒体和细胞释放的主要ROS物种)的传感器灵敏度分别为4.32nA/μM和54.89nA/μM，检测下限分别为2.9nA/μM和58.36nM。对大鼠心脏组织中分离的线粒体(5微克蛋白质)进行的概念验证研究表明，添加呼吸底物后，过氧化氢的释放量预计会比基础速率增加3-4倍，但OCR的变化相对较小。随后添加二磷酸腺苷(ADP)使过氧化氢释放减少了73%(P<0.01)，OCR增加了168%(P<0.01)，这与线粒体膜电位和电子流量从ADP受限(状态4)到ADP刺激(状态3)呼吸状态的既定转变是一致的。这些研究验证了一种新的多传感器设备的使用结果，该设备能够同时监测稀缺生物样本中的OCR和H2O2，在细胞代谢和线粒体功能的非破坏性综合研究中具有潜在的应用价值。
Mitochondria are the primary sites of oxygen (O2) consumption and energy metabolism in most cell types, but also produce reactive oxygen species (ROS) that contribute to a wide array of pathological and physiological processes. Accordingly, simultaneous monitoring of mitochondrial ROS release and oxygen consumption rate (OCR) from cells and mitochondrial preparations is an attractive investigative approach in biological research, particularly when sample quantity is scarce. This paper presents the development of a sensitive multi-sensor device capable of measuring ROS production and OCR from biological samples in a single micro-chamber assay. Sensor sensitivities for O2and hydrogen peroxide (H2O2; the major ROS species released by mitochondria and cells) are 4.32 nA/μM and 54.89 nA/μM, respectively, with limits of detection of 2.9 μM and 58.36 nM, respectively. Proof-of-concept studies in isolated mitochondria from rat cardiac tissue (5 µg protein) demonstrate an expected 3 – 4 fold increase in H2O2release over the basal rate following addition of respiratory substrates, with a comparatively small change in OCR. The subsequent addition of adenosine diphosphate (ADP) decreased H2O2release by 73% (p < 0.01) and increased OCR by 168% (p < 0.01), consistent with established shifts in mitochondrial membrane potential and electron flow from an ADP-limited (State 4) to ADP-stimulated (State 3) respiratory state. These studies validate the results from the use of a novel multi-sensor device capable of monitoring OCR and H2O2simultaneously in scarce biological samples, with potential utility in the non-destructive integrative study of cellular metabolism and mitochondrial function.