Optical sensor for the detection of caspase-9 activity in a single cell
Optical sensor for the detection of caspase-9 activity in a single cell
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DOI：
10.1021/ja037388t
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发表时间：
2004-03-10
影响因子：
15
通讯作者：
Vo-Dinh, T
中科院分区：
文献类型：
--
作者：
Kasili, PM;Song, JM;Vo-Dinh, T
我们首次展示了一种独特的光学传感器的应用和用途，该传感器具有纳米探针，通过检测caspase-9的酶活性来监测单个活细胞中线粒体凋亡途径的开始。使用光学传感器对单个活的MCF-7细胞进行了caspase-9活性的微创分析，该传感器采用了一种用于固定非荧光酶底物Leucine-GlutamicAcid-Histidine-AsparticAcid-7-amino-4-methylcoumarin的免疫化学分析方法(LEHD-AMC)。Lehd-AMC共价连接在光学传感器的纳米探针尖端，在细胞凋亡过程中被caspase-9裂解，产生游离的AMC。使用逝去场激发裂解的AMC，并检测所产生的荧光信号。通过定量监测荧光信号的变化，检测到单个活的MCF-7细胞中caspase-9的活性。通过比较光动力诱导的凋亡细胞和非凋亡细胞的荧光信号，我们成功地检测到caspase-9的活性，这表明细胞发生了凋亡。
We demonstrate for the first time, the application and utility of a unique optical sensor having a nanoprobe for monitoring the onset of the mitochondrial pathway of apoptosis in a single living cell by detecting enzymatic activities of caspase-9. Minimally invasive analysis of single live MCF-7 cells for caspase-9 activity is demonstrated using the optical sensor which employs a modification of an immunochemical assay format for the immobilization of nonfluorescent enzyme substrate, Leucine-GlutamicAcid-Histidine-AsparticAcid-7-amino-4-methylcoumarin (LEHD-AMC). LEHD-AMC covalently attached on the nanoprobe tip of an optical sensor is cleaved during apoptosis by caspase-9 generating free AMC. An evanescent field is used to excite cleaved AMC and the resulting fluorescence signal is detected. By quantitatively monitoring the changes in fluorescence signals, caspase-9 activity within a single living MCF-7 cell was detected. By comparing of the fluorescence signals from apoptotic cells induced by photodynamic treatment and nonapoptotic cells, we successfully detected caspase-9 activity, which indicates the onset of apoptosis in the cells.