State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P. R. China.
Analyst. 2017 Dec 18;143(1):208-214. doi: 10.1039/c7an01515a.
Cytochrome c (Cyt c) and caspase-3 are the key mediators in apoptotic signaling. As is known to all, the release of Cyt c from mitochondria is a vital caspase activation pathway and defines the point of no-return in cell apoptosis. However, it has not been reported that any fluorescence imaging tools could allow simultaneous visualization of Cyt c translocation and caspase-3 activation in apoptotic cells. Here, we develop a sensitive nanosensor that holds the capability of imaging of the released Cyt c from the mitochondria and a caspase-3 activation cascade reaction in apoptotic signaling. The nanosensor is constructed by the assembly of a fluorophore (Cy5)-tagged DNA aptamer on graphene nanosheets that have been covalently immobilized with a FAM-labeled peptide. After a spatially selective delivery into the cytoplasm, the Cy5-tagged DNA aptamer assembled on the nanosensor can bind with Cyt c released from the mitochondria to the cytoplasm and dissociate from graphene, triggering a red fluorescence signal. In addition, the caspase-3 activated by the Cyt c released to the cytoplasm can cleave the FAM-labeled peptide and result in a green fluorescence output. The nanosensor exhibits rapid response, high sensitivity and selectivity for in vitro assays, and high contrast imaging of Cyt c and caspase-3 in living cells. It also provides the method for the study of the kinetic relationship between the Cyt c translocation and caspase-3 activation through simultaneous imaging of Cyt c and caspase-3. The developed nanosensor described here will be an efficient and potential platform for apoptosis research.
细胞色素 c（Cyt c）和半胱天冬酶-3 是凋亡信号转导的关键介质。众所周知，Cyt c 从线粒体中的释放是半胱天冬酶激活的重要途径，并定义了细胞凋亡中不可逆转的点。然而，尚未有报道称任何荧光成像工具可以同时可视化凋亡细胞中 Cyt c 的易位和半胱天冬酶-3 的激活。在这里，我们开发了一种灵敏的纳米传感器，能够对从线粒体释放的 Cyt c 以及凋亡信号转导中的半胱天冬酶-3 激活级联反应进行成像。该纳米传感器通过将荧光染料（Cy5）标记的 DNA 适体组装在石墨烯纳米片上构建而成，石墨烯纳米片通过共价固定带有 FAM 标记的肽。在进行空间选择性递送至细胞质后，组装在纳米传感器上的 Cy5 标记的 DNA 适体可以与从线粒体释放到细胞质中的 Cyt c 结合，并从石墨烯上解离，从而触发红色荧光信号。此外，被释放到细胞质中的 Cyt c 激活的半胱天冬酶-3 可以切割 FAM 标记的肽，导致绿色荧光输出。该纳米传感器在体外试验中表现出快速响应、高灵敏度和选择性，以及对活细胞中 Cyt c 和半胱天冬酶-3 的高对比度成像。它还通过同时对 Cyt c 和半胱天冬酶-3 进行成像，为研究 Cyt c 易位和半胱天冬酶-3 激活之间的动力学关系提供了方法。这里描述的纳米传感器将成为研究凋亡的有效和有潜力的平台。
