Plasma membrane anchored nanosensor for quantifying endogenous production of H2O2 in living cells
Plasma membrane anchored nanosensor for quantifying endogenous production of H2O2 in living cells
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DOI：
10.1016/j.bios.2021.113077
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发表时间：
2021-02-16
影响因子：
12.6
通讯作者：
Puppulin, Leonardo
中科院分区：
文献类型：
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作者：
Hosogi, Shigekuni;Marunaka, Yoshinori;Puppulin, Leonardo
过氧化氢(H_2O_2)是控制细胞代谢的信号通路中的主要第二信使之一。在肿瘤发生过程中，由膜相关的NADPH氧化酶和超氧化物歧化酶在细胞外间隙产生过氧化氢，以刺激细胞增殖和保持转化状态。因此，恶性细胞的一个特征是在细胞外环境中产生过多的过氧化氢，随后在胞浆中吸收。由于内源性细胞外过氧化氢最显著的梯度只能在与质膜接触的流体的非常浅的区域观察到，因此我们在这里展示了一种新设计的纳米传感器的使用，该传感器固定在细胞外表面，能够在与负责产生过氧化氢的膜蛋白的纳米距离处对过氧化氢进行定量。这种生物传感器是建立在金纳米颗粒的基础上的，这种纳米金颗粒带有一种对过氧化氢敏感的硼酸盐化合物，并使用表面增强拉曼光谱(SERS)进行了探测。通过SERS分析获得的细胞表面高度局部化的信息与氧化还原生物学的分析方法相结合，来估计负责细胞信号转导的细胞内过氧化氢的相关水平。从A549肺癌细胞系获得的结果显示，细胞表面的斑点在浓度高达12微米时与细胞内浓度高达5.1微米有关。
Hydrogen peroxide (H2O2) is one of the main second messengers involved in signaling pathways controlling cell metabolism. During tumorigenesis H2O2 is generated on the extracellular space by membrane-associated NADPH oxidases and superoxide dismutase to stimulate cell proliferation and preservation of the transformed state. Accordingly, a characteristic feature of malignant cells is overproduction of H2O2 in the extracellular milieu and the subsequent absorption in the cytosol. Since the most significant gradients of endogenous extracellular H2O2 can be observed only in a very shallow region of the fluid in contact with the plasma membrane, we show here the use of a newly designed nanosensor anchored to the outer cell surface and capable of quantifying H2O2 at nanometer distance from the membrane proteins responsible for its production. This biosensor is built upon gold nanoparticles functionalized with a H2O2-sensitive boronate compound that is probed using surface enhanced Raman spectroscopy (SERS). The highly localized information obtained on the cell surface by SERS analysis is combined with analytical methods of redox biology to estimate the associated levels of intracellular H2O2 responsible for cell signaling. The results obtained from A549 lung cancer cell line show localized spots on the cell surface at concentration up to 12 mu M, associated to intracellular concentration up to 5.1 nM.