Kahali Smitaroopa, Das Sujit Kumar, Kumar Ravinder, Gupta Kunika, Kundu Rajasree, Bhattacharya Baivabi, Nath Arnab, Venkatramani Ravindra, Datta Ankona
Department of Chemical Sciences, Tata Institute of Fundamental Research 1 Homi Bhabha Road Mumbai 400005 India
Department of Developmental Biology and Genetics, Indian Institute of Science Bangalore 560012 India.
Chem Sci. 2024 Jun 13;15(28):10753-10769. doi: 10.1039/d4sc00907j. eCollection 2024 Jul 17.
Central roles of Mn ions in immunity, brain function, and photosynthesis necessitate probes for tracking this essential metal ion in living systems. However, developing a cell-permeable, fluorescent sensor for selective imaging of Mn ions in the aqueous cellular milieu has remained a challenge. This is because Mn is a weak binder to ligand-scaffolds and Mn ions quench fluorescent dyes leading to turn-off sensors that are not applicable for imaging. Sensors with a unique combination of Mn selectivity, μM sensitivity, and response in aqueous media are necessary for not only visualizing labile cellular Mn ions live, but also for measuring Mn concentrations in living cells. No sensor has achieved this combination thus far. Here we report a novel, completely water-soluble, reversible, fluorescent turn-on, Mn selective sensor, M4, with a of 1.4 μM for Mn ions. M4 entered cells within 15 min of direct incubation and was applied to image Mn ions in living mammalian cells in both confocal fluorescence intensity and lifetime-based set-ups. The probe was able to visualize Mn dynamics in live cells revealing differential Mn localization and uptake dynamics under pathophysiological physiological conditions. In a key experiment, we generated an in-cell Mn response curve for the sensor which allowed the measurement of the endogenous labile Mn concentration in HeLa cells as 1.14 ± 0.15 μM. Thus, our computationally designed, selective, sensitive, and cell-permeable sensor with a 620 nM limit of detection for Mn in water provides the first estimate of endogenous labile Mn levels in mammalian cells.
锰离子在免疫、脑功能和光合作用中发挥着核心作用，因此需要开发用于追踪生物系统中这种必需金属离子的探针。然而，开发一种可穿透细胞的荧光传感器，用于在水性细胞环境中对锰离子进行选择性成像，仍然是一项挑战。这是因为锰与配体支架的结合力较弱，并且锰离子会淬灭荧光染料，导致不适用于成像的关闭型传感器。不仅需要可视化活细胞中不稳定的细胞内锰离子，还需要测量活细胞中的锰浓度，因此需要具有独特组合的锰选择性、微摩尔灵敏度和在水性介质中响应的传感器。到目前为止，还没有传感器实现这种组合。在这里，我们报告了一种新型的、完全水溶性的、可逆的、荧光开启的锰选择性传感器M4，其对锰离子的解离常数为1.4 μM。M4在直接孵育15分钟内进入细胞，并应用于共聚焦荧光强度和基于寿命的设置中对活的哺乳动物细胞中的锰离子进行成像。该探针能够可视化活细胞中的锰动态，揭示病理生理条件下不同的锰定位和摄取动态。在一项关键实验中，我们为该传感器生成了细胞内锰响应曲线，从而能够测量HeLa细胞内源性不稳定锰浓度为1.14±0.15 μM。因此，我们通过计算设计的、选择性的、灵敏的和可穿透细胞的传感器，其在水中对锰的检测限为620 nM，首次估计了哺乳动物细胞内源性不稳定锰水平。
