Molecular Sensors and Therapeutics (MST) Research Laboratory, Department of Chemistry, School of Natural Sciences, Shiv Nadar University, Delhi NCR, NH 91, Tehsil Dadri, Greater Noida, Uttar Pradesh 201314, India.
Protein Homeostasis Laboratory, Department of Life Sciences, School of Natural Sciences, Shiv Nadar University, Delhi NCR, NH 91, Tehsil Dadri, Greater Noida, Uttar Pradesh 201314, India.
Anal Chem. 2022 Aug 23;94(33):11633-11642. doi: 10.1021/acs.analchem.2c02177. Epub 2022 Aug 14.
Mitochondrial functions are heavily influenced by acid-base homeostasis. Hence, elucidation of the mitochondrial pH is essential in living cells, and its alterations during pathologies is an interesting question to be addressed. Small molecular fluorescent probes are progressively applied to quantify the mitochondrial pH by fluorescence imaging. Herein, we designed a unique small molecular fluorescent probe, , based on the lipophilic morpholine ligand-conjugated pyridinium derivative of "IndiFluors". The morpholine-conjugated fluorescent probe usually localized the lysosome. However, herein, we observed unusual phenomena of morpholine-tagged that localized mitochondria explicitly. The morpholine ligand also plays a pivotal role in tuning optical properties via photoinduced electron transfer (PET) during internal pH alteration (ΔpHi). In the mitophagy process, lysosomes engulf damaged mitochondria, leading to ΔpHi, which can be monitored using our probe. It exhibited "ratiometric" emission at single wavelength excitation (ex. 488) and is suitable for monitoring and quantifying the ΔpHi using confocal microscope high-resolution image analysis during mitophagy. The bathochromic emission shifts due to intramolecular charge transfer (ICT) in basic pH were well explained by the time-dependent density functional theory (TD-DFT/PCM). Similarly, the change in the emission ratio (green/red) with pH variations was also validated by the PET process. In addition, can quantify the pH change during oxidative stress induced by rapamycin, mutant A53T α-synuclein-mediated protein misfolding stress in mitochondria, and during starvation. Rapamycin-induced mitophagy was further elucidated by the translocation of mCherry Parkin to damaged mitochondria, which well correlates with our probe. Thus, is a valuable probe for visualizing mitophagy and can act as a suitable tool for the diagnosis of mitochondrial diseases.
线粒体功能受酸碱平衡的强烈影响。因此，阐明活细胞中线粒体的 pH 值是至关重要的，其在病理过程中的变化是一个值得探讨的有趣问题。小分子荧光探针逐渐被应用于通过荧光成像来定量测量线粒体 pH 值。在此，我们设计了一种独特的小分子荧光探针 ，基于亲脂性吗啉配体连接的“IndiFluors”吡啶鎓衍生物。吗啉连接的荧光探针通常定位于溶酶体。然而，在此，我们观察到一种不寻常的现象，即吗啉标记的 明确定位于线粒体。吗啉配体在内部 pH 值变化（ΔpHi）过程中通过光诱导电子转移（PET）也对光学性质起着关键作用。在自噬过程中，溶酶体吞噬受损的线粒体，导致 ΔpHi，可以使用我们的探针进行监测。它在单一波长激发（ex. 488）下表现出“比率”发射，适用于在自噬过程中使用共聚焦显微镜高分辨率图像分析来监测和定量测量 ΔpHi。由于质子化，在碱性 pH 值下发生的分子内电荷转移（ICT）导致了红移发射，这可以通过时变密度泛函理论（TD-DFT/PCM）得到很好的解释。同样，发射比（绿/红）随 pH 值变化的变化也通过 PET 过程得到了验证。此外， 可以量化由雷帕霉素诱导的氧化应激、突变 A53T α-突触核蛋白介导的线粒体蛋白错误折叠应激以及饥饿过程中的 pH 值变化。雷帕霉素诱导的自噬通过 mCherry Parkin 向受损线粒体的易位得到进一步阐明，这与我们的探针很好地相关。因此， 是一种用于可视化自噬的有价值的探针，并且可以作为诊断线粒体疾病的合适工具。
