A TAT peptide-based ratiometric two-photon fluorescent probe for detecting biothiols and sequentially distinguishing GSH in mitochondria
A TAT peptide-based ratiometric two-photon fluorescent probe for detecting biothiols and sequentially distinguishing GSH in mitochondria
复制标题
一种基于 TAT 肽的比率双光子荧光探针，用于检测生物硫醇并依次区分线粒体中的 GSH。
DOI：
10.1016/j.talanta.2020.121127
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发表时间：
2020-10-01
期刊：
影响因子：
6.1
通讯作者：
Tang, Yu
中科院分区：
文献类型：
--
作者：
Su, Pingru;Zhu, Zhanwu;Tang, Yu
尽管半胱氨酸(Cys)、谷胱甘肽(GSH)和同型半胱氨酸(Hcy)等生物硫醇在许多生理过程中可以用于疾病诊断和生理代谢研究，但由于它们具有相似的化学性质和分子结构，生物硫醇的原位实时检测和鉴别仍然具有挑战性。在这里，我们利用天然的化学连接(NCL)反应机制，开发了Forster共振能量转移(FRET)策略，用于设计细胞穿透肽TAT修饰的比率双光子生物硫醇探针(TAT-探针)。该TAT探针不仅可以在TAT多肽的辅助下快速进入线粒体，而且可以同时检测生物硫醇，从而区分GSH。当TAT-探针在404/820 nm波长的光激发下，加入生物硫醇后，荧光比率发生了变化，包括红色荧光强度(lambda(Em)=585 nm)和绿色荧光强度增强(lambda(Em)=520 nm)。令人兴奋的是，在545 nm处激发的TAT探针可以向GSH发出红色荧光(λ(Em)=585 nm)，向Hcy或Cys发出猝灭信号。这种特异的荧光反应表明，TAT探针可以有效地检测生物硫醇，并在线粒体中区分GSH和Cys/Hcy。这项工作开创了基于多肽和NCL反应机理设计和合成生物硫醇探针的新途径。
Although biothiols, including cysteine (Cys), glutathione (GSH), and homocysteine (Hcy) can be used to diagnose many diseases and research physiological metabolism in many physiological processes, in situ real-time detection and differentiation of biothiols is still challenging because their similar chemical properties and molecular structures. Herein, we utilized the native chemical ligation (NCL) reaction mechanism to develop a Forster resonance energy transfer (FRET) strategy for designing a cell penetration peptide TAT-modified ratio metric two-photon biothiols probe (TAT-probe). The TAT-probe can not only rapidly enter into mitochondria assisted by TAT peptide, but also simultaneously detect biothiols and sequentially distinguish GSH. When the TAT-probe was excited with 404/820 nm wavelength light, it showed a change in the ratio of fluorescence after adding biothiols, including a quenched red fluorescence intensity (lambda(em) = 585 nm) and an enhanced signal in green fluorescence intensity (lambda(em) = 520 nm). Excitingly, the TAT-probe excited at 545 nm could display a red fluorescence (lambda(em) = 585 nm) towards GSH and a quenched signal towards Hcy or Cys. This specific fluorescence response indicated the TAT-probe could effectively detect biothiols and differentiate GSH from Cys/Hcy in mitochondria. This work pioneered a new approach to design and synthesize biothiol-probes based on peptides and NCL reaction mechanism.