A TAT peptide-based ratiometric two-photon fluorescent probe for detecting biothiols and sequentially distinguishing GSH in mitochondria

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 Förster resonance energy transfer (FRET) strategy for designing a cell penetration peptide TAT-modified ratiometric 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 (λem = 585 nm) and an enhanced signal in green fluorescence intensity (λem = 520 nm). Excitingly, the TAT-probe excited at 545 nm could display a red fluorescence (λem = 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.