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Dual-targeted fluorescent probe or peroxynitrite

已有 1379 次阅读 2022-5-22 11:32 |系统分类:论文交流

Construction of a mitochondria-endoplasmic reticulum dual-targeted red-emitting fluorescent probe for imaging peroxynitrite in living cells and zebrafish

Lingchao He【何令超】1,3, Heng Liu【刘恒】2,3,*, Ziyi Cheng【程子译】2,3, Fabiao Yu【于法标】1,2,3,*

1 College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu 273165, China

2 Department of Radiotherapy, Key Laboratory of Hainan Trauma and Disaster Rescue, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou, 571199, China

3 Key Laboratory of Emergency and Trauma, Ministry of Education, College of Emergency and Trauma, Hainan Medical University, Haikou 571199, China 

 

First published: 06 May 2022   https://doi.org/10.1002/asia.202200388


Keywords: Fluorescent probe; peroxynitrite; dual-targeted; red-emitting; bioimaging


Abstract:
A mitochondria-endoplasmic reticulum dual-targeted red-emitting fluorescent probe MCSA for imaging ONOO in living cells and zebrafish has been designed and reported.


 

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Abstract

Peroxynitrite (ONOO-) is one of the important reactive oxygen species, which plays a vital role in the physiological process of intracellular redox balance. Revealing the biological functions of ONOO- will contribute to further understanding of the oxidative process of organisms. In this work, we designed and synthesized a novel red-emitting fluorescent probe MCSA for the detection of ONOO-, which could rapidly respond to ONOO- within 250 s and exhibited high sensitivity to ONOO- with a low detection limit of 78 nM. Co-localization experiments demonstrated MCSA had the ability to localize into the mitochondria and endoplasmic reticulum. What’s more, MCSA enabled monitoring ONOO- level changes during tunicamycin-induced endoplasmic reticulum stress. We have also successfully achieved the visual detection of exogenous and endogenous ONOO- in living cells and zebrafish. This work presented a chemical tool for imaging ONOO- in vitro and in vivo. 


Scheme 1. Synthetic approaches to MCSA


Figure 1. Spectral properties of MCSA. (a) Absorption spectra of MCSA before and after addition of ONOO-. (b) Fluorescence spectra of MCSA after the reaction with series concentrations of ONOO- (0 - 18 μM). (c) The plot of the linear relationship between the relative fluorescence intensity at 635 nm of MCSA and concentrations of ONOO- (0 - 18 μM). (d) Time-dependent fluorescence intensity of MCSA toward ONOO- (18 μM). (e) Fluorescence intensity at 635 nm of MCSA with and without ONOO- under different pH values. (f) Fluorescence responses of MCSA to different potential interfering species: 1. blank, 2. Na+ (100 μM), 3. Ca2+ (100 μM), 4. Zn2+ (100 μM), 5. Fe3+ (20 μM), 6. NO2- (50 μM ), 7. NO (50 μM), 8. NaClO (20 μM), 9. H2O2 (100 μM), 10. O2•− (100 μM), 11. ·OH (50 μM), 12. Na2S2O3 (200 μM), 13. 1O2 (50 μM), 14. Citric acid (50 μM), 15. Cys (500 μM), 16. GSH (500 μM), 17. ONOO- (18 μM).


Figure 2. Imaging exogenous ONOO in different cells. (a) Confocal laser scanning microscopy of 10 μM MCSA-loaded HeLa, Raw 246.7, PC12 cells only, or treated with SIN-1 (0.5 mM) for 30 min. The red channel was collected in the range of 600−700 nm (λex = 520 nm) and the DAPI channel was collected in the range of 415-550 nm (λex = 405 nm). (b) Relative red fluorescence intensities of MCSA labelled cells in (a) (n = 3).


Figure 3. Imaging endogenous ONOO in RAW264.7 cells. (a) The cells pretreated with LPS, IFN-γ for 4 h, 6 h, 8 h, then PMA for 30 min, were incubated with MCSA (10 μM). (b) Relative red fluorescence intensities of MCSA labelled cells in (a) (n = 3). (c) The cells were pretreated with LPS, IFN-γ for 8 h, PMA for 30 min, UA or AG for 30 min, then incubated with MCSA for 30 min. (d) Relative red fluorescence intensities of MCSA labelled cells in (c) (n = 3). The red channel was collected in the range of 600-700 nm (λex = 520 nm) and the DAPI channel was collected in the range of 415-550 nm (λex = 405 nm).


Figure 4. Co-localization of MCSA and ER-tracker red, Mito-tracker green in HeLa cells. The cells were co-incubated with MCSA (10 μM, λex =520nm, λem = 600-700 nm ), and (a) ER-tracker red (500 nM, λex = 561 nm, λem = 580-630 nm); (d) Mito-tracker green (500 nM, λex = 488 nm, λem = 500-600 nm). (b, e) Intensity correlation plot of MCSA and ER-tracker red, Mito-tracker green. (c, f) Intensity 3D waterfall profiles of MCSA and ER-tracker red, Mito-tracker green within the linear ROI.


Figure 5. Imaging ONOO in HeLa cells during endoplasmic reticulum stress. (a) The cells pretreated with TM (0, 1, 2, 3 μg/ml) for 8 h, were incubated with MCSA (10 μM). (b) Relative fluorescence intensities of MCSA labelled cells in (a) (n = 3).