Real-Time Discrimination and Versatile Profiling of Spontaneous Reactive Oxygen Species in Living Organisms with a Single Fluorescent Probe

Abstract

Fluorescent probes are powerful tools for the investigations of reactive oxygen species (ROS) in living organisms by visualization and imaging. However, the multiparallel assays of several ROS with multiple probes are often limited by the available number of spectrally nonoverlapping chromophores together with large invasive effects and discrepant biological locations. Meanwhile, the spontaneous ROS profilings in various living organs/tissues are also limited by the penetration capability of probes across different biological barriers and the stability in reactive in vivo environments. Here, we report a single fluorescent probe to achieve the effective discrimination and profiling of hydroxyl radicals (•OH) and hypochlorous acid (HClO) in living organisms. The probe is constructed by chemically grafting an additional five-membered heterocyclic ring and a lateral triethylene glycol chain to a fluorescein mother, which does not only turn off the fluorescence of fluorescein, but also create the dual reactive sites to ROS and the penetration capability in passing through various biological barriers. The reactions of probe with •OH and HClO simultaneously result in cyan and green emissions, respectively, providing the real-time discrimination and quantitative analysis of the two ROS in cellular mitochondria. Surprisingly, the accumulation of probes in the intestine and liver of a normal-state zebrafish and the transfer pathway from intestine-to-blood-to-organ/tissue-to-kidney-to-excretion clearly present the profiling of spontaneous •OH and HClO in these metabolic organs. In particular, the stress generation of •OH at the fresh wound of zebrafish is successfully visualized for the first time, in spite of its extremely short lifetime.

Illuminating the Function of the Hydroxyl Radical in the Brains of Mice with Depression Phenotypes by Two‐Photon Fluorescence Imaging

“…[6] Excess COH leads to irreparable damage to neural cells and potentially even to neurological disease. [11][12][13][14][15][16][17][18][19] Given the very high reactivity and low concentration of COH and the particularly complicated construction of the brain, two-photon (TP) fluorescence imaging is the most appropriate for brain imaging because it provides ah igher signal-tobackground ratio,d eeper tissue imaging, higher spatialtemporal resolution, and less specimen photodamage than one-photon (OP) fluorescence imaging. [8][9][10] Fluorescent probes have been developed to reveal the biological functions of COH in living cells,i nz ebrafish, and in the abdomens of mice.…”

Two-photon dual imaging platform for in vivo monitoring cellular oxidative stress in liver injury

“…This sensing platform has advantages over small-molecule fluorescent-based probes for in vivo ROS or GSH imaging reported to date. Conventional emission-based detection methods are influenced by probe concentration and the biomolecular microenvironment103637, or have only been employed under one-photon model938. We found metal complex-based probes can uniquely overcome current limitations to improve detection accuracy in quantitative visualization of oxidative stress by FLIM.…”

“…Methods to detect oxidative stress in vivo have encountered technical challenges, which prevented implementation of this method for preclinical drug efficacy screening29. A number of GSH and ROS-detection probes have been developed910111213.…”

“…A number of GSH and ROS-detection probes have been developed910111213. Most of them are single-mode intensity-based probes, which can provide quantitative results, but may often be influenced by fluid optical properties, endogenous fluorophores, probe concentration, and other in vivo environmental or instrumental factors.…”

Light-up probe based on AIEgens: dual signal turn-on for caspase cascade activation monitoring

“…3 Recently, Zhang et al developed a single fluorescent probe with dual reactive sites that can be used to detect two different reactive oxygen species. 4 It remains challenging to develop a single fluorescent probe that can be used for multiple enzyme detection with a single-wavelength excitation.…”