A NIR fluorescence probe having significant fluorescence turn-on signal at 700 nm and large Stokes shift for rapid detection of HOCl in vivo
Graphical abstract
Introduction
Hypochlorous acid (HOCl) is known as a very important reactive oxygen species (ROS) in the life systems (including human beings), produced from the myeloperoxidase (MPO)-catalyzed reaction of H2O2 and Cl− [1]. Studies have shown that HOCl plays an essential role in various physiological and pathological processes including aging, anti-inflammation, immune regulation, and pathogen response and so on [[2], [3], [4], [5]]. However, because of its strong capability to oxidize biomolecules, in vivo generation of excess HOCl can cause cell and inflammation-associated tissue damage and lead to various diseases [[6], [7], [8]]. These findings suggest that HOCl may have dualistic effects, but the exact intracellular and molecular mechanism of HOCl remains unclear [9]. A possible main reason is the lack of reliable detection tools to real-time capture and study this elusive and highly active species. Because of this, it is urgent and significant to develop an effective HOCl detection tool in the life system [[10], [11], [12], [13], [14], [15], [16], [17], [18]].
Fluorescent probes are valuable tools that possess high sensitivity, high convenience, nondestruction, and high temporal-spatial resolution. Such probe-based imaging has been proved to be a very attractive detection technology for reactive oxygen species [[19], [20], [21], [22]]. Particularly, near-infrared (NIR) emissive probes have excellent characteristics, which can be used for imaging applications both in vitro (including cell imaging) and in vivo due to their low biological background fluorescence interference, very little or no photodamage, and deep tissue penetration depth [23]. Over the past years, based on different fluorophores and response sites, many probes have been designed for HOCl detection [[24], [25], [26]]. However, the emission wavelength of most probes located in the visible region [[27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37]], which hinders their application for in vivo imaging. So far, the reports of NIR probes for both cell and in vivo imaging of HOCl are still limited [[38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], [53]]. Moreover, most of these probes have some shortcomings, such as showing fluorescence turn-off signals in the NIR region, small Stokes shift, long response time and low sensitivity etc., which seriously limit their practical applications [see Table S1]. Hence, the development of new NIR fluorescent probes for HOCl with a better fluorophore and a more efficient reaction site is still necessary.
Recently, we reported a novel NIR fluorescent fluorophore CDCI-OH (Scheme 1), which has an easily obtained coumarin-dicyanoisophorone hybrid structure and shows strong fluorescence around 700 nm with a massive Stokes shift [54]. With these attractive optical properties, we thought that this fluorophore might be good to construct a new NIR fluorescence probe to detect HOCl. Dimethylthiocarbamate (DMTC) was then selected as the reaction site because it was recently found to be able to effectively and selectively recognize HOCl [55,56]. Thus, probe CDCI-HClO (Scheme 1) was prepared and studied. Herein, we report that CDCI-HClO can be applied as a desirable new probe for the selective detection of HOCl, featuring with short response time (<2 s), high sensitivity, large fluorescence turn-on signals at 700 nm, and huge Stokes shift (190 nm). Besides, we also show that CDCI-HClO has great potential for imaging of HOCl both in live cells and in vivo.
Section snippets
Materials and methods
Basic experimental materials and methods are attached in the Supplementary Data. Fluorophore CDCI-OH was prepared from compound 1 and 2 (Scheme 2) in 60% yield according to the reported method [54].
The synthesis of CDCI-HClO
CDCI-HClO was synthesized in moderate yield by reacting CDCI-OH with dimethylthiocarbamoyl chloride (Scheme 2). Pure CDCI-HClO was characterized by 1H and 13C NMR, and MS including HR-MS (see Supplementary material).
Spectral characteristics
After obtaining probe CDCI-HClO, we first measured its optical spectra and compared them with that of CDCI-OH. As can be seen in Fig. S1, CDCI-HClO is almost non-fluorescent (Ф < 0.01) and has a maximum absorption at 416 nm. In comparison, CDCI-OH emits strong fluorescence at 700 nm
Conclusions
In this work, we have attached the reaction site dimethylthiocarbamate into a hybrid fluorophore of coumarin-dicyanoisophorone and successfully developed a new probe (CDCI-HClO) for HOCl. CDCI-HClO showed high sensitivity (~4 nM of detection limit) and high selectivity for HOCl with a rapid response (<2 s), large turn-on NIR fluorescence signal at 700 nm, and a massive Stokes shift (190 nm). Besides, the probe also showed low cytotoxicity and excellent cell imaging and in vivo imaging
Credit Author Statement
Haiyan Zhang: Methodology, Formal analysis, Writing - original draft. Xinyu Yin: Carried out the experiments, Formal analysis, Writing - original draft. Jiaxin Hong: Carried out the experiments, analyzed the data. Yingzhen Deng: Carried out the experiments, analyzed the data. Guoqiang Feng: Supervision, Reviewed and edited the manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
We thank the National Natural Science Foundation of China (21672080) and the Science and technology research project of Hubei Education Department (B2019063) for financial support.
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These authors contributed equally to this work.