Short Communication
Selective detection of peroxynitrite in living cells by a near-infrared diphenyl phosphinate-based dicyanoisophorone probe

https://doi.org/10.1016/j.saa.2020.118890Get rights and content

Highlights

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    A novel sensitive and selective NIR fluorescent probe NR-ONOO for detecting peroxynitrite in vitro and in vivo was developed.

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    Peroxynitrite can be selectively and sensitively recognized via a turn-on NIR fluorescence.

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    The probe works in the presence of various ROS/RNS and the detection limit can be as low as 78.70 nM.

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    Probe NR-ONOO realized peroxynitrite monitoring in living HeLa cells.

Abstract

A new NIR fluorescent probe for detection of ONOO− has been developed, which possesses a large Stokes shift, good selectivity and low cytotoxicity. This NR-ONOO probe exhibits a strong turn-on near-infrared fluorescence response toward ONOO− ion under excitation at 560 nm and has been successfully applied in detecting ONOO− in living HeLa cells.

Introduction

Peroxynitrite (ONOO−) acts as a reactive oxygen species (ROS) in various pathological and physiological processes, which was endogenously formed by the combination reaction of superoxide (O2radical dot−) and nitric oxide (NOradical dot) [[1], [2], [3]]. In living biosystem, low level of peroxynitrite plays a critical role in signal transduction, but high level of peroxynitrite could induce damage of some certain biomolecules, such as lipids, proteins, and nucleic acids, etc [4,5]. The abnormal fluctuations of the peroxynitrite concentration eventually cause various diseases including inflammation, cancers, cardiovascular diseases, atherosclerosis, ischemia-reperfusion injury, and diabetes [6]. Thus, the development of peroxynitrite detection methods in living biosystem has become urgently needed.

Compared with other techniques, fluorescent probe-based imaging technology has higher sensitivity, less invasiveness and greater convenience, thus it attracted a great deal of attention [7,8]. However, the excitation and emission wavelengths of most previously reported probes for detection of peroxynitrite just cover ranges within 650 nm, which render them not suitable for detecting peroxynitrite in vivo due to the autofluorescence interferences and insufficient penetration depth in the visible region [[9], [10], [11], [12]]. In contrast, the use of near-infrared fluorescence (650–900 nm) avoids the influence of background bio-autofluorescence and allows deep penetration into tissues [[13], [14], [15]]. However, to date, only a few probes of NIR fluorescence for detecting peroxynitrite have been reported [[16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27]], and it is severely desired to develop new fluorescent probes for the detection of peroxynitrite with NIR emission.

Thus, we herein developed a new NIR florescent probe NR-ONOO for detection of peroxynitrite by using dicyanoisophorone as fluorophore, which was widely utilized in NIR fluorescence because of its excellent photophysical property including large Stokes shift, good photostability and lower biotoxicity. Besides, diphenylphosphinic group was chosen as a sensitive and selective recognition group for the detection of peroxynitrite. The fluorescence of probe NR-ONOO was inhibited by the blocking of the intramolecular charge transfer (ICT). After reaction with peroxynitrite, phosphinate group departed and released the free phenolic hydroxyl group as a strong electron-donating group, which recovers the ICT effect and the generated dicyanoisophorone exhibits the NIR fluorescence emission (Scheme 1). The probe NR-ONOO could selectively and sensitively detect peroxynitrite with a large Stokes shift (133 nm) and a low detection limit (78.70 nM). Besides, this probe also has good membrane penetrability as well as low cell toxicity, making it suitable for the detection of peroxynitrite in living HeLa cells by using confocal microscope.

Section snippets

Materials and instruments

Unless specifically mentioned, all used chemical reagents were obtained from Sigma-Aldrich chemical company without further purification. Deionized water was used for preparing phosphoric acid buffer solution. UV–Vis-NIR absorption spectra and fluorescence spectra were obtained with LAMBDA 1050+ UV/Vis/NIR and Horiba Scientific Spectrofluorometer. Bruker 400 MHz spectrometer is used for 1H NMR and 13C NMR experiments. Nikon A1MP confocal laser scanning microscope was used for living cell

Design and synthesis

Fluorophore is an important member of fluorescent probes. In recent years, isophorone-fused fluorophore (compound 1) has attracted wide attention due to its excellent optical property such as near-infrared fluorescence emission [29], large Stokes shift and the convenience in modifying phenolic hydroxyl group. Besides, diphenylphosphinate group has been reported as ONOO−-specific reaction model [30].Thus, it is highly probable to regulate the switching of fluorescence of probe NR-ONOO by the

Conclusion

A new NIR fluorescent probe for detection of ONOO− through an ICT process has been developed and successful applied in detecting ONOO− in living HeLa cells. This probe has a large Stokes shift, good selectivity and low cytotoxicity. The addition-elimination reaction process by nucleophilic attack of ONOO− to diphenyl phosphinate motif of the probe yielding free phenolic hydroxyl-containing compound 1 generated the fluorescence emission locating at NIR region. We anticipate that the probe might

CRediT authorship contribution statement

Yibin Zhang: Conceptualization, Methodology, Software, Data curation, Writing - original draft. Dongge Ma: Visualization, Investigation, Supervision, Validation, Writing - review & editing.

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

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.

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