Peroxynitrite (ONOO), a highly reactive nitrogen species, which plays a crucial role in numerous physiological and pathological processes of cell functionalization. The anomalous concentration of ONOO may result in a range of diseases, such as arthritis, neurological disorders, and even cancer. Therefore, it is urgent to develop a simple and effective tool to monitor the fluctuation of ONOO levels in biological systems. Herein, an ultrafast fluorescent probe (HND-ONOO) is proposed to detect ONOO, which displays brilliant fluorescence in less than 30 s with a large Stokes shift. Furthermore, the probe exhibited the lower detection limit (48 nM) and satisfactory results in differentiating ONOO from other related species. The probe that possesses good biocompatibility and low toxicity was employed to monitor the level of exogenous and endogenous ONOO in living cells. Thus, the probe HND-ONOO could be served as a potential imaging tool to visualize intracellular ONOO and understand the relationship between ONOO and inflammation.

1. Introduction

Reactive nitrogen species (RNS), extremely significant intracellular signaling molecules, are irreplaceable in various physiological and pathological processes, such as cell growth, differentiation, and apoptosis [1, 2]. However, external stimuli may cause abnormal levels, leading to a range of diseases, including arthritis, high blood pressure, and neurological diseases [35]. Peroxynitrite (ONOO), a common type of RNS, is generated from superoxide anion (O2) and nitric oxide (NO) by intracellular diffusion [68]. As a strong oxidative and nitrative reagent, ONOO can react with biological macromolecules such as proteins and DNA and is thought to be involved in signal transduction. [9,10] However, elevated levels of ONOO may affect the normal activities of cells and organ aging, which in turn may lead to inflammatory diseases, autoimmune diseases, and cancer. [1113] For example, rheumatoid arthritis served a general chronic inflammatory disease that may induce joint swelling, pain, and damage, bringing a lot of inconvenience to human life. [14, 15] Therefore, the development of a simple and effective tool to detect ONOO in real-time in biological systems to understand its role in related diseases is urgently needed to understand the progress of the disease.

Over the decades, various analysis techniques have been employed to sense ONOO, such as UV-vis spectroscopy, electrochemical analysis, and electron spin resonance. [1620] However, although these methods can be used for the detection of ONOO, they still have certain deficiencies due to the short half-life and low content of ONOO in biological systems. In order to further understand the regulation mechanism of ONOO in vivo, it is urgent to develop effective techniques to realize the rapid and precise detection of ONOO in cells. The fluorescence probe and imaging method have attracted wide attention because of its noninvasiveness, excellent sensitivity and significant spatiotemporal resolution, and real-time visual inspection. [7, 2125] So far, more and more fluorescent probe is constructed for the detection of ONOO. [2628] These reported probes are mainly based on ONOO reaction with specific groups, including the cleavage of C=C, trifluoromethyl ketone, α-ketoamide, and the oxidation of hydrazides, N-dearylation and boronate. [2936] However, although some probes are proposed to sense trace amounts of ONOO, they are disturbed by other substances, such as hydrogen peroxide (H2O2) or hypochlorous acid (HClOH). [3739] Furthermore, slow reaction speed, poor water solubility, and low biocompatibility limit the application of probes in biological systems. Therefore, there are still some challenges in developing fluorescent probes with good biocompatibility to achieve the rapid monitoring of ONOO in living cells.

In this work, we propose a reasonable fluorescent probe (HND-ONOO) for rapid monitoring of intracellular ONOO. The probe consists of hemicyanine dye and a phenylboronic acid ester derivative as a specific response group for ONOO. Notably, this probe displays high selectivity and specificity for ONOO, especially when not disturbed by active species such as H2O2 and HClO. In addition, the probe cleaved the boronic ester group within 30 s, releasing the naked hydroxyl group and causing the turn-on of the fluorescent signal. HND-ONOO also exhibits a great linear relationship and lower detection limit (LOD) toward ONOO. Furthermore, the probe HND-ONOO was successfully utilized to monitor and image exogenous and endogenous of ONOO in cells. We believe that this probe has great potential for discussing the relationship between ONOO and inflammatory diseases and is helpful for the diagnosis of inflammation.

2. Experimental Section

2.1. Apparatus and Reagents

The UV-vis and fluorescence spectra were carried out on the UV-2910 and F-7000 fluorescence spectrophotometers, respectively. 1H NMR (500 MHz) and 13C NMR (125 MHz) were implemented on a Bruker nuclear magnetic resonance spectrometer. The fluorescence images of living were performed on an Olympus FV1000. The chemical reagents come from Maclean, and the main biological reagents come from Biyuntian.

2.2. Synthesis of Compound 2

We added 4.5 mL of iodoethane and 2.2 g of 2,3,3-trimethyl-3H-indole into a 100 mL round bottom flask, and then, 30 mL of acetonitrile was added to the mix. After refluxing for 12 h, a large amount of pink solids were generated. The solid obtained by filtration is used directly in the next step without further purification.

2.3. Synthesis of HND

The compound 2 (0.63 g) and 2-Hydroxy-6-naphthaldehyde (0.38 g) were put in a round bottom flask. Then, 8 mL of absolute ethanol was added to the mixture, and further heated to reflux for 9 h. The mixture was cooled to room temperature, and then, placed in ice water to cool; a lot of dark red solid was obtained. Subsequently, the solid was filtered by suction and washed with ice absolute ethanol to obtain compound HND. 1H NMR (500 MHz, DMSO-d6) δ 9.56 (s, 1H), 7.93–7.80 (m, 2H), 7.61–7.50 (m, 2H), 7.43 (d, J = 8.9 Hz, 1H), 7.37–7.35 (m, 1H), 7.12–7.06 (m, 1H), 6.99–6.92 (m, 2H), 6.86 (s, 1H), 6.40–6.33 (m, 2H), 4.26–4.02 (m, 2H), 1.64 (s, 3H), and 1.21 (s, 6H).

2.4. Synthesis of HND-ONOO

To a 50 mL round bottom flask was added 220 mg of compound 2 (0.66 mmol), 300 mg of 4-bromomethyl-phenylboronic acid pinacol ester (1 mmol), 276 mg of anhydrous K2CO3 (2 mmol), and 20 ml of acetonitrile. The mixture was heated to 80°C and refluxed overnight. The reaction was stopped, it was cooled to room temperature, and the solvent was removed under reduced pressure to obtain the crude product. The obtained crude product was purified by silica gel column chromatography (CH2Cl2 : CH3OH = 20:1, v/v) to obtain 150 mg of a solid compound (yield: 41%). 1H NMR (500 MHz, CDCl3) δ 8.92 (s, 1H), 8.36–8.30 (t, J = 14.8 Hz, 3H), 8.15–8.14 (d, J = 8.9 Hz, 1H), 7.89–7.88 (d, J = 7.9 Hz, 3H), 7.64–7.58 (t, J = 14.6 Hz, 4H), 7.52–7.51 (d, J = 7.9 Hz, 2H), 7.32–7.32 (d, J = 2.2 Hz, 1H), 7.23–7.23 (d, J = 2.2 Hz, 1H), 5.27 (s, 4H), 1.89 (s, 3H), 1.60 (s, 6H), 1.38 (s, 12H). 13C NMR (125 MHz, CDCl3) δ 156.04 (s), 143.32 (s), 139.29 (s), 135.17 (s), 132.30 (s), 129.73 (s), 129.55 (d, J = 8.9 Hz), 128.87 (s), 128.41 (s), 126.91 (s), 126.69 (s), 122.63 (s), 120.37 (s), 114.57 (s), 112.05 (s), 83.89 (s), 70.19 (s), 52.10 (s), 44.17 (s), 27.29 (s), 24.88 (s), and 14.48 (s). HRMS: m/z C37H41BNO3 calcd, 558.3180; found [M]+, 558.3181.

2.5. Cell Culture and Confocal Imaging

The HeLa and RAW 264.7 cells were incubated in DMEM containing with 10% FBS and 1% antibiotics (streptomycin/penicillin). The tested cells were placed at 37°C in a 95% humidified atmosphere with 5% CO2. The cytotoxicity of HND-ONOO was assessed by the CCK-8 experiment. Those cells were put in a 96-well plate (10000 cells/well) for about 24 h. Subsequently, various levels of probe were added and then incubated for 24 h. After washing with DMEM medium, 10 μL CCK-8 was added to the cells. After incubating for about 4 h, the optical density was measured at 450 nm via a microplate reader (Tecan, Austria). The fluorescent image was carried out on the Olympus FV1000. The cells seeded in the culture dish and incubated for 24 h. Then, the cells stained by the probe were imaged after being washed with PBS for three times. Fluorescence collection window is 560-640 nm.

3. Results and Discussion

3.1. Design and Synthesis of HND-ONOO

The structure and the proposed response mechanism of probe HND-ONOO for ONOO are shown in Scheme 1. In order to construct a sensitive and special fluorescent probe for ONOO, hemicyanine dye was chosen as the fluorophore due to its large Stokes shift, long emission properties, and great biocompatibility. Recent studies indicated that benzeneboronic acid and its ester are able to more quickly and efficiently respond to ONOO; therefore, it can serve as good candidate reaction site for the sensitive and fast detection of ONOO. [4042] 4-(bromomethyl) benzene-boronic acid pinacol ester was introduced as response group of ONOO and the hemicyanine dye act as the light-emitting group. The probe HND-ONOO was nonfluorescent because of the 4-(bromomethyl) benzeneboronic acid pinacol ester inhibited the intramolecular charge transfer (ICT) process. The presence of ONOO can first convert the phenylboronic ester to phenol, and then, the phenol derivative undergoes 1,6-self-elimination to release the fluorophore HND. The detailed synthetic route and 1H NMR (Figure S1), [13]C NMR (Figure S2), and HRMS (Figure S3) characterizations of the probe are depicted in Supplementary Materials.

3.2. Spectral Properties of HND-ONOO toward ONOO

The spectral characteristics of HND-ONOO were discreetly discussed in PBS-DMSO buffer solution (pH = 7.4, PBS/DMSO = 9 : 1, v/v). The probe HND-ONOO exhibited an absorption centered at 450 nm. After reacting with ONOO, this absorption peak was significantly increased (Figure 1(a)). Furthermore, this absorption peak reduced with increasing ONOO concentrations (0–20 μM). Subsequently, the fluorescence signal of HND-ONOO to variable levels of ONOO was assessed. In the initial state, the probe HND-ONOO exhibited negligible fluorescence at the excitation of 440 nm. However, this probe HND-ONOO displayed a significant fluorescence emission centered at 590 nm in the presence of ONOO. The fluorescence intensity of the probe HND-ONOO obviously improved with the level of ONOO elevated (Figure 1(b)), which should be ascribed to the rapid and specific reaction of ONOO and (bromomethyl) benzeneboronic acid pinacol ester, resulting in the release of fluorescent signal. It also demonstrated that the probe HND-ONOO was capable of detecting ONOO. Furthermore, there obtained a great linear relationship between the fluorescence intensity of HND-ONOO and ONOO content ranging from 0 to 20 μM (Figure 1(c)). The regression equation was established as F590 nm = 12.9843[ONOO] + 31.6068 with a satisfying linear relationship (R2 = 0.9863). The detection limit (LOD) was affirmed as 48 nM according to the standard method of 3σ/k, which suggested that proposed probe HND-ONOO was sufficient to identify trace ONOO. In addition, this property of HND-ONOO provided a possibility for further detection of intracellular ONOO.

3.3. Selectivity

Significant selectivity is one of the excellently pivotal factors in evaluating the merits of fluorescent probes. We first evaluated active species with similar properties to ONOO. As shown in Figure 1(d), significant fluorescence intensity enhancement was observed in the presence of ONOO. It was delightful that only H2O2 caused the probe fluorescence intensity to fluctuate slightly, while other reactive species, such as tBuOO•, •OH, NO2-, NO; O2, HNO, and HClO, cannot significantly alter the signal of the probe HND-ONOO. Furthermore, we tested whether common anions or cations (Na+, K+, Ca2+, HCO3, Br, and HS) and amino acid (glutamic acid (Glu), tryptophane (Trp), serine (Ser), methionine (Met), aspartic acid (Asp), threonine (Thr), and lysine (Lys) would respond to the probe (Figure S4(b)). Although previous studies [43] have shown that the probe may be destroyed by high concentrations of HClO, higher concentrations are required. As shown in Figure S4(c), the results demonstrated that low concentrations of HClO had only negligible effects on the probe. As we expected, there was negative fluorescence was obtained, further declaring that the probe HND-ONOO owned brilliant selectivity and specificity for ONOO.

3.4. The Response Speed and pH Effect

The reaction kinetics of HND-ONOO to ONOO was further accessed in PBS buffer (DMSO/PBS = 1 : 9, v/v, pH = 7.4). As shown in Figure 2(a), the fluorescence intensity was quickly improved and then achieved a plateau within 30 s, suggesting that HND-ONOO can fastly react with ONOO. Next, we added 20 µM ONOO to verify, and similar experimental phenomena have been obtained. These results demonstrated the strong potential of the probe for real-time monitoring of ONOO in cells. Subsequently, we investigated the effect of pH on HND-ONOO via fluorescence intensity fluctuations of HND-ONOO in the absence or presence of ONOO ranging from 3.0 to 10.0. In the absence of ONOO, the fluorescence signal of HND-ONOO was weak and remained steady under the tested pH range. However, the fluorescence signal of HND-ONOO increases rapidly and remains constant under physiological pH (Figure 2(b)). The above result revealed that the probe HND-ONOO suitable for detecting trace amounts of ONOO in physiological environments.

3.5. Imaging of ONOO in Living Cells

Encouraged by the excellent spectral properties of HND-ONOO, the ability of the probe HND-ONOO to trace ONOO in biological systems was further investigated. Initially, the Cell Counting Kit-8 (CCK-8) experiment was carried out to evaluate the cytotoxicity of the probe HND-ONOO. Herein, RAW 264.7 cells and HeLa cells were checked as study subjects. As shown in Figure S5, even when the concentration of the probe was as high as 50 µM, the cell viability was still more than 85%, which suggested the probe HND-ONOO had lower cytotoxicity. It can be applied to detect the content of ONOO in living cells because of probe exhibits low cytotoxicity, high sensitivity, and specificity. Therefore, we further accessed the application of the probe for detecting ONOO in HeLa cells and RAW 264.7 cells. Herein, the HeLa cells were pretreated with 3-morpholino-sydnonimine (SIN-1, an ONOO donor, 100 μM, 250 μM, and 500 μM) for about 30 min before imaging. A weak fluorescence signal was obtained for the control group that has not been processed. However, we observed the obvious fluorescence signal enhancement when these cells that were stained by the probe were treated with 100 μM SIN (Figure 3). Furthermore, to verify whether the fluorescence fluctuation with the change of ONOO content, we added different concentrations of SIN to study the changes in the fluorescence signal. As we expected, the fluorescence intensity enhanced in existence 250 μM and 500 μM of SIN, which indicated that the probe can be employed to analyze fluctuations in intracellular ONOO levels. This content has been added accordingly to the manuscript and Supplementary Materials.

Next, we investigated whether the probes responded to possible interferers in living cells. The RAW 264.7 cells were only treated with HND-ONOO, and the cells had no obvious changes in fluorescence signal. However, when the RAW 264.7 cells were pretreated with 3-morpholino-sydnonimine (SIN-1, an ONOO donor) for around 30 min and then incubated with HND-ONOO (10 µM) before imaging, a significantly enhanced fluorescence signal was obtained (Figure 4). Subsequently, the cells were pretreated with H2O2, HClO, and NOC-18 (a NO donor) and then incubated with HND-ONOO before imaging, respectively. As expected, a weak fluorescence signal was found, once again proving that the change in the fluorescence signal of the probe was caused by ONOO rather than other species. The abovementioned experiments showed that the probe could be used for the detection of exogenous ONOO in cells.

Since the probe displayed the capability of detecting exogenous ONOO, it had the potential to image endogenous ONOO. The application of the probe HND-ONOO for monitoring endogenous ONOO was further researched. The assay was performed in RAW 264.7 cells because of the cells could generate high levels of inducible nitric oxide synthase (iNOS) after being stimulated by lipopolysaccharide (LPS) and interferon-γ (INF-γ) [44,45]. As the control group, the RAW 264.7 cell was pretreated with probe, and an inert fluorescent was observed (Figure 5(a)). However, a splendid fluorescence enhancement signal was found in RAW264.7 cells, after co-incubating with LPS/INF-γ, which suggested that the level of intracellular ONOO was increased in the stimulation of oxidative stress (Figure 5(b)). Next, the RAW 264.7 cell line induced by LPS/INF-γ was pretreated with aminoguanidine (a nitric oxide synthase (NOS) inhibitor AG, 0.5 mM), then treated with HND-ONOO. The falling fluorescence signal was found, which revealed that AG inhibited the generation of NOS and further reduced the level of ONOO (Figure 5(d)). Similarly, the superoxide scavenger 2,2,6,6-tetramethylpiperidine-N-oxyl (Tempol) was used to verify whether its fluorescence signal was caused by ONOO because the generation of ONOO is inseparable from the participation of O2 (Figure 5(c)). A weaker fluorescence experimental phenomenon was obtained, suggesting the levels of ONOO was inhibited. Meanwhile, it also proved that intracellular fluorescence signal caused ONOO rather instead of ROS. Finally, the intracellular fluorescence signal was obviously inhibited when an ONOO scavenger (uric acid) was added (Figure 5(e)). All the above result evidenced that HND-ONOO can monitor fluctuations of endogenous and exogenous ONOO in living cells.

4. Conclusions

In summary, we proposed a novel fluorescent probe HND-ONOO was employed to track intracellular ONOO contents. The probe has excellent specificity and sensitivity and responds rapidly to ONOO over various relevant analytes. The lower LOD and great linearity of the probe to ONOO provide a certain basis for the analysis and detection of ONOO. Furthermore, its low biocompatibility cytotoxicity will benefit the monitoring of ONOO under imaging studies and complex biological conditions. Furthermore, the proposed probe HND-ONOO was applied for imaging and detecting exogenous and upregulation of ONOO via LPS stimulation in living cells. Therefore, these favorable properties of HND-ONOO to ONOO make it promising for bioimaging research, in particular, to study its relationship with inflammatory diseases.

Data Availability

The all data used to support the findings of this study are included within the article.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Xiaofeng Wang and Shu Xiao contributed equally to this work.


This work was supported by the Natural Science Foundation of Hainan Province (820MS141).

Supplementary Materials

Structure characterizations of the probe, additional fluorescence spectra, and images. (Supplementary Materials)