Fluorescent probes for sensing peroxynitrite: biological applications

ABSTRACT

Peroxynitrite (ONOO^-) is a quintessential reactive oxygen species (ROS) and reactive nitrogen species (RNS), renowned for its potent oxidizing and nitrifying capabilities. Under normal physiological conditions, a baseline level of ONOO^- is present within the body. However, its production escalates significantly in response to oxidative stress. ONOO^- is highly reactive with various biomolecules in vivo, particularly proteins, lipids, and nucleic acids, thereby playing a role in a spectrum of physiological and pathological processes, such as inflammation, cancer, neurodegenerative diseases, and cardiovascular diseases. Consequently, detecting ONOO^- in vivo is of paramount importance for understanding the etiology of various diseases and facilitating early diagnosis. Fluorescent probes have become a staple in the identification of biomolecules due to their ease of use, convenience, and superior sensitivity and specificity. This review highlights the recent advancements in the development of fluorescent probes for the detection of ONOO^- in diverse disease models and provides an in-depth examination of their design and application.

1. Introduction

Peroxynitrite (ONOO^-) is a typical reactive oxygen species (ROS) or reactive nitrogen species (RNS) with strong oxidizing as well as nucleophilic properties. As one representative ROS, ONOO^- has high activity in living systems. It is part and parcel of the physiological and pathological processes of living organisms. ONOO^- is primarily derived in mitochondria, and is a downstream toxic product converted from the combination of nitric oxide (NO) and superoxide anion (O₂•^-), which is characterized by a high reactivity and a short life span (∼10 ms) [1–4]. On the one hand, it is attributed to the outstanding oxidizing and nitrifying ability of ONOO^- itself, which can diffuse freely through the bilayer of phospholipid membranes and react directly with target molecules, resulting in mitochondrial dysfunction and ultimately leading to cell death. On the other hand, ONOO^- can be protonated to produce peroxynitrous acid (ONOOH), which further breaks down to form highly reactive •OH and NO₂•, in most cases hazardously. Besides, ONOO^- reacts with CO₂ to form the unstable intermediate nitrosoperoxycarbonate (ONOOCO₂^-), which is further regenerated into CO₃^-, which then reacts with the target molecule to cause apoptosis or programed cell death.

ONOO^- can cause harm to various important biomolecules such as proteins, lipids, and nucleic acids, bringing on DNA single-strand breaks and base modifications, protein oxidation and nitration, and lipid peroxidation [5–11]. Therefore, ONOO^- has become an essential susceptibility contributor to many diseases, and the overproduction of peroxynitrite can lead to different diseases, including inflammatory diseases, cancers, neurodegenerative diseases, and cardiovascular diseases. But then, the presence of ROS in vivo is critical for organismal function, and the hyperresponsiveness of ONOO^- is also instrumental in cellular signaling processes, e.g. regulating signaling pathways through the nitration of key proteins. In addition, ONOO^- has antimicrobial properties that can be proliferated when macrophages resist invading pathogens (e.g. bacteria), thus conducive to killing these pathogens and serving to protect cells and organs. Hence, it is important to monitor ONOO^- change in biological systems [12–14].

Conventional ONOO^--detection methods, such as electrochemistry [15], UV-Vis absorption spectroscopy [16], electron paramagnetic resonance spectroscopy [17], and chemiluminescence [18], are analytical tools that usually do not allow direct measurements in living systems due to some of the characteristics of ONOO^-, such as immediacy, low lifetime, and low in vivo concentrations. In addition, diagnostic indicators for some diseases, such as blood test markers, may be affected by other diseases, and thus have no specificity and are not able to provide real-time early detection of the disease as a manifestation of the disease [19,20]. Conventional imaging methods such as magnetic resonance imaging (MRI) [21], computed tomography (CT) [22], and ultrasound [23] have limited application due to their high cost, time-consuming, and low sensitivity. Currently, fluorescent probes have increasingly attracted the attention of researchers and turned into an expedient tool for the research of biologically active molecules because of their non-invasive, highly sensitive, highly selective, real-time detection in situ, simple and easy-to-operate, and rapid imaging advantages [24–28]. Three general principles are used for the development of fluorescent probes, which are near-infrared (NIR) fluorescent probes, two-photon (TP) fluorescent probes, and ratiometric fluorescent probes. NIR fluorescent probes are characterized by good photostability, deeper tissue penetration, minimized background fluorescence, and less photodamage [29–32]. High resolution, high throughput, and deep imaging depth show TP fluorescent probes to have good advantages [33–36]. Ratiometric fluorescent probes have a better ability to avoid interference and have better selectivity and sensitivity [37–40]. ONOO^- plays a part in multifarious diseases, and in recent years, miscellaneous fluorescent probes for recognizing ONOO^- have been created and constructed. This review will summarize the ONOO^- probes that have been used in different disease models in recent years (Table 1).

Table 1.

Probes characteristics in this review.

Probes	λex /λem (nm)	Feature	Recognition group/ fluorophore	Emitting color	LOD (nM)	Cell/animal models	Ref.
1	λex = 525 λem = 590	Turn-on	Diphenyl phosphate Resorufin	Red	238	RAW264,7 Inflammation	[41]
2	λex = 620 λem = 660	NIR Turn-on Two-photon	Rhodamine analogue	Red	15	HeLa RAW 264.7 Inflammation	[44]
3	–	CL	Borate	–	201	RAW 264.7 Inflammation	[45]
4	λex = 400/440 λem = 450/520	Ratiometric (ONOO-/ ClO-)	Borate	Blue/Green	21/17	RAW 264.7 Inflammation	[51]
5	λex = 375 λem = 548/662	NIR Ratiometric	Rhodamine	Red/Green	47	RAW 264.7/HepG-2 Inflammation/peritonitis/tumor	[22]
6	λex = 610/670 λem = 705	NIR Turn-on	Borate Hemicyanine dye	Red	56	RAW 264.7/HepG2 Inflammation/tumor	[57]
7	λex = 450/615 λem = 530/670	Turn-on Dual-channel (ONOO-/GSH)	1,8-naphthalimide	Green/red	89.2/5.36 × 103	RAW 264.7/MCF-10A/MCF-7/MDA-MB-231	[58]
8	λex = 645 λem = 696	NIR Turn-on	Borate Si-rhodamine	Red	10	U251/HT22/U87-MG Gliomas	[61]
9	λex = 380/340 λem = 606/418	Two-photon (ONOO-/Aβ)	Oxindole Naphthalimide	Blue/green	–	PC12 AD	[62]
10	λex = 445 λem = 565	Two-photon Turn-on	Oxindole Naphthalimide	Green	–	PC12 AD	[64]
11	λex = 572 λem = 700	NIR Turn-on	Aminophenol Rhodol	Red	3.4	PC12/bEnd.3 AD	[65]
12	λex = 511 λem = 670	NIR Turn-on	Aminophenol Dicyanoisophorone	Red	4.59	PC12/SH-SY5Y PD	[70]
13	λex = 345 λem = 538/656	Ratiometric Turn-on	Borate	Green	270	PC12 Epilepsy	[78]
14	λex = 520 λem = 685	Two-photon NIR Turn-on	Diphenyl phosphinamide Dicyanomethylene-benzopyran	Red	151	RAW 264.7/HT22 Epilepsy	[79]
15	λex = 650 λem = 690	NIR Turn-on	Borate Methylene blue	Red	149	HepG2 AMI	[85]
16	λex = 670 λem = 725	Nanoprobe NIR Turn-on Ratiometric	Xanthene	Red	85	H9c2 IR injury	[86]
17	λex = 800 λem = 470/660	Two-photon (ONOO-/O2•-)	Cy5	Blue/red	6.09/6.38	HL-7702 HIRI	[81]
18	λex = 580 λem = 710	NIR Turn-on	Diphenyl phosphinate Cyanine	Red	78.40	HepG2 DILI	[88]
19	λex = 432 λem = 560	Turn-on	Aminophenol Dicyanoisophorone	Green	130	LX-2 ALI	[91]
20	λex = 355 λem = 560	Turn-on	Phenylglyoxylic acid Chalcone	Green	31	MCF-7 Drug-damaged liver	[92]
21	λex = 405/600 λem = 460/644	Two-photon (ONOO-/GSH)	CD	Red/green	15/840	HepG2 ALI	[94]
22	λex = 465/590 λem = 505/710	Two-photon (ONOO-/ Viscosity)	Coumarin	Red/green	67	Hela/RAW264.7/HepG2 NAFLD	[98]
23	λex = 683 λem = 766	NIR Turn-off Reversible	Imine	Red	418/280 × 103	HK-2 AKI	[101]
24	λex = 490 λem = 632	AIE Turn-on	Borate	Red	10	HeLa RA	[106]

2. ONOO^- fluorescence probes for inflammation

Peroxynitrite interferes with downstream signaling by oxidizing or nitrating receptors and molecules, which is likely to play a part in cellular inflammatory responses. Furthermore, the inflammatory environment favors the biological generation of ONOO^-, which reacts with inflammatory mediators, interleukins for instance, during inflammation and thus modulates the inflammatory response [2].

Su H, Wang N, Wang J, et al. devised and synthesized a newfangled resorufin-based fluorescent probe 1 with a phosphate recognition group for the sense of ONOO^- [41]. Probe 1 is comprised of three parts: (1) the resorufin part, acting as a fluorophore; (2) diphenyl phosphate, a specific trigger that reacts with ONOO^-, and (3) benzyl ether, which is a self-immolation linker, quenching the fluorescence of the resorufin (Scheme 1). Resorufin has great water solubility, featuring with long wavelength, and its 7-hydroxy alkylation induces efficient fluorescence quenching [42]. The absorption of 1 reaches a peak at 469 nm, while there is minimal fluorescence intensity at 590 nm. When treated with ONOO^-, there could be seen a red shift of the maximum absorption band from 469 nm to 585 nm. Probe 1 showed high selectivity to ONOO^-, especially compared to NaClO and H₂O₂. With a concentration of 20 μM, probe 1 displayed low cytotoxicity in living cells. Then 1 took effect in endogenous ONOO^- imaging in RAW264.7 cells (mouse leukemia cells of monocyte-macrophage) and lipopolysaccharide (LPS)-induced inflammation in mice. RAW264.7 cells can be stimulated by LPS and the pro-inflammatory cytokine interferon-gamma (IFN-γ) to produce endogenous ONOO^- [43]. RAW264.7 cells showed relatively weak red fluorescence when incubated with 1 alone, however, probe 1 showed significant red fluorescence after LPS and INF-γ treatment, suggesting that probe 1 can detect endogenous ONOO^- at the cellular level. Then, mice were injected LPS in the left leg subcutaneously to conduct an inflammation mice model. After 12 h, probe 1 was injected subcutaneously, incubating for another 0.5 h. The fluorescence intensity of LPS-induced inflammation tissues was significantly enhanced compared with normal tissues. Moreover, the fluorescence intensity was up to a maximum within 15 min, implying that probe 1 can rapidly monitor endogenous ONOO^- in inflammatory mice.

Scheme 1.

Structures of ONOO^- fluorescence probes for inflammation.

Mao GJ, Gao GQ, Dong WP, et al. developed a rhodamine analog that owns near-infrared emission together with a TP absorption cross-section (54 GM) by extending the conventional rhodamine π-conjugated system, based on which a NIR fluorescent probe featuring with two-photon excitation for detecting ONOO^- was developed [44] (Scheme 1). Probe 2 was eminently selective and sensitive to ONOO^-. The cell survival rate was above 89% after treatment with a concentration gradient of probe 2 (0–30 μM) for 24 h, denoting that 2 has good biocompatibility. Next, utilizing SIN-1 (3-morpholinosydnonimine, an ONOO^- generator) and UA (uric acid, an ONOO^- scavenger) to image exogenous ONOO^- in HeLa cells (human cervical cancer cells). Enhanced fluorescence signals were observed in HeLa cells with SIN-1 treatment, and the fluorescence signals decreased in UA-treated HeLa cells. Similarly, RAW 264.7 cells were monitored for endogenous ONOO^- with LPS and IFN-γ treatment. The probe 2-treated RAW 264.7 showed very weak fluorescence, which was gradually enhanced after treatment with different doses of LPS and IFN-γ. Moreover, the mice pretreated with LPS and phorbol 12-myristate 13-acetate (PMA) showed elevated fluorescence, suggesting that 2 can track endogenous ONOO^- and assess inflammation in animal models.

Deng Y, Shi X, Hu X, et al. designed a chemiluminescent probe 3 [45]. Since the microenvironment of inflammatory tissues is relatively acidic, it is worth designing a tool to imagine ONOO^- under weakly acidic conditions [46]. The borate structure, which has excellent responsiveness to ROS/RNS, is extensively used in designing probes. ONOO^- is a powerful oxidizing agent, reacting with borates much faster than other oxidizing agents [47]. Probe 3 is a borate-based chemiluminescence (CL) probe that is utilizable for the efficient detection of ONOO^- in both vitro and vivo. Probe 3 includes three portions, (1) a specialized site for ONOO^-: 2-hydroxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, (2) p-hydroxybenzyl alcohol (HBA), and (3) acrylonitrile-substituted Schaap's adamantylidene dioxetane (SAD). These three components enable 3 to differentiate between inflamed and surrounding normal tissues, increasing the quantum yield. Besides, they contribute to prolonging the luminescence time of 3 [48–50]. The cytotoxicity of 50 μM probe 3 remained low. The monitoring of endogenous ONOO^- level was also studied in RAW264.7 cells. LPS and IFN-γ pre-stimulated RAW264.7 cells for 12 h, then PMA was added to generate endogenous ONOO^-, and after incubating for 30 min, 10 μM of 3 was added. The real-time CL signal showed that LPS/IFN-γ and PMA significantly enhanced the CL signal of 3, which was 3.2 times stronger than that of the blank control group. Thus, probe 3 was able to monitor endogenous ONOO^-, which enabled the effective detection of inflamed tissues. Similarly, an inflammatory mouse model was established to verify that probe 3 could rapidly localize early inflammatory tissues.

The redox environment in organisms is very complex, and many highly reactive ROS and RNS interact with each other. Huang T, Yan S, Yu Y, et al. designed and synthesized one dual-responsive ratiometric fluorescent probe 4 to detect ONOO^- and hypochlorite (ClO^-) separately and simultaneously [51]. The two major parts of 4 are phenothiazine-based coumarin, responsive to ClO^-, and the precursor of 2-(benzo[d]thiazol-2-yl) aniline, a specific detector for ONOO^- (Scheme 1). Probe 4 itself emits red fluorescence at 640 nm, however, after treatment of ONOO^- and ClO^-, the original fluorescence peak at 640 nm disappears and transforms into green at 450 nm and blue at 520 nm, so the fluorescence intensity ratio of ONOO^- and ClO^- can be determined based on the 450/640 nm and 520/640 nm fluorescence intensity ratio for ONOO^- and ClO^- detection. At a high concentration of 10 μM, probe 4 was almost non-toxic after 24 h of interaction with RAW 264.7 cells. LPS and PMA were used to establish an inflammation cell model likewise. The control group showed obvious red fluorescence signals, whereas the inflammatory cell group showed strong blue and green fluorescence, and there was no red fluorescence, suggesting that 4 can visualize ONOO^- and ClO^- in inflammatory cells. Next, to further confirm that the changes in fluorescence signaling of inflammatory cells were caused by overexpression of ONOO^- and ClO^-, inflammatory cells were pretreated with the ROS scavenger, N-Acetylcysteine (NAC). The results showed only significant red fluorescence without blue and green signals, suggesting that probe 4 can track endogenous ONOO^- and ClO^- individually and concurrently by different fluorescent channels without any mutual interference. Finally, the LPS/PMA-induced inflammation zebrafish model was established to monitor endogenous ONOO^- and ClO^-, and the experimental results denoted that 4 can monitor the flux of ONOO^- and ClO^- in vivo.

3. ONOO^- fluorescence probes for tumor

Malignant tumors are notoriously lethal diseases, and the redox microenvironment of tumor tissues is much more complex compared to normal tissues, and their pathological processes are closely related to ROS overexpression [52]. ONOO^- is one of the important elements of the tumor microenvironment playing a part in tumor development as well as the redox environment and a preponderance of evidence suggests that ONOO^- is a potential biomarker for tumors [53,54]. ONOO^- is abundant in tumors and acts as an important tumorigenic molecule, consequently, in vivo monitoring of ONOO^- holds great importance for the diagnosis and treatment of tumors and, furtherly, for the improvement of survival rate. Due to the insidious symptoms of early-stage tumors, the application of some traditional detection methods is often limited as a result, and the detection of early-stage tumors is greatly hindered. Fluorescence imaging, as a non-invasive and highly sensitive technical tool, can be used for diagnostic localization of tumors by identifying specific molecules. (Scheme 2)

Scheme 2.

Structures of ONOO^- fluorescence probes for tumor.

Li Z, Lu J, Pang Q, et al. designed and constructed a NIR ratio-based fluorescent probe 5, which is non-destructive, featuring low background fluorescence interference and deep-tissue penetration, for monitoring fluctuations of ONOO^- during tumor progression [22]. Probe 5 is based on a modified rhodamine scaffold and 4-(methylthio)benzaldehyde. Probe 5 has a large Stokes shift, which is more than 150 nm, reducing crosstalk and quenching caused by overlapping of excitation and emission spectra. HepG-2 cells (human hepatocellular carcinomas) were implanted into the axilla of nude mice to establish a xenograft tumor mouse model. Probe 5 (1 mM, 10 μL) was injected intratumorally on days 5, 8, 11, and 14 of HepG-2 loaded tumor mice, respectively. The results showed that the tumor region emitted a 4.5-fold higher fluorescence ratio value than the control group 5 days after HepG-2 cell implantation. In addition, the green fluorescence brightened with tumor growth. Within the size of 0.463∼3.053 cm, the specific fluorescence signal in the tumor area also enhanced with the development of tumor volume. These results confirm that endogenous ONOO^- levels are significantly elevated during tumorigenesis. Probe 5 can quantify and visualize the elevation of ONOO^- in tumors. Therefore, probe 5 has great potential for tumor diagnosis and evaluation of the efficacy of tumor therapy.

ONOO^- is closely related to breast cancer, one of the most pervasive invasive cancers, as well as the second leading cause of cancer deaths in women, and its incidence and mortality rates are rising rapidly [55,56]. Accordingly, the development of a technique to effectively recognize ONOO^- is crucial to understanding its role in breast cancer. Xu Z, Xu Z, and Zhang D presented an ultrafast NIR fluorescent probe 6, which can detect ONOO^- in less than 30 s [57]. Probe 6 consists of two parts, (1) hemicyanine dye (Cy-OH) and (2) phenylboronic acid ester derivative, a specific recognition moiety for ONOO^-. After the treatment of ONOO^-, probe 6 rapidly cleaves the boronic ester group and shows an NIR fluorescent signal at 705 nm due to the ICT mechanism. The 4T1 cells (murine mammary carcinoma) were implanted in situ into mice to establish an implant tumor model. After injection of the probe solution, there was stronger fluorescence in the tumor site than in the normal site, indicating that 6 has great performance in tumor imaging. Probe 6 can be used to evaluate ONOO^- content at the cellular level, additionally, it can be considered to be a potential imaging method to perceive the pathophysiological role of ONOO^- in breast cancer.

Luo X, Zhang C, Yuan F, et al. designed a new dual-channel fluorescent probe 7 with mitochondria-targeted ability, which is capable of tracking ONOO^- and glutathione (GSH) to precisely differentiate between non-tumorigenic, malignant as well as metastatic breast cells when co-cultured [58]. Three moieties were used to synthesize probe 7, namely (1) 1,8-naphthalimide derivative with 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-ylidene)malononitrile (TCF), as an ONOO^- responsive site, (2) and 2,4-dinitrobenzenesulfonyl, as a GSH responsive site, and (3) triphenylphosphine, as a mitochondrial targeting group. Under different excitation wavelengths, probe 7 exhibits different emission wavelengths for GSH and ONOO^- (green at 530 nm, and red at 630 nm, respectively). MCF-10A (normal breast cells, non-tumorigenic), MCF-7 (malignant breast cancer cells, non-invasive), and MDA-MB-231 (metastatic cells, highly invasive) were selected for this study. Then the three types of cells were co-cultured and the fluorescence imaging showed that the blend of the three cell lines had similar results as every cell line alone, suggesting that probe 7 can recognize different mammary cell lines in the mixture. Therefore, probe 7 could be perceived as a fluorescent tool for the early diagnosis of breast cancer.

Although it has been reported that ONOO^- is a reliable biomarker for tumors and may act as an important tumorigenic molecule, it remains to be investigated whether ONOO^- is upregulated in gliomas. As a principal primary brain tumor in adults, gliomas have a poor prognosis [59]. Although the presence of ONOO^--modifying protein footprints has been observed in human malignant gliomas [60], there has been no direct observation of ONOO^-. To track ONOO^- in gliomas, it is a prerequisite to develop a tool able to detect ONOO^- at multiple scales in gliomas. No probe has been used for glioma pathology, not even for multiscale practicals in the past. To predict blood–brain-barrier permeability and screen for Si-rhodamine fluorophores, Wu XY, Shen YK, Tan SY, et al. evaluated the CNS MPO (Central Nervous System Multiparameter Optimization) scores of serial NIR fluorophores, employing the arylboronate structure to be an ONOO^--response trigger and synthesizing a NIR fluorescent probe 8 to track ONOO^- in a variety of glioma-related samples efficiently and selectively [61]. Probe 8 has negligible cytotoxicity and good biocompatibility. In in vitro experiments, the fluorescence response was observed to stabilize within seconds. Besides, half of the maximal response could be reached in 18 s. First, this study compared ONOO^- levels in different cells, including primary astrocytes, HT22 cells (hippocampal neurons), U87-MG cells (glioma cells, low-grade malignancy), and U251 cells (glioma cells, high-grade malignancy). Those four cell types all exhibited significant fluorescence, among which U87-MG and U251 showed pronouncedly radiant expression and co-localization with mitochondria. Next, a glioma model of nude mice was established with in situ implantation of U87-MG, and glioma imaging was performed in live mice. The fluorescence signals were considerably elevated in the tumor-occupied part contrasting with the normal part. In addition, the fluorescence of 8 in live mouse gliomas was sufficiently stable for more than 12 h without significant attenuation, providing a basis for the follow-up application in multiscale imaging. Finally, the potentiality of probe 8 to inspect ONOO^- in different clinical specimens was investigated. Probe 8 was injected into freshly excised glioma specimens, and the fluorescence persisted for 3 days at least. The multiscale imaging and dynamic monitoring of ONOO^- by probe 8 in different glioma-related specimens can be used for the detection of gliomas.

4. ONOO^- fluorescence probes for neurodegenerative disease

In addition to inflammation and tumors, ONOO^- is also closely related to neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), etc. due to ONOO^- contributes to inducing neurotoxicity and neuroinflammation [62].

As the most prevalent form of dementia, AD is a classic neurodegenerative disease. With the aging of the population, its prevalence and impact on society are increasing worldwide and thus receiving more and more attention. The clinical distinctiveness of AD is reflected in loss of memories, gradual mental decline, verbal communication disorders, disorientation disorders, ambulation disorders, and personality and behavioral changes. Apart from amyloid-β (Aβ) and Tau aggregates, ONOO^- is also a biomarker of AD [63]. In recent years, fluorescence imaging has increasingly drawn researchers’ attention due to its high sensitivity, noninvasiveness, and real-time response. Fluorescent probes targeting AD biomarkers will be of great significance for AD diagnosis and pathology analysis. (Scheme 3)

Scheme 3.

Structures of ONOO^- fluorescence probes for neurodegenerative disease.

Xie XL, Liu GZ, Niu YX, et al. utilized oxindole-functionalized benzothiazole-naphthalene derivative, BTNP, to design a fluorescent probe 9 to simultaneously detect and distinguish ONOO^- and Aβ [62]. Probe 9 consists of an ONOO^- response trigger (oxindole moiety) and a structure similarity to thioflavin T binding with Aβ plaque, thus having a dual-channel (blue for Aβ plaque, green for ONOO^-) for monitoring the distribution and changes of ONOO^- and Aβ plaque. This study verified three points: (1) Aβ aggregation induced neuronal ONOO^- overproduction; (2) ONOO^-accelerated Aβ aggregation; (3) ONOO^- appeared earlier than Aβ plaque. First, PC12 cells (pheochromocytoma) with Aβ treatment emitted brighter green fluorescence than untreated cells. The enhanced fluorescence of Aβ treatment PC12 cells decreased after incubation of the ONOO^- scavenger UA, suggesting that ONOO^- levels are upregulated in neurons during Aβ treatment. The green fluorescence emission of PC12 cells gradually increased over 12 h as the Aβ incubation time increased. This suggests that intracellular ONOO^- is overproduced when Aβ aggregates. On the other hand, intracellular green fluorescence was also amplified with the rise of Aβ concentration, indicating that the ONOO^- level was gradually up-regulated. Accordingly, the ONOO^- stress of neurons was positively correlated with Aβ stimulation. Next, to verify whether the overproduced ONOO^- collection would in turn accelerate Aβ aggregation, ONOO^- was incubated with Aβ monomers, and probe 9 was utilized to detect Aβ aggregation in the blue channel. The results showed that ONOO^--treated Aβ aggregated faster than untreated Aβ. The Aβ tyrosine residues were nitrated by ONOO^-, thus promoting Aβ aggregation. From the above, it can be seen that ONOO^--stress and Aβ aggregation can work together and there is a positive feedback mechanism between the two, boosting the occurrence and development of AD. Finally, the practicability of 9 in detecting ONOO^- and Aβ plaque was tested in the brain tissue of animal models. The log P value of probe 9 was 3.80, indicating its good blood–brain barrier penetration. Isolating and sectioning the hippocampal region of mice injected with 9, the results showed that there were relatively low levels of ONOO^- in the brain hippocampus of healthy mice, however, ONOO^- levels were elevated in mice with 2-month AD in the identical region and gradually increased with the age of the mice and the progression of AD. On the other hand, healthy mice as well as 2- and 3-month-old AD mice did not have significant Aβ plaque. In AD mice, Aβ plaque of the hippocampus began to appear at 4 months of age, which was markedly later than the increase of ONOO^-. Besides, both the size and number of Aβ plaque increased significantly with the mice's age. In conclusion, ONOO^- can be considered a promising AD biomarker that appears ahead of Aβ plaque during AD, which perhaps gives a new perception of AD early diagnosis.

Based on a naphthalimide fluorophore, Xie XL, Liu YW, Liu GZ, et al. designed an oxindole-functionalized two-photon fluorescent probe 10 in the following year [64]. Using this probe, Aβ stimulation-induced mass production of ONOO^- was observed in neurons, meanwhile, the connection between neuronal ferroptosis and ONOO^- stress was investigated. The experimental data implied that without Aβ treatment the fluorescence of PC12 cells was obviously weakened, and the fluorescence signal was enhanced by degrees with the addition of the Aβ dose. In addition, the fluorescence signal of cells pre-incubated with Aβ at separate time points progressively brightened in a time-dependent manner and then reached stable. These results showed that during the process of Aβ treatment, the ONOO^- level of neurons increased significantly, besides, the abnormal ONOO^- content counted on the dose and stimulation time of Aβ, suggesting neuronal oxidative stress was manner positively correlated with Aβ stimulation. Oxidative stress is a feature of ferroptosis, and to further verify the relationship between Aβ-provoked neurotoxicity and ferroptosis, antioxidant ferrostatin-1 (Fer-1) and iron chelator deferiprone (DFP), two specific inhibitors of ferroptosis, were used to explore whether Aβ provokes neuronal ferroptosis. The results showed that Aβ-induced fluorescence of PC12 cells was markedly declined after Fer-1 or DFP pretreatment, implying that the ONOO^- upregulation and oxidative stress induced by Aβ are related to ferroptosis.

Based on the reaction between 4, 4’-azanediyldiphenol and the NIR emissive rhodol fluorophore, Wang PZ, Yu L, Gong JK, et al. developed an ONOO^--activated NIR emitting probe 11 to explore the association of ONOO^- and AD for diagnosis and assessment of AD progression [65]. Probe 11 is the first reported fluorescent probe that can track ONOO^- fluctuations in the AD mouse brain. Probe 11 has a maximum absorption at 564 nm and shows noticeable NIR emission at 698 nm. The Stokes shift is 134 nm. Metal ions can induce oxidative stress, making disruption of metal ion homeostasis in vivo affiliated with the development of AD [66]. By adding Fe²⁺ to PC12 cells, AD cell models were formed. With the increase of Fe²⁺ concentration (0-8 mM), the fluorescence was gradually enhanced, indicating that ONOO^- was produced during Fe²⁺-induced oxidative stress. Moreover, the fluorescence signal was dramatically decreased after the addition of UA, suggesting that UA effectively reduced the intracellular ONOO^- level. Thus, probe 11 could monitor the changes in ONOO^- level in the AD cell model. The log P value of 11 was 2.1, suggesting that it has a good blood–brain barrier (BBB) penetration. Clioquinol (CQ) and curcumin (CUR), common chemicals for AD treatment, were used to alter the expression levels of ONOO^- in APP/PS1 mutated transgenic mice brains. The fluorescence intensity of APP/PS1 mice was significantly elevated from 0–6 h and reached a peak and then remained stable from 6–11 h. On the contrary, only slight fluorescence signal enhancement was noted in the wild-type (WT) mice, thus suggesting that aggregation of ONOO^- is a trait of AD. Additionally, in the CQ or CUR-treated APP/PS1 mice, the weaker fluorescence signals of brains indicated that regulated ONOO^- level. Ex vivo near-infrared imaging of AD and WT brain showed that there was a much higher fluorescence in the AD brain. In addition, this study verified that there was Aβ plaque formation in AD brain tissue by thioflavin S (ThS) staining. TNF-α (tumor necrosis factor-alpha), IL-6 (interleukin 6), and IL-1β (interleukin-1β) were up-regulated in AD brain tissues compared to WT brain tissues as detected by ELISA (enzyme-linked immunosorbent assay), confirming the presence of neuroinflammation in AD brain. Western Blot results implied that iNOS (inducible nitric oxide synthase) expression was much higher in the APP/PS1 mice brains. It was then hypothesized that the agglomeration of Aβ in the AD brain contributed to neuroinflammation and then fostered iNOS activity, resulting in ONOO^- bursts in the AD brain. Moreover, age factors also matter in AD pathogenesis. Administration of 11 to 3-, 8-, and 12-month-old APP/PS1 mice resulted in a noteworthy elevation in fluorescence signaling with age increasing, providing proof for the link of accumulation of ONOO^- and age in AD brains. The development of probe 11 provides new insights for ONOO^- probe development and AD diagnosis.

Parkinson's disease (PD) is another common neurodegenerative disease that seriously hampers patients’ lives and is still incurable. Increasingly declined dopaminergic (DA) neurons in the substantia nigra (SN) region are the pathological badge of PD [67]. The mechanism of DA neuron deletion remains unclear, but past researches suggest that the neurotoxicity of overproduced ROS and RNS may be the cause of dopaminergic deficits [68]. Therefore, the monitoring of ROS and RNS helps the diagnosis and treatment of PD. Excess ONOO^- is reckoned as a key neurotoxic factor, which was associated with oxidative stress and neurodegeneration. Accordingly, ONOO^- is notable in the pathogenesis of PD, serving as a promising hallmark for early diagnosis of PD [69].

Sun Q, Xu J, Ji C, et al. designed and synthesized serial novel NIR fluorescent probes, of which probe 12 has outstanding sensing properties and BBB penetration, enabling the first visualization of the dynamics of ONOO^- in a variety of PD models [70]. Probe 12 consists of two parts: (1) dicyanoisophorone, a NIR fluorophore; (2) p-aminophenol, a common ONOO^- receptor, was integrated into the fluorescent moiety as an ONOO^- responsive site. The fluorescence of the dicyanoisophorone is properly quenched by the photoinduced electron transfer (PET) mechanism because of the p-aminophenol moiety, which is electron-rich. Upon treatment with ONOO^-, the p-aminophenol moiety is reacted to form benzoquinone so that the fluorescence recovered. Drosophila serves as a model for PD because of the absence of endogenous parkin (an E3 ubiquitin ligase) [71,72]. This study used the Drosophila PD model to investigate whether 12 can detect the connection between ONOO^- and parkin via tissue imaging. The fluorescence signal of parkin null Drosophila was much increased than that of WT Drosophila, indicating that parkin null Drosophila brains had higher ONOO^- levels. In addition, the fluorescence intensity was significantly enhanced after SIN-1 pre-stimulated WT Drosophila brain tissue, whereas the fluorescence intensity was significantly reduced after UA pre-stimulated parkin null Drosophila brain tissue. Next, to test the feasibility of 12 to discern ONOO^- in mammalian tissues, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model was created. Probe 12 was confirmed to be able to penetrate the blood–brain barrier by determining 12 concentrations in plasma and brain. To detect the level of ONOO^- in the SN region, brain slices of the substantia nigra region were imaged. Control mice showed weak fluorescence in the substantia nigra, whereas the nigrostriatal fluorescence of PD mouse slices was 7.14-fold stronger. After treatment with UA, the slices showed very weak fluorescence, indicating a decrease in ONOO^- levels. Finally, using WLZ3 C. elegans strains to establish PD models via LRRK2 (a signaling component, leading to neurodegeneration) [73] overexpression, indicating that the fluorescence of WLZ3 C. elegans was much stronger. After being treated with UA, the fluorescence was weaker than that of WLZ3 C. elegans. In conclusion, probe 12 is capable of recognizing ONOO^- in vivo, which can be used to understand the biological action of ONOO^- in PD.

Epilepsy is a complex chronic disease closely related to neurodegenerative pathology, which poses a great threat to human health [74]. The abundance of proof suggests the pathological progression of epilepsy is closely related to oxidative stress-generated ROS and RNS [75]. Therefore, it’s necessary to investigate the role of oxidative stress in epilepsy to fully understand the development of epilepsy. As an important reactive oxygen/nitrogen, ONOO^- overexpression can be a key marker for the early diagnosis of epilepsy [76]. The ONOO^- concentration in healthy brains is about 300 nM, while in the brain of epileptic patients, the concentration of ONOO^- is significantly elevated [77]. The role of ONOO^- in epilepsy has also attracted increasing attention from researchers.

Lu G, Fan H, Wang K, et al. developed fluorescent probe 13 which can rapidly and stably cross BBB for ONOO^- monitoring in kainite (KA)-induced epileptic brain [78]. Introducing an amphiphilic molecule, probe 13 was designed with a pyridine head and a dimethylamine rotor in the tail, followed by a relatively well-established borate as the recognizing moiety. Probe 13 is a ratiometric probe, and itself showed an absorption peak around 280 nm, while after being treated with ONOO^-, there are two distinct peaks at 275 nm and 450 nm. As excited at 457 nm, there was an emission peak of 13 at 656 nm. After reacting with ONOO^-, there was an emission peak at 538 nm when the excitation wavelength was 345 nm. The ratio of the fluorescence at 538 nm and 656 nm is an important mark of this probe. The study used a KA-induced mouse epileptic brain model. Confocal fluorescence images showed that 13 could be aggregated in the brains of mice in the KA-induced group 1 h after tail vein injection. The degree of fluorescence intensity at 2 h was higher than that at 1 h. This confirms that 13 can quickly and stably cross the blood–brain barrier in this model. In addition, whole rat brains were removed for imaging. The epilepsy group showed significantly higher fluorescence than the control group. In summary, probe 13 can effectively monitor ONOO^- levels in the epileptic mice brains, which can help better comprehension of epilepsy pathological mechanisms.

Luo X, Cheng Z, Wang R, et al. developed a NIR TP fluorescent probe 14, which tracked ONOO^- in an epilepsy rat model in real-time with good sensitivity and selectivity [79]. Probe 14 involves two parts, (1) dicyanomethylene-benzopyran fluorophore and (2) diphenylphosphinamide, a recognition structure. Probe 14 has a large Stokes shift, and 10 min after ONOO^- treatment, the phosphoramidic bond of the probe is interrupted, and the released amino group emits intense fluorescence at 685 nm owing to the resumption of the intramolecular charge transfer (ICT) process. Resveratrol (Res) is a small molecule with strong antioxidant properties [80]. HT22 cells (mouse hippocampal neurons) were separated into five groups, the control group, the KA group, and the KA + Res group treated with low, medium, and high doses of Res, respectively, and each group of HT22 cells was imaged after incubation for 12, 24, and 72 h, severally. The control group provided a weaker fluorescence signal, whereas there was a stronger fluorescence signal in the KA group, further indicating the increase of ONOO^- level after KA stimulation. When treated with different concentrations of Res, the fluorescence was almost invisible contrasted with the KA group, implying the lower content of ONOO^-. Moreover, the antioxidant effect of Res increased as the dose of Res increased. Flow cytometry analysis was subsequently performed, and the results were also consistent with the above. Next, the study established a KA-induced epilepsy rat model. The fluorescence signals in the control group were weak, indicating that the low-level ONOO^- in normal rats. Comparatively, the KA group emitted significantly enhanced fluorescence intensity at 12 h, showing the ONOO^- content was higher and confirming that there is an abnormal increase of ONOO^- level in epilepsy. The Res-treated groups showed relatively weaker fluorescence intensity than the KA group, indicating decreased ONOO^- level after being treated with Res. The above results indicate that probe 14 is capable of tracking ONOO^- flux in epilepsy rat model, which is helpful to further assist in the diagnosis of epilepsy. The Res has an effective inhibit ability on the overexpression and neuronal damage of ONOO^-, and has the potential to alleviate epilepsy.

5. ONOO^- fluorescence probes for ischemia-reperfusion injury

After an ischemic/hypoxic injury, once blood flow and oxygen supply are restored, reactive oxygen species increase dramatically, attacking biomolecules and causing cell death, and tissues and organs will be further injured, resulting in ischemia-reperfusion injury (IRI) [81]. Oxidative stress acts as a pivotal part of IRI [82]. Monitoring molecular components in IRI utilizing highly sensitive and selective fluorescence imaging has also become popular in recent years. (Scheme 4)

Scheme 4.

Structures of ONOO^- fluorescence probes for ischemia-reperfusion injury.

A sharp decrease in coronary blood supply leading to acute ischemia can cause myocardial necrosis, thus resulting in acute myocardial infarction (AMI) [83]. One optimum method to constrain the myocardial infarction size and ameliorate cardiac function at present is reperfusion to restore coronary blood flow in a timely manner [84]. However, myocardial IRI often occurs after blood supply is restored to the infarcted vessel. Therefore, real-time diagnosis at the early stage is significant to recognize and assess the gravity of myocardial IRI. After AMI, there are markedly increased ONOO^- levels in the myocardium. Hence, Lu JL, Wu YR, Zhan SY, et al. designed the NIR fluorescent probe 15 to track endogenous ONOO^- content in AMI [85]. Probe 15 uses methylene blue (MB), which has remarkable photophysical properties, good biocompatibility, and good pharmacokinetic properties, as a fluorophore. The borate acts as a recognition group for ONOO^-, which was attached to reduced 3,7-bis(diethylamino)phenothiazin-5-ium (REB). The weak absorption and emission signals of REB are restored upon reaction with ONOO^-. In this study, the cardiomyocyte ischemia/reperfusion model was conducted via oxygen-glucose deprivation/reperfusion (OGD/R). H9c2 rat cardiomyocytes were treated with hypoxia following reperfusion at different times. After being treated with probe 15, there was significant fluorescence in the OGD/R cells, indicating the overproduction of ONOO^- at different reperfusion time and ONOO^- overexpressing at the early stage of IRI. In addition, after treatment with NAC, H9c2 cells emitted weak red fluorescence, indicating the decrease of intracellular ONOO^- level. Next, the IR animal model was established. The fluorescence intensity of AMI rats increased in certain regions of the heart, demonstrating probe 15 can specifically recognize AMI injury. Subsequently, isolated hearts of IR rats with different reperfusion times were imaged, which showed that the fluorescence of the IR rat group was markedly enhanced, indicating that 15 can visualize early myocardial injury.

Shi AP, Zeng YL, Xin DX, et al. designed a highly sensitive ratiometric fluorescent nanoprobe 16 based on a NIR fluorophore xanthene, NOF5, to detect ONOO^- in situ for real-time assessment of antioxidant therapy for cardiac IR injury [86]. The NOF5 fluorescent dye was attached to silica cross-linked micelles (SCLMs), and Cy3 acted as an internal reference. Carvedilol (Car), atorvastatin (Ato), and resveratrol (Res), which were antioxidants, were used as model drugs to synthesize Car@SCLMs, Ato@SCLMs, and Res@SCLMs. In this study, a rat myocardial ischemia/reperfusion model was established. The sham group showed weak fluorescence, whereas, the reperfused heart of rats with ligated left anterior descending (LAD) emitted significant fluorescence, which was enhanced with time. The values of F_NOF5/F_Cy3 raised sharply within 10 min and reached stable after 60 min. Subsequently, the drugs were loaded in the nanoprobes, and the results implied the F_NOF5/F_Cy3 values were kept at a low level, indicating that all three drugs were able to prevent the ROS burst. Therefore, probe 16 can be used to evaluate the antioxidant effects of drugs.

Hepatic ischemia/reperfusion injury (HIRI) is a serious but inevitable complication of liver resection and liver transplantation [82]. Zhang W, Liu J, Li P, et al. designed a dual-color TP fluorescence probe 17 to monitor ONOO^- and O₂•^- in HIRI and revealed the relationship between O₂•^- and ONOO^- and arginase 1 in IR injury [81]. Probe 17 linked the caffeic acid group (CA) and Cy5 group with a piperazyl linker. Upon reaction with O₂•^-, the catechol of the CA residue was converted to benzoquinone, then, hydrogen supply of the reducing substance reduced the quinone to a phenolic structure, causing an enhancement of fluorescence. In addition, Cy5, which has a near-infrared-emitting, mitochondria-targeting property, reacts with ONOO^- and causes a fluorescence quenching. At 800 nm excitation, the CA and Cy5 moieties showed distinct dual-channel fluorescence, indicating O₂•^- and ONOO^-, respectively. HIRI cell and animal models were established via OGD/R. Compared with normal cells, there was a higher blue signal and a lower red signal in IR cells. In addition, the results of IR mice and normal mice were in accordance with that of the cell experiments. The above results imply that ONOO^- and O₂•^- show synergistic elevation during IR at the cellular level and in vivo. Moreover, since ELISA assay revealed that the increase of ONOO^- in IR injury contributed to the inactivation of arginase 1. Nomega-hydroxy-nor-arginine (nor-NOHA) was an arginase 1 inhibitor used to study the action of arginase 1 inactivation on ONOO^- levels. The Arginase 1-inhibited group showed a lower red signal than normal cells. Moreover, the signal of normal cells was significantly weaker after nor-NOHA treatment in IR cells. It was shown that inhibiting arginase 1 activity can help the increase of ONOO^- level.

6. ONOO^- fluorescence probes for other diseases

The liver is a multifunctional metabolic factory of the human body, while excessive or long-lasting use of drugs can result in liver damage. Drug-induced liver injury (DILI) is the injury caused to liver cells by drugs and their metabolites and is the main side effect associated with most drugs [87]. During DILI, ONOO^- levels are upregulated in the liver. The recognition of ONOO^- is important for liver injury treatment. Therefore, ONOO^- in DILI can be monitored with a simple fluorescent probe for early diagnosis of DILI in situ. Chai XZ, Li BH, Chen C, et al. reported a novel NIR probe 18, which is capable of efficiently monitoring the endogenous ONOO^- production in acetaminophen (APAP)-induced liver injury mice model [88]. Probe 18 contains a cyanine skeleton with diphenylphosphine (Scheme 5). Under physiological conditions, ONOO^- mediates hydrolytic cleavage of diphenylphosphine through an enhanced ICT mechanism, and the near-infrared emission at 710 nm of 18 was enhanced by about 200 folds. The absorption and emission peaks of the probe were 580 nm and 710 nm, respectively, and the Stokes shift was 130 nm, enabling noninvasive real-time monitoring of ONOO^- production in a DILI mice model.

Scheme 5.

Structures of ONOO^- fluorescence probes for other diseases.

Acute injury to liver tissue is common, and acute liver injury (ALI) manifests itself as a dramatic failure of hepatocyte function out of the past medical history of the liver [89,90]. To realize timely diagnosis of ALI has been a research hotspot these years. By combining p-aminophenol as the ONOO^- reaction site and dicyanoisophorone as the fluorophore, Jin C, Wu PF, Yang YS, et al. designed a fluorescent probe 19 for recognizing ONOO^- in ALI [91]. In this study, a CCl₄-induced ALI model mice as well as an APAP-induced hepatotoxicity model were established, which proved that probe 19 could stably reflex ONOO^- content in ALI.

Using chalcone fluorophore, Ling C, Cui MY, Chen JR, et al. synthesized a new type of fluorescent probe 20 to monitor ONOO^- ex vivo. With phenylglyoxylic acid as an ONOO^- recognition moiety and chalcone analog as the fluorophore, probe 20 showed a rapid response, maximum fluorescence intensity within 15 min, outstanding sensitivity, and good selectivity [92]. Probe 20 was effectively used to monitor ONOO^- in mice liver tissues with drug-induced hepatotoxicity.

In an earlier study, Jing CL, Wang YZ, Song XR, et al. synthesized 7-(4-diethylamino)-4-(2-carboxy) -phenyl)-2-(4-(piperazin-1-yl) phenyl) chromium (CD) as an ONOO^- recognition site and mitochondria targeting group [93]. The sulfoxide structure (NA) can be readily reduced by GSH independently of other thiols. Based on the above, Liu Y, Zhao JJ, and Wang YZ developed a TP fluorescent probe 21 to detect ONOO^- and GSH using a dual fluorophore and dual-site strategy [94]. Upon reaction with ONOO^- and GSH, the red signal of ONOO^- was diminished and the green signal of GSH was enhanced. This study produced ONOO^- indirectly by stimulating HepG2 cells with APAP to cause acute liver injury. Control cells incubated with 21 showed higher signals in both green and red channels. However, the red fluorescence was almost completely cleared and the green fluorescence was almost unchanged in the APAP group. In addition, a clear enhancement of red fluorescence and a stronger green emission were observed in the NAC group. Probe 21 provides an effective method to understand the oxidative-antioxidant equipoise and related functions of GSH and ONOO^- in living systems.

Nonalcoholic fatty liver disease (NAFLD) is becoming a paramount cause of liver disease [95], without timely diagnosis and treatment, NAFLD may develop into cirrhosis and even liver cancer. Studies have shown that ONOO^- and viscosity are highly correlated with liver injury and NAFLD. ONOO^- and viscosity are considered the potential biomarkers of NAFLD [96,97].

Liu YJ, Feng SM, Gong SY, et al. report a new probe 22 that can simultaneously recognize ONOO^- and viscosity in two channels with no crosstalk [98]. Probe 22 consists of (1) a benzo-quinoline coumarin unit, acting electron donor, (2) a quinoline cation unit, acting electron acceptor, and (3) a vinyl bond to connect (1) and (2). Probe 22 could co-localize with mitochondrion, featuring outstanding selectivity, quick response, and low cytotoxicity. Feeding mice via a high-fat diet and intraperitoneal injection of dexamethasone, an NAFLD model was established. The fluorescence reached its maximum in the hepatic region at about 2 min. After being treated with 22, there were stronger fluorescence signals of adipose tissue in both ONOO^- and viscosity channels than controls, showing that adipose tissue has more ONOO^- and higher viscosity.

Drug-induced acute kidney injury (DIAKI) is identified by rapid loss of organ function and high morbidity and mortality rates [99]. Oxidative stress is one principal factor leading to DIAKI [100]. Ding YT, Zhong RB, Jiang RF, et al. reported a reversible NIR probe 23 featuring with large Stokes shift. Probe 23 includes (1) a hydrogenated quinoxaline moiety and (2) an imine unit, Nile blue (NB), as the recognition site. Probe 23 is used to track ONOO^- and GSH in DIAKI stimulated with cisplatin (CP) only and CP combined with APAP and evaluate the efficacy of the therapeutic drug L-Carnitine (LC) [101]. Probe 23 itself had strong fluorescence, which was oxidized by ONOO^- after reaction with ONOO^-, and the fluorescence was weakened. Meanwhile, GSH could reduce it and restore fluorescence.

Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disease related to a variety of complicated factors, which is characterized by joint tumescence, acute ache, stiffness, synovitis, etc. [102–104]. Timely diagnosis and screening of RA are essential for the treatment of RA. ONOO^- has strong oxidizing and nitrating properties, and is associated with inflammation, tumorigenesis, autoimmune diseases, and other pathological processes [105], and may be closely associated with RA. Wang Z, Gong JK, Wang PZ, et al. developed the novel fluorescent probe 24 with aggregation-induced emission (AIE) property to detect ONOO^- in RA [106]. Binding phenyl-boronate, the ONOO^- sensitive reaction triggers the AIE part, and the excited-state intramolecular proton transfer (ESIPT) part to provide a new red-emitting fluorophore. Within 30 s, a markedly far-infrared fluorescence increase was observed at 632 nm under 490 nm excitation. A mouse model of rheumatoid arthritis was established by continuous administration of LPS or Complete Freund's Adjuvant (CFA) to mice, and probe 24 was injected subcutaneously. A rapid and notable fluorescence intensification was observed in RA mice 5 min after injection of 24, and reached a maximum at 15 min, followed by a gradual decrease after 30 min. This research showed that 24 can easily detect ONOO^- level in RA, providing a practical method for studying the critical function of ONOO^- in RA.

7. Summary and outlooks

In conclusion, the abnormal accumulation of ONOO^- in vivo is linked to a variety of pathophysiological processes and diseases. This review presents an overview of ONOO^- fluorescent probes that have been applied across various disease models in recent years, including inflammation, tumors, neurodegenerative diseases, ischemia-reperfusion injuries, hepatic and renal injuries, rheumatoid arthritis, etc. The probes are based on diverse response mechanisms, including ratiometric, two-photon, and near-infrared technologies. We provide a detailed account of their design principles, excitation and emission wavelengths, detection thresholds, and their application in biological contexts. (Table 1)

Although fluorescent probes exhibit good biocompatibility, high sensitivity, and selectivity, can be successfully applied to intracellular and extracellular ONOO^- imaging, and can be a potential method for studying the physiological and pathological role of ONOO^-, it seems that the development and design of ONOO^- probes still face big challenges. For example, the short emission wavelength. The fact that majority of fluorescent probes reported show short-wavelength fluorescence (<650 nm), in which case these probes are vulnerable to interference from the biomolecule's fluorescence. Moreover, the application of probes with small Stokes shifts is limited due to unavoidable spectral crosstalk, low resolution, and incompetence for deep tissue imaging. Other ROS including HOCl and H₂O₂ are also strongly oxidizing, making the detection of ONOO^- susceptible to other ROS. In addition, owing to the very short half-life of ONOO^-, there are strict requirements for the sensitivity of the probes to effectively monitor ONOO^- in real-time in a real physiological environment. Furthermore, for some brain diseases, such as brain tumors, neurodegenerative lesions, etc., it is required that the probes should feasibly cross the BBB to accomplish imaging in the specific brain region. Therefore, the design and synthesis of fluorescent probes with relatively large Stokes shift and fast response time for highly selective and sensitive monitoring of ONOO^- is essential for a thorough comprehension of the biochemical function of ONOO^- in vivo.

Abbreviations

AD

Alzheimer's Disease

AIE

Aggregation-Induced Emission

ALI

Acute Liver Injury

AMI

Acute Myocardial Infarction

APAP

Acetaminophen

Aβ

Amyloid-Βeta

BBB

Blood–Brain Barrier

CA

Caffeic Acid

CL

Chemiluminescence

ClO^-

Hypochlorite

CNS MPO

Central Nervous System Multiparameter Optimization

CP

Cisplatin

DA

Dopaminergic

DFP

Deferiprone

DIAKI

Drug-Induced Acute Kidney Injury

DILI

Drug-Induced Liver Injury

ESIPT

Excited-State Intramolecular Proton Transfer

Fer-1

Ferrostatin-1

GSH

Glutathione

HIRI

Hepatic Ischemia/Reperfusion Injury

ICT

Intramolecular Charge Transfer

IELISA

Enzyme-Linked Immunosorbent Assay

IFN-γ

Interferon-Gamma

IL-6

Interleukin 6

iNOS

Inducible Nitric Oxide Synthase

IRI

Ischemia-Reperfusion Injury

KA

Kainite

IL-1β

Interleukin-1 Beta

LAD

Left Anterior Descending

LC

L-Carnitine

LPS

Lipopolysaccharide

MB

Methylene Blue

MPTP

1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine

NAC

N-Acetylcysteine

NAFLD

Nonalcoholic Fatty Liver Disease

NB

Nile Blue

NIR

Near Infrared

NO

Nitric Oxide

O₂•^-

Superoxide Anion

OGD/R

Oxygen-Glucose Deprivation/Reperfusion

ONOO^-

Peroxynitrite

ONOOH

Peroxynitrous Acid

PD

Parkinson's Disease

PET

Photoinduced Electron Transfer

PMA

Phorbol 12-Myristate 13-Acetate

RA

Rheumatoid Arthritis

Res

Resveratrol

RNS

Reactive Nitrogen Species

ROS

Reactive Oxygen Species

SIN-1

3-Morpholinosydnonimine

SN

Substantia Nigra

ThS

Thioflavin S

TNF-α

Tumor Necrosis Factor Alpha

TP

Two-Photon

UA

Urine Acid

WT

Wild-Type

Cell lines

RAW264.7

Mouse Leukemia Cells Of Monocyte Macrophage

HepG-2

Human Hepatocellular Carcinomas

4T1

Murine Mammary Carcinoma

MCF-10A

Normal Breast Cells

MCF-7

Malignant Breast Cancer Cells

MDA-MB-231

Metastatic Cells

HT22

Hippocampal Neurons

U87-MG

Glioma Cells

U251

Glioma Cells

PC12

Pheochromocytoma

HT22

Mouse Hippocampal Neurons

H9c2

Rat Cardiomyocytes

HeLa

Human Cervical Cancer Cells

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.