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Abbreviation (ISO4): Prog Chem      Editor in chief: Jincai ZHAO

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Review

Construction and Application of ONOO- Small Molecule Fluorescent Probes in Pathophysiological Processes

  • Ting Ma ,
  • Chunyu Deng ,
  • Jie Li ,
  • Zhouyu Wang , * ,
  • Qian Zhou , * ,
  • Xiaoqi Yu , *
Expand
  • School of Science,Xihua University Sichuan Engineering Research Center for Molecular Targeted Diagnostic & Therapeutic Drugs Asymmetric Synthesis and Chiral Technology Key Laboratory of Sichuan Province,Chengdu 610039,China
*(Zhouyu Wang);
(Qian Zhou);
(Xiaoqi Yu)

Received date: 2024-09-04

  Revised date: 2025-01-20

  Online published: 2025-03-20

Supported by

the National Natural Science Foundation of China(22307104)

Sichuan Science and Technology Program(2023NSFSC0637)

Sichuan Science and Technology Program(2023NSFSC1977)

Abstract

ONOO-,produced by the diffusion-controlled reaction of nitric oxide and superoxide radicals,is a strong oxidizing and nitrating agent that causes damage to DNA,proteins,and other biomolecules in cells. Since ONOO- is characterized by its short lifetime,high reactivity,and low concentration under physiological conditions,and the pathophysiological roles it plays in biological systems are not yet fully understood,it is of great significance to develop highly sensitive and selective detection technologies to achieve real-time dynamic monitoring of ONOO-. In this paper,we review the research progress of ONOO- fluorescent probes in disease-related processes in the recent 5 years,revealing the potential role of ONOO- in various diseases,such as inflammation,tumors,liver injury,and brain diseases. Finally,the bottlenecks in the development of ONOO- probes and future trends are discussed,which will promote the application of ONOO- probes in chemistry,biology,and pharmacology.

Contents

1 Introduction

2 Design strategies of ONOO- fluorescent probe

3 Detection and imaging of ONOO- by fluorescent probes in disease-related processes

3.1 Detection and imaging of ONOO- in inflammation

3.2 Detection and imaging of ONOO- in tumors

3.3 Detection and imaging of ONOO- in liver injuries

3.4 Detection and imaging of ONOO- in brain diseases

3.5 Detection and imaging of ONOO- in other disease models

4 Conclusion and outlook

Cite this article

Ting Ma , Chunyu Deng , Jie Li , Zhouyu Wang , Qian Zhou , Xiaoqi Yu . Construction and Application of ONOO- Small Molecule Fluorescent Probes in Pathophysiological Processes[J]. Progress in Chemistry, 2025 , 37(4) : 519 -535 . DOI: 10.7536/PC240815

1 Introduction

Reactive oxygen species (ROS) are a series of oxygen-containing active metabolites that participate in maintaining redox homeostasis and regulating signal transduction, playing important roles in physiological and pathological processes. As one of the intracellular ROS, peroxynitrite (ONOO-) is a stronger oxidant and nucleophile than hydrogen peroxide (H2O2). It is generated in situ through a diffusion-controlled reaction between nitric oxide radical (·NO) and superoxide radical (O2·-) at a 1:1 stoichiometric ratio with a rate constant of approximately 1×1010 mol-1·s-1[1]. The generation rate of ONOO- in vivo can reach up to 50–100 μM·min-1. Due to its high chemical reactivity and tendency to protonate and rapidly decompose under physiological pH conditions (pKa = 6.8), the steady-state concentration of ONOO- is only within the nanomolar range[2]. Although ONOO- has a short half-life (< 20 ms), it can still cross cell membranes and affect microenvironments within a radius of 5–20 μm around its source.
As shown in Fig. 1, ONOO- directly or indirectly participates in the radical oxidation and nitration of biomolecules[3-4]. The conjugate acid of ONOO-, ONOOH, can directly decompose into OH and NO2, or convert thiol groups or cysteine residues of proteins into sulfenic acids through a two-electron oxidation mechanism, which represents a detoxification pathway for cells against oxidative damage caused by ONOO-. The metal centers of metalloproteins (e.g., cytochrome c, Mn/Fe SOD) can react with ONOO- to form highly oxidizing high-valent M-O complexes. Additionally, the high concentration of CO2 (~1.2 mM) inside cells can undergo nucleophilic addition with ONOO- to generate an unstable intermediate, ONOOCO2- (k = 5.8×104 mol-1·s-1), which rapidly homolyzes into NO2 and CO3•- radicals, or isomerizes into nitrate (NO3-). These secondary derivatives (M-O complexes, OH, NO2, CO3•-, etc.) can mediate further oxidation or nitration reactions, leading to protein modifications, DNA mutations, lipid peroxidation, and other outcomes.
图1 过氧化亚硝酰反应途径

Fig.1 Peroxynitrite reaction pathways

In the past few decades, it has been widely accepted in the field that under both physiological and pathological conditions, ONOO- can profoundly affect cellular metabolism by modifying specific biological targets and play a role in the onset and progression of various diseases, including cancer, neurodegenerative disorders, and cardiovascular diseases[4]. Tyrosine nitration is a classic example of ONOO--dependent protein modification. The nitration process involves the attack of tyrosine aromatic rings by one-electron oxidants (OH, CO3•-), forming tyrosyl radicals, which rapidly react with NO2 to generate 3-nitrotyrosine. It has been confirmed that protein tyrosine nitration can promote metabolic changes related to bioenergetics, proliferation, and stress responses. Signaling mediated by 3-nitrotyrosine involves disruption of specific metabolic pathways (e.g., blocking tyrosine phosphorylation cascades) or generation of conformational isomers with novel catalytic activities. Another possible mechanism is that 3-nitrotyrosine can mimic phosphorylated tyrosine and recruit SH2 domains to nitrated proteins, thereby promoting downstream events without requiring phosphorylation[5]. During tumor progression, nitrosative stress can regulate cell proliferation and chemoresistance or enable immune evasion by altering antigen presentation and cytokine signaling[6-8]. Recently, researchers from Oregon State University in the United States discovered that selective nitration of Hsp90 promotes tumor cell proliferation, and nitrated Hsp90 plays a key role in schwannoma metabolic reprogramming and cell proliferation, potentially serving as a novel target for tumor-targeted therapy[9]. Moreover, nitrated proteins have also been detected in multiple tumor types, including pancreatic ductal adenocarcinoma, breast cancer, colon cancer, bladder cancer, glioblastoma, and metastatic melanoma. In motor neurons, nitration of Hsp90 at either Y33 or Y56 activates the FAS/FAS-L-dependent apoptotic pathway; in undifferentiated PC12 cells, nitration of Hsp90 at Y56 promotes apoptosis by activating the purinergic receptor P2X7 (P2X7R) and downstream homologous phosphate and tensin homolog (PTEN), indicating differences in target sites and outcomes of ONOO- across different cell types[10-11]. In addition, ONOO⁻ also participates in processes such as aging, cardiovascular disease, microbial defense, and lung injury[3].
It is worth noting that pharmacological inhibition of ONOO- generation or degradation has been proven beneficial for various diseases, including inflammation, drug-induced liver injury, stroke, atherosclerosis, ischemia-reperfusion injury, and Alzheimer's disease[12-15]. Meanwhile, ONOO- is increasingly regarded as a biomarker for early disease diagnosis[16]. Nevertheless, the complex associations between ONOO- and various diseases remain incompletely understood, primarily due to the lack of accurate and reliable detection tools for in situ, real-time quantitative monitoring of ONOO- in specific pathological environments.
Compared with traditional analytical methods such as spectrophotometry[17-18], high-performance liquid chromatography[19], and electrochemical techniques[20], fluorescence imaging technology based on fluorescent probes offers prominent advantages including high sensitivity, rapid response, good selectivity, high spatiotemporal resolution, and non-invasiveness[21-24], and has gradually become one of the important tools for molecular monitoring in complex biological processes. In 2006, Yang et al.[25] reported the first highly selective ONOO- fluorescent probe based on the characteristic that aryl methyl ether-derived activated ketones can form dioxetane epoxide structures upon oxidation by ONOO-. A significant fluorescence enhancement was observed when the probe was co-incubated with neuronal cells and an ONOO- releasing agent; however, slight interference from OH still existed. Subsequently, numerous studies have focused on designing and synthesizing novel ONOO- molecular probes[26], improving their ONOO- detection performance through structural modifications, and applying them to bioimaging studies[27-29]. In recent years, using ONOO- probes as reliable tools, fluorescence imaging has been employed for in situ and real-time monitoring of ONOO- levels, exploring its complex roles in various pathological processes, which has significantly advanced research into disease pathogenesis and provided new insights for early diagnosis and treatment. To date, numerous excellent reviews on ONOO- fluorescent probes have been published, mostly focusing on aspects such as probe structure and response mechanisms, lacking specificity in elucidating the close relationship between ONOO- and disease progression. In view of this, this review categorizes recent developments according to different pathological models and summarizes small-molecule fluorescent probes for ONOO- reported over the past five years from perspectives including molecular design, detection performance, and biological applications. Finally, we discuss current limitations of existing ONOO- fluorescent probes and provide an outlook on future development trends, aiming to guide the design and application of probes for other biologically active small molecules and promote the clinical translation of fluorescent probes in disease diagnosis and therapy.

2 Design Strategies for ONOO- Fluorescent Probes

Fluorescent probes typically consist of three components: a recognition group (Receptor), a fluorophore (Fluorophore), and a linker (Linker) (Figure 2). The recognition group, also known as the receptor, mainly influences the selectivity and anti-interference ability of the probe molecule; the fluorophore is responsible for generating the response signal and is the primary determinant of sensitivity; while the linker serves as a molecular recognition hub. An excellent fluorescent probe design involves a clever combination of the recognition group, fluorophore, and linker. It not only requires the probe to possess excellent detection performance and potential for biological applications, but also demands ease of preparation and purification to meet market requirements.
图2 荧光探针结构示意图

Fig.2 Structure diagram of fluorescent probe

Under physiological conditions, ONOO- is characterized by low concentration, high reactivity, short lifespan, and susceptibility to interference from coexisting ROS, posing high demands on the sensitivity, response rate, selectivity, and anti-interference capability of probes. Meanwhile, guided by biomedical imaging requirements, near-infrared emission, large Stokes shift, high photostability, and low toxicity are also important considerations in probe design. Benefiting from the strong oxidizing, nucleophilic, and nitrating properties of ONOO-, combined with fluorescent mechanisms such as ICT, PET, ESIPT, FRET, and AIE, various specific responsive sites have been "anchored" within fluorophores for designing highly selective and sensitive ONOO- fluorescent probes. Currently reported recognition mechanisms capable of visualizing ONOO- levels in pathological models mainly include (see Figure 3): (1) Oxidative cleavage of C ̿         C bonds. When the reactive C ̿         C bonds in the probe are oxidized by ONOO-, the C ̿         C bonds break, generating corresponding aldehydes and carboxylic acids; (2) N-dearylation reaction. These probes contain para-aminophenol-derived structures, where electron-donating groups are introduced to increase the electron density of the benzene ring, thereby facilitating oxidation of phenolic hydroxyl groups. Subsequently, the oxidation product, an imine ion, undergoes hydrolysis and bond cleavage, forming corresponding amines and quinones; (3) Oxidative hydrolysis of boronic acid (ester). Boronic acid (ester) recognition sites are also widely used in the design of H2O2 fluorescent probes; however, the reaction rate between boronic acid (ester) and ONOO- is nearly six orders of magnitude faster than that with H2O2[30]. After nucleophilic attack of ONOO- on the boron center, a peroxyboronic acid (ester) intermediate forms, which subsequently undergoes electron migration and hydrolysis to yield the corresponding phenol; (4) Oxidative cleavage of spirohydrazide. This mechanism commonly uses rhodamine, fluorescein, and their derivatives as fluorophores, where the spiro ring opens upon oxidative bond cleavage induced by ONOO-, resulting in significantly enhanced fluorescence. In addition, α-ketoamides, α, β-unsaturated ketones and enolates, diaryl phosphinite esters, chalcogens, among others, can also serve as recognition moieties for ONOO- fluorescent probes. In different physiological and pathological contexts, the response characteristics of various ONOO- recognition groups show significant differences, which will be discussed in detail in Section 3 below.
图3 ONOO-响应机理图

Fig.3 ONOO- response mechanism diagrams

3 Detection and Imaging of ONOO- in Disease-Related Processes

3.1 Detection and Imaging of ONOO- in Inflammation

Inflammation is an innate protective response of organisms to infection or injury, involving resident immune cells releasing soluble mediators such as cytokines, chemokines, and ROS, aiming to promote healing or eliminate pathogens[31-32]. Studies have shown that excessive ONOO- is produced during the inflammatory process. Therefore, in situ monitoring of the dynamic changes of ONOO- during inflammation through effective means is of great significance for the diagnosis and treatment of related diseases.
In 2016, Wu et al.[33] designed a near-infrared probe 1 based on xanthene fluorescent dyes, which could image endogenous ONOO- generated by lipopolysaccharide (Lipopolysaccharides, LPS) stimulation in a live mouse inflammation model. In 2019, Wu et al.[34] synthesized a near-infrared phosphorescent iridium(III) complex probe 2 with excellent photostability and successfully achieved visualization of ONOO- in live cells and mouse inflammation models. This study provided important data and established a platform foundation for designing other phosphorescent metal complex probes for in vivo imaging (Figure 4).
图4 探针1、2对ONOO-的检测机理[33-34]

Fig.4 The detection mechanism of probe 1 and 2 for ONOO-[33-34]

In 2020, Huang et al.[35] synthesized a long-wavelength emitting fluorescent probe 3, composed of 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran and phenylboronic ester linked by triphosgene. Probe 3 exhibited advantages including high specificity, high sensitivity, rapid response, and long-wavelength emission. Fluorescence imaging demonstrated that probe 3 could dynamically monitor intracellular ONOO- levels in zebrafish larvae, with fluorescence mainly concentrated in the brain and yolk regions, indicating higher ONOO- content and associated inflammatory responses in these areas (Figure 5).
图5 探针3对ONOO-的检测机理[35]

Fig.5 The detection mechanism of probe 3 for ONOO-[35]

In 2021, Wang et al.[36] developed an infrared fluorescent "turn-on" probe 4 based on Resorufin and diphenylphosphinate. This probe exhibited excellent selectivity and anti-interference ability towards ONOO-, and enabled naked-eye detection of ONOO-. Probe 4 was successfully applied for high-resolution imaging of endogenous ONOO- in RAW 264.7 living cells and an inflammatory mouse model. In 2022, Zhang et al.[37] designed fluorescent probe 5 using Resorufin as the fluorophore and α-ketoamide as the recognition group. This probe enables rapid imaging (within 5 min) of ONOO- in inflammatory regions, providing a new experimental tool for the study of inflammation-related diseases (Figure 6).
图6 探针4、5的结构[36-37]

Fig.6 Structure of probes 4 and 5[36-37]

In 2023, Luo et al.[38] covalently linked a hydrophobic ONOO- small molecule probe with a hydrophilic natural polymer, glycol chitosan, via amide bonds. Subsequently, a highly selective nanosensor 6 was constructed based on self-assembly, thereby enhancing its water solubility, reducing renal clearance, optimizing biodistribution, and prolonging blood circulation for the diagnosis and evaluation of inflammation. Experimental results demonstrated that probe 6 successfully achieved efficient fluorescent imaging of ONOO- in an LPS-induced inflammatory mouse model, and effectively monitored intracellular ONOO- levels in drug-induced inflammatory mice. This study provides an experimental basis for the application of nanoprobes in the early diagnosis and assessment of inflammation-related diseases (Figure 7).
图7 探针6对ONOO-的检测机理[38]

Fig.7 The detection mechanism of probe 6 for ONOO-[38]. Copyright 2023,Elsevier

In 2024, Li et al.[39] developed a novel fluorescent probe (probe 7) using a dual reaction unit strategy. Probe 7 exhibited excellent sensitivity (8.03 nM), rapid response (< 2 min), large Stokes shift (150 nm), and high selectivity. This probe enables fluorescence imaging of endogenous ONOO- to diagnose inflammation and evaluate the anti-inflammatory effects of traditional Chinese medicine components. Such dual reaction unit-based probes are expected to become the preferred tools for detecting inflammatory diseases and screening anti-inflammatory drugs (Figure 8).
图8 探针7的结构及生物成像[39]

Fig.8 Structure and bioimaging of probe 7[39]. Copyright 2023,Elsevier

3.2 Detection and Imaging of ONOO- in Tumors

Cancer has become a significant public health issue worldwide. Early diagnosis and treatment can markedly improve patient survival rates. However, the mechanisms underlying its onset and recurrence remain incompletely understood, posing major challenges for modern medical technologies in accurately distinguishing tumor tissues from healthy tissues at the cellular level. Recent studies have shown that ROS can promote tumor development by driving various pro-carcinogenic pathways or causing genomic DNA mutations. For example, ONOO- can post-translationally modify tyrosine residues of the tumor suppressor protein p53 through nitration reactions, leading to loss of its regulatory function over the cell cycle and resulting in uncontrolled cell proliferation and ultimately tumor formation[40]. Therefore, elevated expression of ONOO- is regarded as one of the hallmarks of the tumor microenvironment. Monitoring ONOO- levels in vivo holds great significance for understanding tumor formation and progression, as well as for diagnosis and treatment[41-42].
As mentioned above, the pathological process of malignant tumors is closely related to the excessive expression of ROS. You et al.[43] designed a near-infrared ratiometric fluorescent probe (probe 8) in 2021 based on the rhodamine fluorescence core and a thioether oxidation mechanism. The fluorophore part of this probe is similar to that of probe 1; however, replacing the diphenylphosphinoester recognition site with a thioether endows probe 8 with excellent photostability, high quantum yield, rapid response (< 5 s), high selectivity, and good biocompatibility. After reacting with ONOO-, it exhibits a significant ratiometric fluorescent signal change. Using a mouse acute peritonitis model, they demonstrated the potential of probe 8 in monitoring endogenous ONOO- and evaluating inflammation in vivo. Furthermore, using a xenografted tumor model constructed with HepG2 cells as an example, they quantitatively visualized the levels of ONOO- during tumor progression, showcasing the probe's promising application as a molecular tool for tumor diagnosis and therapeutic evaluation (Figure 9). Compared to probe 1, probe 8 further enables visualization of tumor progression.
图9 a)探针8对ONOO-的检测机理;b)探针8的生物成像[43]

Fig.9 a) The detection mechanism of probe 8 for ONOO-;b) Biological imaging of probe 8[43]. Copyright 2021,Royal Society of Chemistry

In 2021, Zhu et al.[44] constructed a “dual-targeting” ONOO- fluorescent probe 9 by conjugating rhodamine hydrazide with biotin. In this design, biotin serves as the “active” targeting moiety that binds to the sodium-dependent multivitamin transporter (SMVT), which is overexpressed on the membrane of tumor cells, while rhodamine hydrazide functions as the “passive” targeting component that reacts with excessive ONOO- in tumor cells to activate fluorescence. This dual-targeting strategy enhances the probe's ability to recognize tumor cells and has been applied for visualization and detection of ONOO- in HNSCC cells and in vivo tumors. Furthermore, they evaluated the probe's potential application in cytokine therapy using a 3D cell spheroid model, aiming to facilitate preoperative and even intraoperative tumor diagnosis (Figure 10).
图10 探针9对ONOO-的检测机理[44]

Fig.10 The detection mechanism of probe 9 for ONOO-[44]

In 2020, Mao et al.[45] developed a rhodamine-based two-photon near-infrared fluorescent probe (probe 10). This probe employs 1,4-diethyltetrahydroquinoline-fused rhodamine as the fluorophore and is modified with 1-methylindole-2,3-dione as the recognition group. It rapidly reacts with ONOO- in PBS solution, emitting near-infrared fluorescence at 654 nm with a 17-fold enhancement. Excess ONOO- increases the probe's two-photon activity cross-section (Фδ, 820 nm) from 0.7 GM to 119 GM, enabling two-photon imaging of exogenous and endogenous ONOO- in living cells. Furthermore, imaging results in zebrafish larvae demonstrated that the probe can specifically localize in the pancreas and image intracellular ONOO- levels. Notably, this probe is one of the rare two-photon "NIR-to-NIR" probes currently applied for ONOO- imaging within mouse tumors, revealing fluctuations in ONOO- levels during tumor initiation and progression (Figure 11). As a molecular tool for tumor diagnosis, compared with other probes discussed in this section, near-infrared two-photon probes offer distinct advantages: not only do they provide high resolution and a high signal-to-noise ratio, but they also overcome limitations of one-photon probes such as high phototoxicity and susceptibility to photobleaching, making them more suitable for biological detection and imaging.
图11 a)探针10对ONOO-的检测机理;b)探针10的生物成像[45]

Fig.11 a) The detection mechanism of probe 10 for ONOO-;b) Biological imaging of probe 10[45]. Copyright 2020,American Chemical Society

In 2018, Zeng et al.[46] reported a hemicyanine-based reaction-activated probe 11 for near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging of ONOO- in tumor-bearing mice. After recognition of ONOO-, the probe exhibited significant enhancement in both fluorescence (59-fold) and PA signal (5.1-fold). Imaging results from a 4T1 cell-induced breast cancer mouse model demonstrated that the probe specifically accumulated at the tumor site after tail vein injection. The NIRF and PA signals gradually increased after injection and reached peak levels at 3 h, where the NIRF and PA signals were enhanced by 2.1-fold and 5.3-fold, respectively, compared to the tumor background. Overall, these experimental results demonstrated that probe 11 can be selectively activated by ONOO- in tumors and enables in vivo dual-modal NIRF/PA imaging (Figure 12).
图12 a)探针11对ONOO-的检测机理;b)探针11的生物成像[46]

Fig.12 a) The detection mechanism of probe 11 for ONOO-;b) Biological imaging of probe 11[46]. Copyright 2018,American Chemical Society

In 2023, Zhao et al.[47] designed a red fluorescent probe 12 for endogenous ONOO- imaging. This probe exhibited characteristics including large Stokes shift, high signal-to-noise ratio, excellent selectivity, and high sensitivity. Based on this, probe 12 was combined with hyaluronic acid synthesis inhibitor 4-Methylumbelliferone (4-MU) through nanoprecipitation to form a nanomaterial, which not only preserved the ONOO- imaging capability of probe 12 but also demonstrated anti-proliferative effects against cancer cells (Figure 13). This multifunctional "Probe-drug" nano-assembly model provides a novel strategy for the design of new ONOO- probes, although further improvements are still required in terms of particle size control and imaging signal-to-noise ratio.
图13 探针12对ONOO-的检测机理[47]

Fig.13 The detection mechanism of probe 12 for ONOO-[47]. Copyright 2023,Elsevier

Chemiluminescence does not require an excitation light source, offering certain advantages in eliminating background interference caused by autofluorescence, light scattering, and thermal effects. In 2024, Xu et al.[48] designed a chemiluminescent probe 13 based on adamantyl-1,2-dioxetane, while also designing a comparative fluorescent probe Flu-1 using coumarin as the fluorophore. Compared to Flu-1, probe 13 exhibited a lower detection limit (9.8 nM), higher signal-to-noise ratio (502), and superior bioimaging capability. Finally, this probe was successfully applied for the detection and imaging of ONOO- in tumor cells under paraquat stimulation, live animals, and human hepatocellular carcinoma tissues (Figure 14). Such chemiluminescent probes hold promise as valuable tools for visualizing tumors and investigating the pathological roles of ONOO- in tumors.
图14 探针13和Flu-1的结构[48]

Fig.14 The structure of probe 13 and Flu-1[48]. Copyright 2024,Elsevier

3.3 Detection and Imaging of ONOO- in Liver Injury

The liver is an essential metabolic organ in the human body, performing functions such as detoxification, glycogen storage, and synthesis of secretory proteins. Liver injury is a clinical disease that includes drug-induced liver injury (DILI)[49], alcoholic liver disease (ALD)[50], and nonalcoholic fatty liver disease (NAFLD)[51]. Among these, DILI is mostly acute liver injury. During DILI, drugs are enzymatically converted in the liver into polar, water-soluble, excretable metabolites, accompanied by the generation of reactive free radicals or reactive electrophilic species such as ROS. Lipid peroxidation caused by excessive ROS production is one of the main factors leading to liver injury[52]. Therefore, ROS have become a class of biologically active molecules for evaluating DILI, including ONOO-. To further understand the association between ONOO- and DILI, researchers have developed a series of fluorescent probes with potential and promising applications for clinical diagnosis of DILI.
In 2021, Mao et al.[53] developed a two-photon excited near-infrared fluorescent probe, designated as 14. This probe exhibited high selectivity, a low detection limit (15 nM), and significant fluorescence enhancement (340-fold). It enabled visualization of endogenous ONOO- levels in living cells and in inflammatory mouse models, and could also monitor changes in ONOO- levels during drug-induced liver toxicity and the subsequent repair process (Figure 15).
图15 探针14对ONOO-的检测机理[53]

Fig.15 The detection mechanism of probe 14 for ONOO-[53]

In 2016, Ma et al.[54] developed a peroxisome-targeted two-photon ONOO- fluorescent probe 15. This probe utilizes 1,8-naphthalimide as the two-photon fluorophore, electron-rich para-diphenol as the recognition site for ONOO-, and a peptide (HLKPLQSKL) as the peroxisome-targeting unit. Based on the high specificity, sensitivity, and low detection limit of probe 15 towards ONOO-, they successfully visualized endogenous and exogenous ONOO- in peroxisomes of living cells and monitored a significant upregulation of ONOO- in the livers of mice with CCl4-induced acute liver injury (Figure 16Figure 16).
图16 探针15对ONOO-的检测机理[54]

Fig.16 The detection mechanism of probe 15 for ONOO-[54]

Asialoglycoprotein receptor (ASGPR) is a receptor highly expressed on the surface of hepatocytes, capable of specifically recognizing, binding, and mediating endocytosis of molecules terminated with galactose residues. Based on this characteristic, modifying natural ligands of ASGPR (e.g., galactose) to construct liver-targeted drug delivery systems has received widespread attention. In 2019, Li et al.[55] introduced galactose into a coumarin-benzopyrylium fluorophore to develop a liver-targeted near-infrared ratiometric fluorescent probe 16. The emission wavelengths of this probe before and after reacting with ONOO- were 720 nm and 500 nm, respectively, with an emission shift of nearly 220 nm, providing high sensitivity and selectivity. Using HepG2, HCT116, HeLa, and MCF-7 cells as examples, they confirmed the selective targeting of probe 16 toward hepatocytes via fluorescence imaging. After confirming that probe 16 could detect both endogenous and exogenous ONOO- in HepG2 cells, they further applied it to monitor ONOO- changes during APAP-induced hepatotoxicity and the subsequent repair process in live cells and mice (Figure 17). In the design of several probes described in this section, benzopyrylium structures were commonly employed, while differences existed in their innovative strategies, such as utilizing natural ligands (e.g., galactose) to construct liver-targeted drug delivery systems or enhancing probe sensitivity, response rate, and selectivity through structural modifications. These probes have been applied to deeply elucidate different liver pathological mechanisms, real-time monitoring of dynamic processes of liver injury and repair, and systematic evaluation of therapeutic effects of hepatoprotective drugs.
图17 a)探针16对ONOO-的检测机理;b)探针16的生物成像[55]

Fig.17 a) The detection mechanism of probe 16 for ONOO-;b) Biological imaging of probe 16[55]. Copyright 2019,Royal Society of Chemistry

During DILI, drug metabolism not only generates excessive ROS but is also accompanied by changes in other microenvironmental parameters, such as increased viscosity. Monitoring multiple targets simultaneously can effectively reduce the high false-positive risk associated with relying on a single biomarker for early DILI diagnosis. In 2020, Feng et al.[56] designed and synthesized a near-infrared dual-responsive fluorescent probe 17, which utilizes a boronic acid group as the responsive moiety to detect ONOO- (λex = 488 nm, λem = 600-660 nm), while simultaneously monitoring viscosity changes through a twisted intramolecular charge transfer (TICT) mechanism in another imaging channel (λex = 633 nm, λem = 670-800 nm). Using the APAP-induced mouse liver injury model as an example, significant fluorescence enhancement was observed in both the ONOO- and viscosity channels with probe 17 compared to the control group, whereas the fluorescence intensity decreased in samples pretreated with N-acetylcysteine. These results confirm that probe 17 can be used to simultaneously investigate the levels of ONOO- and viscosity changes during the generation and repair processes of drug-induced hepatotoxicity, providing new insights into the pathological progression of DILI (see Figure 18).
图18 探针17对黏度和ONOO-的检测机理[56]

Fig.18 The detection mechanism of probe 17 for viscosity and ONOO-[56]

In 2024, Dong et al.[57] designed a near-infrared fluorescent probe 18 based on coumarin π extension for simultaneously monitoring intracellular ONOO- fluctuations and lipid droplet (LDs) state changes. The results indicated that the probe could sensitively respond to endogenous and exogenous ONOO- in the cytoplasm, and its moderate ClogP value enabled it to accumulate within lipid droplets as well. Using probe 18, they observed significant upregulation of ONOO- and LDs accumulation in an APAP-induced DILI cell model, showing time-dependent and dose-dependent effects. Following GSH repair, ONOO- levels decreased, and both the number and size of lipid droplets reduced, confirming the potential of probe 18 for diagnosing, preventing DILI, and evaluating the efficacy of GSH repair (Figure 19).
图19 a) 探针18的生物成像;b)探针18对ONOO-的检测机理[57]

Fig.19 a) Biological imaging of probe 18;b) The detection mechanism of probe 18 for ONOO-[57]. Copyright 2024,Elsevier

Hydrogen peroxide (H2O2) and HClO are common interfering substances in ONOO- detection, although their oxidizing capacity is lower than that of ONOO-. The carbonyl group within the benzopyran molecular structure has been reported as one of the responsive sites for ONOO-, and by modulating the electron-donating or electron-withdrawing ability of substituents adjacent to this carbonyl group, an effective balance between sensitivity, response rate, and selectivity of ONOO- probes can be achieved. In 2020, Yuan et al.[58] developed a highly specific ratiometric fluorescent probe 19 based on the above strategy by introducing a weak electron-withdrawing 7-(diethylamino)coumarin group at the ortho-position of the carbonyl. In the presence of ONOO-, the ratiometric fluorescence signal (F469/F703) was enhanced by 130-fold, with a detection limit as low as 4.1 nM. Using this probe, the variation of ONOO- in mixed cell models of free fatty acid-induced NAFLD and APAP-induced DILI was observed, revealing for the first time the correlation between NAFLD/DILI liver injury and CYP2E1 enzyme activity. Moreover, ONOO- detection in liver tissues of high-fat diet-induced NAFLD mice was also achieved (Figure 20).
图20 a) 探针19的生物成像;b)探针19对ONOO-的检测机理[58]

Fig.20 a) Biological imaging of probe 19;b) The detection mechanism of probe 19 for ONOO-[58]. Copyright 2020,American Chemical Society

Similarly, in 2023, Liu et al.[59] introduced para-substituted benzene rings at the ortho position of the carbonyl group in benzopyran, regulating the emission wavelength and ONOO- selectivity of the probe by altering the electron-donating or electron-withdrawing abilities of the para-substituents. Probe 20 with piperazine substitution was selected as the optimal candidate, which was further utilized to evaluate the severity of DILI and the therapeutic effects of hepatoprotective drugs. However, compared to probe 19, probe 20 exhibited fluorescence quenching response toward ONOO-, which is unfavorable for bioimaging. Nevertheless, the retained "-NH-" reactive site in the probe structure can be further linked with other fluorophores or detection groups, providing new insights for the design of novel ONOO- probes (Figure 21).
图21 探针20对ONOO-的检测机理[59]

Fig.21 The detection mechanism of probe 20 for ONOO-[59]

Reversible fluorescent probes are essential tools for real-time monitoring of the dynamic processes of liver injury and repair; however, currently there are still few applicable for in vivo imaging. In 2022, Yuan et al.[60] developed a highly sensitive and selective ONOO-/GSH reversible nanosensor (probe 21) by conjugating small-molecule probe 21 with PEI-coated core-shell upconversion nanoparticles. Moreover, this probe was successfully applied for reversible imaging of ONOO- and GSH in live cells and animals, and was used to investigate various degrees of liver damage and the in vivo repair process (Fig.22).
图22 a)探针21对ONOO-的检测机理;b)探针21的生物成像[60]

Fig.22 a) The detection mechanism of probe 21 for ONOO-;b) Biological imaging of probe 21[60]. Copyright 2022,Chinese Chemical Society

In 2024, Kan et al.[61] designed and synthesized fluorescent probe 22 for ferroptosis-mediated DILI research. This probe, based on the ICT mechanism, exhibited high sensitivity towards ONOO- and was successfully applied to monitor ONOO- levels in LO2 cells, zebrafish, and mouse models under stimulation with Erastin, APAP, APAP/Fer-1, INH, and INH/Fer-1, indicating that ferroptosis plays an important role in the progression of DILI and that the ferroptosis inhibitor Fer-1 can effectively alleviate DILI (Figure 23).
图23 探针22对ONOO-的检测机理[61]

Fig.23 The detection mechanism of probe 22 for ONOO-[61]

3.4 Detection and Imaging of ONOO- in Brain Diseases

ROS plays a key regulatory role in brain metabolism and neuronal function, primarily originating from mitochondria[62]. Imbalance of ROS can lead to brain damage, subsequently causing ischemic and hemorrhagic strokes, and exacerbating neurodegenerative diseases through oxidative stress reactions[63]. Therefore, real-time monitoring of ONOO- fluctuations in the brains of patients with brain disorders is crucial for early diagnosis and treatment of cerebral dysfunction.
Alzheimer's Disease (AD) is a neurodegenerative disorder that causes cognitive dysfunction. In 2018, Sessler et al.[64] designed and synthesized an excited-state intramolecular proton transfer (ESIPT)-type fluorescent probe 23 based on 3-hydroxyflavone and boronic ester. After activation by ONOO-, this probe displays different aggregation states of beta-amyloid protein (Amyloid beta-protein, Aβ) through host-guest interactions and simultaneously provides a ratiometric fluorescent signal for quantifying ONOO-. To evaluate the practical application of the probe in vivo, fluorescent imaging was performed using brain slices from AD transgenic mice, showing good overlap between the probe fluorescence before and after ONOO- treatment and anti-Aβ antibody fluorescence (Figure 24). Although the short excitation and emission wavelengths of the probe prevent its use in in vivo imaging, this study provides guidance for developing other bioactive small molecule probes by demonstrating a method to monitor AD progression and oxidative status changes in the brain through hybrid design of small molecular probes and peptides.
图24 探针23对ONOO-的检测机理[64]

Fig.24 The detection mechanism of probe 23 for ONOO-[64]

In 2024, Sun et al.[65] developed a novel fluorescent probe 24 for monitoring endogenous ONOO- in AD model mice. Cellular imaging results indicated that when the probe was co-incubated with LPS/IFN-· pretreated SH-SY5Y cells, significant fluorescence attenuation was observed. Further treatment of the cells with the NO synthase inhibitor TEMPO along with LPS/IFN-· resulted in enhanced fluorescence, confirming the probe's capability to image endogenous ONOO- at the cellular level. Moreover, by evaluating the probe's ClogP value (3.397) and measuring fluorescence intensity in mouse cerebrospinal fluid, it was confirmed that the probe can penetrate the blood-brain barrier. Finally, using a commercial APP/PS1 transgenic AD mouse model and the AD therapeutic drug curcumin, the probe was proven capable of monitoring ONOO- flux in the brains of AD mice ( Figure 25).
图25 a) 探针24的生物成像;b)探针24对ONOO-的检测机理[65]

Fig.25 a) Biological imaging of probe 24;b) The detection mechanism of probe 24 for ONOO-[65]. Copyright 2024,Elsevier

Parkinson's disease (PD), also known as "paralysis agitans," is a central nervous system degenerative disorder, in which ONOO- plays a key role in its pathogenesis. In 2020, Zhang et al.[66] synthesized an ultrafast, highly selective, near-infrared ONOO- probe (compound 25) using dicyanide isoindole as the fluorophore and para-aminophenol as the recognition group. The design strategy of this molecule was similar to that of probe 22; both achieved significant improvement in probe performance through precise modulation of molecular structure and rational selection of recognition groups. Fluorescence imaging results demonstrated that this probe could monitor dynamic changes of ONOO- in multiple PD models, including live cells, fruit flies, Caenorhabditis elegans, and mouse brains. This probe holds promise as an imaging agent for studying the biological roles of ONOO- in PD, potentially contributing to early diagnosis and treatment of PD (see Figure 26).
图26 探针25对ONOO-的检测机理[66]

Fig.26 The detection mechanism of probe 25 for ONOO-[66]

Endoplasmic reticulum (ER) stress is a mechanism for maintaining ER homeostasis and has complex interactions with oxidative stress, potentially contributing together to disease pathogenesis. In 2021, Wang et al.[67] designed and synthesized an endoplasmic reticulum-targeted two-photon fluorescent probe 26 for visualizing ONOO- in PD models. Similar to probe 15, this probe utilizes 4-amino-2-methoxyphenol, 1,8-naphthalimide, and para-toluenesulfonamide as the ONOO- responsive site, two-photon fluorophore, and ER-targeting group, respectively, enabling rapid detection of ONOO- based on the ICT mechanism, with high sensitivity and specificity. Initially, the probe was co-incubated with ER Tracker Red in PC12 cells, confirming its ER-targeting capability through a Pearson correlation coefficient as high as 0.95. Subsequently, imaging of intracellular and extracellular ONOO- was conducted in PC12 cells, and finally, the probe was applied for ONOO- imaging in a PD model (WLZ3 Caenorhabditis elegans) (Figure 27). In contrast, probe 15, which has a similar structure, employs a peptide as a peroxisome-targeting unit for monitoring ONOO⁻ levels in the liver of mice with CCl₄-induced acute liver injury.
图27 探针26对ONOO-的检测机理[67]

Fig.27 The detection mechanism of probe 26 for ONOO-[67]

In 2023, Bai et al.[68] developed a symmetrical molecular probe 27 using α-ketoamide as the ONOO- recognition site. This probe possesses two-photon imaging capability, and using this probe, significant upregulation of ONOO- was observed in SH-SY5Y cells and zebrafish PD models induced by MPP+ and rotenone, respectively (Figure 28;Fig. 28).
图28 探针27对ONOO-的检测机理[68]

Fig.28 The detection mechanism of probe 27 for ONOO-[68]. Copyright 2023,World Scientific

Epilepsy is a chronic neurodegenerative disease affecting 50 million people worldwide, characterized by recurrent physical convulsions, sometimes accompanied by loss of consciousness and urinary or fecal incontinence. Epileptic seizures are spontaneous and recurrent, and are closely associated with oxidative stress and mitochondrial dysfunction. ONOO- is considered an important biomarker for the early diagnosis of epilepsy; real-time monitoring of ONOO- changes in cells and living organisms contributes to elucidating its underlying mechanisms in epileptic diseases. In 2021, Wang et al.[69] established a rat model of epilepsy and developed a near-infrared two-photon fluorescent probe (compound 28) based on the ICT mechanism for sensing and imaging ONOO- within this model. This probe demonstrates excellent endoplasmic reticulum targeting capability, enabling effective observation of endogenous ONOO- levels in the hippocampal region of kainate (KA)-induced epileptic seizure rats. Furthermore, it confirms a positive correlation between abnormally elevated ONOO- levels and both seizure activity and neuronal damage. Resveratrol was shown to effectively inhibit excessive ONOO- expression and alleviate neuronal injury, suggesting its potential as a promising antiepileptic agent. In conclusion, this probe holds considerable potential for clinical translation and can serve as a chemical tool for tracking ONOO- fluctuations, thereby aiding in the diagnosis of epilepsy (Figure 29).
图29 a) 探针28对ONOO-的检测机理;b) 探针28的生物成像[69]

Fig.29 a) The detection mechanism of probe 28 for ONOO-;b) Biological imaging of probe 28[69]. Copyright 2021,American Chemical Society

Glioblastoma (GBM) is the most lethal and common malignant primary brain tumor in adults. Due to the highly aggressive and heterogeneous nature of GBM, the median survival period for GBM patients is only 15 months, with an average 5-year survival rate of less than 8%. In 2023, based on the reduced extracellular pH and elevated ROS levels in the tumor microenvironment caused by aerobic glycolysis, Wang et al.[70] designed and synthesized ONOO- specific fluorescent probe 29, aiming to apply it in the early clinical diagnosis of GBM. This probe exhibits advantages including a low detection limit (0.9 μM), rapid response (< 2 s), and significant fluorescence enhancement upon binding to ONOO- (up to 1700-fold). Moreover, this probe demonstrates minimal cytotoxicity and excellent cellular uptake capability, having been successfully applied for ONOO- imaging in U87MG cells, 3D tumor spheres, and an orthotopic xenograft GBM nude mouse model. Cancer cell screening experiments revealed that probe 29 shows high specificity towards U87MG cells, effectively distinguishing between different cancer cell lines. Furthermore, confocal imaging of 3D tumor spheres at varying depths indicates the probe's tissue penetration capacity up to 70 μm. Following tail vein injection in nude mice, accumulation of the probe within GBM tissues could be observed within 30 minutes, highlighting its potential for clinical diagnostics (Figure 30).
图30 探针29对ONOO-的检测机理[70]

Fig.30 The detection mechanism of probe 29 for ONOO-[70]

3.5 Detection and Imaging of ONOO- in Other Disease Models

Studies have shown that ONOO- overexpression is not only a marker of various diseases such as inflammation, tumors, drug-induced liver injury, and brain disorders, but also closely related to cardiovascular diseases, such as atherosclerosis (Atherosclerosis, AS). In AS, the formation and progression of arterial wall plaques lead to vascular stenosis and restricted blood flow, potentially causing severe cardiovascular events. Researchers have observed ONOO- overexpression in foam cells of human atherosclerotic tissues, which is considered an important pathological factor in the progression of AS. Therefore, ONOO- has become one of the biomarkers for diagnosing atherosclerotic plaques.
In 2021, Fisher et al.[71] synthesized a FRET-based ratiometric fluorescent probe (probe 30) by linking a rhodamine derivative (FRET acceptor) and a quinoline derivative (FRET donor) via an amide bond. This probe responded rapidly to ONOO- (< 2 min), with the oxidation of ONOO- causing the spirolactam ring to open, thereby activating the FRET process and shifting the fluorescence emission wavelength from 474 nm to 574 nm. To investigate the regulatory mechanism of arginase 1 (Arginase 1, Arg-1) on ONOO-, probe 30 was used to monitor changes in ONOO- levels in RAW 264.7 cells and mouse inflammation models after different pretreatments. It was found that ONOO- levels were negatively correlated with Arg-1 activity, revealing that Arg-1 downregulates ONOO- by competing with iNOS for the substrate arginine. Furthermore, this probe enabled visualization of ONOO- fluctuations during the development and regression of atherosclerotic plaques in atherosclerotic mice (Figure 31).
图31 a) 探针30对ONOO-的检测机理;b) 探针30的生物成像[71]

Fig.31 a) The detection mechanism of probe 30 for ONOO-;b) Biological imaging of probe 30[71]. Copyright 2021,American Chemical Society

In 2023, Dai et al.[72] developed a fluorescent probe 31 for the sequential detection of ONOO- and LDs based on the abundant presence of LDs and ONOO- in atherosclerotic plaques. After entering cells, this probe initially localizes in mitochondria and undergoes intramolecular charge rearrangement upon reaction with ONOO-, generating a coumarin derivative. This structure exhibits a strong solvent effect, enabling the probe to recognize intracellular LDs and emit a blue-shifted fluorescence. Using this fluorescent signal change, they demonstrated that oxidized low-density lipoprotein (Oxidized LDL, Ox-LDL) can stimulate RAW 264.7 cells to form foam cells, accompanied by abnormal accumulation of both ONOO- and LDs. Compared with the commercial ONOO- probe hydroxyphenyl fluorescein (HPF), probe 31 exhibited superior selectivity and signal-to-noise ratio. Furthermore, using a mouse model of carotid atherosclerotic plaque established by carotid ligation combined with high-fat diet in ApoE-deficient mice, imaging results showed that probe 31 could not only provide morphological and distributional information of aortic plaques but also reveal lipid accumulation and oxidative stress levels within these plaques. Therefore, this probe enables accurate detection of ONOO- and lipid content in biological samples, which is beneficial for the diagnosis and treatment of AS-related diseases. Moreover, the "dual-lock" mechanism of this probe provides a new strategy for designing efficient fluorescent tools (Figure 32).
图32 a)探针31对ONOO-的检测机理;b)探针31的生物成像[72]

Fig.32 a) The detection mechanism of probe 31 for ONOO-;b) Biological imaging of probe 31[72]. Copyright 2023,American Chemical Society

4 Conclusion and Prospect

In summary, this paper classifies different pathological models and reviews ONOO- small molecule fluorescent probes reported in the past five years from the perspectives of molecular design, optical properties, and biological applications. The testing solvent systems, excitation/emission wavelengths, detection ranges, detection limits, and response times are listed in Table 1. Additionally, detailed information regarding cell imaging, tissue imaging, and disease modeling applications is summarized in Table 2. In terms of probe construction, various functional groups can be specifically recognized by ONOO-. Among them, fluorescent probes based on recognition sites such as electron-rich para-aminophenol, electrophilic unsaturated C̿    C bonds, and spirolactams generally exhibit lower detection limits (0–100 nM) and faster response times (2–120 s), performing particularly well in disease models. Moreover, besides traditional fluorophores such as rhodamines, coumarins, and triphenylamines, near-infrared and two-photon fluorescent dyes like dicyanoisophorone, benzopyran, and novel oxazanthrene derivatives have attracted increasing attention. The introduction of construction methods such as self-assembly and nanoprecipitation has further enhanced the multifunctionality of these probes. Regarding biological applications, the case studies reviewed here demonstrate that ONOO- fluorescent probes have directly or indirectly revealed significant correlations between ONOO- and various disease processes at cellular, tissue, and organism levels. For instance, during inflammation, elevated ONOO- levels can be observed through fluorescent histopathology and in vivo imaging, enabling subsequent screening for anti-inflammatory drugs. Two-photon imaging of intratumoral ONOO- levels in tumor-bearing mouse models reveals dynamic changes in ONOO- during tumor initiation and progression, which can distinguish tumor tissues from surrounding healthy tissues, facilitating early cancer diagnosis and surgical navigation. By establishing a DILI mouse model, fluctuations in ONOO- levels during DILI progression and recovery were detected, providing insights into the pathogenesis of DILI and aiding in drug development. Furthermore, abnormal ONOO- levels have also been monitored in various animal models of neurodegenerative diseases such as AD and PD (including transgenic mice, fruit flies, and Caenorhabditis elegans). Overall, these probes have achieved significant progress in sensitive detection and real-time monitoring of ONOO- in vivo and have been preliminarily applied in early prediction and auxiliary treatment of diseases including inflammation, tumors, liver injury, AD, and PD, promoting interdisciplinary integration among chemistry, biology, and medicine.
表1 ONOO-荧光探针的相关参数

Table 1 Related parameters of ONOO- fluorescent probes

Probe Year Test solution λex/λem (nm) Response time (s) Detection range (μM) LOD (nM) Ref
1 2016 DMSO/PBS = 1:1 (v/v,pH = 7.4) 670/742 / 0~40 400 33
2 2019 DMSO/PBS = 1:19 (v/v,pH = 7.4) 500/660 / 0~110 600 34
3 2020 ACN/PBS = 3:20 (v/v,pH = 7.4) 540/604 40 0~10 21 35
4 2021 PBS (pH = 7.4,1% DMSO,3 mM CTAB) 525/590 600 0~35 238 36
5 2022 PBS (pH = 7.4,1% DMSO,3 mM CTAB) 525/590 < 300 0~30 220 37
6 2023 PBS (pH = 7.4) 570/628 60 0~1 33 38
7 2024 PBS (pH = 7.4,1% DMSO) 381/518 120 0~5×107 8.03 39
8 2021 PBS (pH = 7.4,1% DMSO) 548/662 < 5 0~26 2.6×104 43
9 2021 PBS (pH = 7.4) 530/545-750 5 0~10 7 44
10 2020 PBS (pH = 7.4,10% DMSO) 560/652 < 180 0.5~20 72 45
11 2018 PBS (pH = 7.4) 675/712 / 50~100 53 46
12 2023 ACN/PBS = 4:6 (v/v,pH = 7.4) 450/555 / 0~14,0~90 123,40 47
13 2024 DMSO/PBS = 1:9 (v/v,pH = 7.4) 400/550 600 0~2.5×105 9.8,98 48
14 2021 PBS (pH = 7.4) 620/660 10 0.05~10 15 53
15 2016 PBS (pH = 8.2) 405/553 / 0~10 6.2 54
16 2019 PBS (pH = 7.4) 440/500,720 / 500~1.5×104 1.7×105 55
17 2020 PBS (pH = 7.4,40% DMSO) 420/635 300 0~1 1.69 56
18 2024 PBS (pH = 7.4,30% DMSO) 518/655 2 0~28 27 57
19 2020 EtOH/PBS = 2:9 (v/v,pH = 7.4) 405/469,703 25 0~7 4.1 58
20 2023 Purified water 580/638 19 / 9.36 59
21 2022 PBS (pH = 7.4,1% Ethanol) 550/600 < 10 / / 60
22 2024 PBS (pH = 7.4,20% DMSO) 500/655 600 20~40 65.8 61
23 2018 Tris-HCl (pH = 7.4) 365/420,530 / 0~2 65.5 64
24 2024 PBS (pH = 7.4,1% DMSO) 474/544 3 0~38 11 65
25 2020 PBS (pH = 7.4,0.4% Tween 80) 511/670 < 5 0~15 3.3 66
26 2021 PBS (pH = 7.4,2% DMSO,0.4% Tween 80) 450/540 25 0~5 8.3 67
27 2023 PBS (pH = 7.4) 350/490 60 0~3 9.89 68
28 2021 PBS (pH = 7.4,30% DMSO) 520/685 600 0~20 96 69
29 2023 PBS (pH = 7.4,1% DMSO) 425/548 < 2 0~10 900 70
30 2021 PBS (pH = 7.4,10% CH3CN) 580/770 < 120 0~10 330 71
31 2023 PBS 365/474,574 120 4~10 1.33 72
表2 ONOO-荧光探针的病理信息

Table 2 Pathological information of ONOO- fluorescent probes

Probe Cell imaging Tissue imaging Disease models Disease Ref
1 RAW 264.7 / Mice were intraperitoneally injected with LPS and PMA Inflammation 33
2 MCF-7 / Mice were intraperitoneally injected with LPS and PMA Inflammation 34
3 CHO-KE / Zebrafish larvae were immersed in LPS Inflammation 35
4 HepG2,RAW 264.7 / LPS was injected subcutaneously into the left leg of nude mice Inflammation 36
5 HepG2,RAW 264.7,HeLa / LPS was injected subcutaneously into the right leg of nude mice Inflammation 37
6 RAW 264.7 / LPS was injected subcutaneously into the left leg of mice Inflammation 38
7 LO2,MCF-7 / LPS was injected subcutaneously into the hind legs of nude mice Inflammation 39
8 RAW 264.7 Tumor tissues LPS induced acute peritonitis in mice
Xenotransplantation of HepG2 tumor cells in nude mice
Tumor 43
9 Cal-27,HSC-2,MC3 / Xenotransplantation of HSC-2 tumor cells in nude mice Tumor 44
10 HeLa,RAW 264.7 Zebrafish pancreatic tissue Subcutaneous transplantation of 4T1 tumor cells in nude mice Tumor 45
11 RAW 264.7 / Subcutaneous transplantation of 4T1 tumor cells in nude mice Tumor 46
12 RAW 264.7 / / Tumor 47
13 HepG2,A549,
SH-SY5Y,LO2
Human liver cancer tissue Xenotransplantation of HepG2 tumor cells into nude mice Tumor 48
14 HeLa,RAW 264.7 Mice liver tissue APAP induced hepatotoxicity in mice Liver injury 53
15 SMMC-7721,HL-7702 Mice liver tissue CCl4 induced acute liver injury in mice Liver injury 54
16 HepG2,HCT116,HeLa ,MCF-7 Mice liver tissue APAP induced hepatotoxicity in mice Liver injury 55
17 HeLa Mice liver tissue APAP induced liver injury in mice
LPS and IFN-γ stimulated zebrafish
Subcutaneous injection of exogenous ONOO- mice
Liver injury 56
18 HepG2 / / Liver injury 57
19 HepG2,LO2 / HFD induced nonalcoholic fatty liver mice Liver injury 58
20 HepG2 Mice liver tissue APAP induced liver injury in mice Liver injury 59
21 HepG2,RAW 264.7 Mice liver tissue CCl4 induced acute liver injury in mice Liver injury 60
22 LO2 Mice liver tissue APAP and INH induced ferroptosis-mediated drug induced liver injury in zebrafish and mice Liver injury 61
23 / Brain tissue of AD AD transgenic mice Neurodegenerative diseases 64
24 SH-SY5Y Murine brain APP/PS1 transgenic mice Neurodegenerative diseases 65
25 PC12 Drosophila brain tissues MPTP induced Parkinson 's disease in mice
LRRK2 overexpression constructed WLZ3 C.elegans Parkinson's disease
Neurodegenerative diseases 66
26 PC12 / LRRK2 overexpression constructed WLZ3 C.elegans Parkinson's disease Neurodegenerative diseases 67
27 SH-SY5Y / SIN-1 and MPP+ stimulated zebrafish Neurodegenerative diseases 68
28 RAW 264.7,HT22 Brain sections of epileptic rats KA induced epilepsy in rats Neurodegenerative diseases 69
29 U87MG Glioblastoma tissue Xenotransplantation of U87MG cells into the brain of nude mice Neurodegenerative diseases 70
30 RAW 264.7 Mouse aortic tissue High fat fed low-density lipoprotein receptor knockout (Ldlr-/-) mice Cardiovascular 71
31 A549,RAW 264.7 / Carotid artery ligation combined with high-fat diet feeding ApoE gene-deficient mice Cardiovascular 72
Nevertheless, the biological environment is extremely complex, and ONOO- probes applicable to biology, medicine, and even clinical practice still face significant challenges. A comprehensive understanding of the pathological and physiological roles of ONOO- requires collaborative efforts from researchers in chemistry, biology, and medicine: (1) Near-infrared emitting ONOO- probes can partially overcome the depth limitations of fluorescence imaging, providing more accurate sample information and making them particularly suitable for in vivo imaging. Especially, probes operating in the second near-infrared window show promising clinical application potential and have become a research hotspot for future studies; (2) During circulation in the animal body, probes may bind with proteins in the blood, affecting their response characteristics and leading to false-positive or false-negative results. Combined with the inherently low concentration, short lifetime, and high reactivity of ONOO-, further improvements in the selectivity and sensitivity of probes toward ONOO- in vivo are required; (3) The complex biological environment causes ONOO- concentrations to vary unpredictably across different times and conditions. Most currently reported ONOO- probes are irreversible types that become ineffective once reacting with ONOO-. This kind of "one-time-use" tool can only provide ONOO- information at specific times and spaces, failing to achieve process monitoring. Therefore, reversible ONOO- probes remain the pursuit goal for scientists; (4) The targeting capability of probes toward diseased tissues needs further enhancement, especially their performance in animal models. Addressing these scientific challenges will contribute to the development of superior and efficient ONOO- fluorescent probes, bringing new breakthroughs to biochemical research and clinical applications.
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