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.

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.

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.

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 \stackrel{\mathrm{ }\mathrm{ }\mathrm{ }\mathrm{ }}{̿} C bonds. When the reactive C \stackrel{\mathrm{ }\mathrm{ }\mathrm{ }\mathrm{ }}{̿} C bonds in the probe are oxidized by ONOO^-, the C \stackrel{\mathrm{ }\mathrm{ }\mathrm{ }\mathrm{ }}{̿} 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.

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 C\stackrel{\mathrm{ }\mathrm{ }\mathrm{ }\mathrm{ }}{̿}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.

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.