Recent Progress of Spectroscopic Probes for Peroxynitrite and Their Potential Medical Diagnostic Applications

Peroxynitrite (ONOO−) is a crucial reactive oxygen species that plays a vital role in cellular signal transduction and homeostatic regulation. Determining and visualizing peroxynitrite accurately in biological systems is important for understanding its roles in physiological and pathological activity. Among the various detection methods, fluorescent probe-based spectroscopic detection offers real-time and minimally invasive detection, high sensitivity and selectivity, and easy structural and property modification. This review categorizes fluorescent probes by their fluorophore structures, highlighting their chemical structures, recognition mechanisms, and response behaviors in detail. We hope that this review could help trigger novel ideas for potential medical diagnostic applications of peroxynitrite-related molecular diseases.


Introduction
Peroxynitrite (ONOO − ) is a kind of reactive oxygen species (ROS) generated by the rapid reaction of nitric oxide (NO) and a superoxide anion free radical (O 2 • − ) in the absence of enzyme catalysis, which has strong oxidation, nucleophilic, and nitration properties [1].It occupies crucial roles in the transformations of other major reactive species.(Scheme 1) Its pKa value is 6.8 [2], and the half-life is approximately 1 s [3,4] at pH 7.4.ONOO − can react with a variety of bioactive substances (such as protein, nucleic acid, lipid, etc.) with very high reactivity.In addition to its oxidation, nucleophilic, and nitration properties, ONOO − can also be converted into higher activity secondary free radicals, including hydroxyl radicals (•OH), nitro radicals (•NO 2 ), and carbonate radicals (CO 3 • − ), which further react with biomolecules and ultimately lead to cell death.

Introduction
Peroxynitrite (ONOO − ) is a kind of reactive oxygen species (ROS) generated by the rapid reaction of nitric oxide (NO) and a superoxide anion free radical (O2• − ) in the absence of enzyme catalysis, which has strong oxidation, nucleophilic, and nitration properties [1].It occupies crucial roles in the transformations of other major reactive species.(Scheme 1) Its pKa value is 6.8 [2], and the half-life is approximately 1 s [3,4] at pH 7.4.ONOO − can react with a variety of bioactive substances (such as protein, nucleic acid, lipid, etc.) with very high reactivity.In addition to its oxidation, nucleophilic, and nitration properties, ONOO − can also be converted into higher activity secondary free radicals, including hydroxyl radicals (•OH), nitro radicals (•NO2), and carbonate radicals (CO3• − ), which further react with biomolecules and ultimately lead to cell death.Scheme 1.The biogenesis of peroxynitrite and its transformations with other major reactive species.
Based on these properties, peroxynitrite exhibits two effects with different directions.In the living system, when the ONOO − remains at a level which is under normal physiological conditions, it serves as an indispensable physiological activator and signaling molecule.However, when the concentration of ONOO − elevates, the excess ONOO − will turn the redox state of the cell to a pro-oxidant state [5,6].Eventually, serious inflammation and disease will be induced, for example, rheumatism, hepatic disease, neurodegenerative disease, cancer, and so on [7][8][9][10].Therefore, it would be of great significance to develop a method which could accurately detect ONOO − and explore the physiological role of ONOO − in living systems.
So far, a variety of reviews on ONOO − fluorescent probes have been published [12][13][14][15].This review focuses on the fluorophore structure in the ONOO − fluorescent probe molecules with their potential medical diagnostic applications.Herein, we categorized, analyzed, and discussed the recently reported organic probes according to their fluorophore core, including xanthene (rhodamine, rhodol, and fluorescein), cyanine (and hemicyanine), coumarin, malononitrile-based [Dicyanomethylene-4H-pyrans (DCM), dicyanoisophorone (DCI or DCO) dyes, quinoline-malononitrile (QM)], 2-benzothiazoleacetonitrile-based dyes, and naphthalimide.In particular, we summarized the key factors of the ONOO − -responsive probes, such as chemical structures, responsive pathways, emission wavelength, dynamic range of fluorescence response, response time, ONOO − detection range, detection limit, ONOO − production pathways in the biosystem, and bioimaging objects.We believe that researchers will benefit from this review when they rationally design ONOO − fluorescent probes, thus contributing more excellent theranostic studies in relating areas.We will review other spectroscopic probes for the detection of ONOO − .

Fluorescent Probes 2.1. Xanthene as Fluorophore Core
Xanthene dyes can be categorized as fluorescein, rhodol, and rhodamine based on the type of the substituents on the 3-and 6-position [16].They are well known because of their switchable fluorescent off-on flexibility.Xanthene dyes can produce fluorescence wavelengths above 510 nm, reaching far-red areas depending on the conjugative substituents.Thus, they are of widespread use in optical diagnostic research [17].

Hydrazide Oxidative Xanthene Probes
In 2002, Guo et al. reported a spiro form hydrazide rhodamine (Xan 1) [18] as the ONOO − fluorescent probe.The hydrazide probe was colorless and non-fluorescent.Upon treating with ONOO − , the spiro hydrazide group was oxidized, releasing a highly fluorescent rhodamine B. The response finished in as fast as 30 s.Meanwhile, the detection limit was only 24 nM.The response avoids interference from the 10 −5 M Cu(II) ion.Thus, it represents the rapid, sensitive, and specific fluorescent detection of ONOO − .
Based on the recognized pattern and the easy structurally modification character of rhodamine, a series of related probes were developed, aiming to improve the performance of different aspects of the response (Figure 1).Longer emissive wavelengths (up to the NIR range) were obtained with more conjugate groups installed in Xan 2-5 [19][20][21][22], Xan 8 [25], and Xan 10 [27].Dual-channel fluorescence was afforded when coumarins were introduced to the rhodamine ring (515/700 nm for Xan 5 and 631/669 nm for Xan 10), making the response produce more information.Ratiometric fluorescence was realized in Xan 6 [23] and Xan 7 [24] with the introduction of a 2-(2 -hydroxyphenyl)benzothiazole group and a 4-hydroxycarbazole group, respectively, in which the intensity of the original band disappeared with the generation of a new band with a longer wavelength.Large Stokes shift and excellent lysosome-targeting ability were achieved with the engineering of a fused tetrahydroquinoxaline ring, making Xan 9 [26] capable of detecting both peroxynitrite and lysosomal pH.Sodium-dependent multivitamin transporter (SMVT)-targeted ability was acquired by introducing the biotin group for Xan 9, making it possible to detect the peroxynitrite in head and neck cancer cells.
NIR range) were obtained with more conjugate groups installed in Xan 2-5 [ 8 [25], and Xan 10 [27].Dual-channel fluorescence was afforded when coum introduced to the rhodamine ring (515/700 nm for Xan 5 and 631/669 nm for Xa ing the response produce more information.Ratiometric fluorescence was real 6 [23] and Xan 7 [24] with the introduction of a 2-(2′-hydroxyphenyl)benzothia and a 4-hydroxycarbazole group, respectively, in which the intensity of the or disappeared with the generation of a new band with a longer wavelength.L shift and excellent lysosome-targeting ability were achieved with the engin fused tetrahydroquinoxaline ring, making Xan 9 [26] capable of detecting bot trite and lysosomal pH.Sodium-dependent multivitamin transporter (SMV ability was acquired by introducing the biotin group for Xan 9, making it possib the peroxynitrite in head and neck cancer cells.If a phenyl group was introduced into the hydrazide (Xan 11-Xan 12) [28,29], the response time would prolong to 10 or more minutes, presumably due to the steric-hindrancecaused decreased reactivity.It should be noted that, with alkyl substituent groups in both nitrogens of the hydrazide, the cyclic hexahydropyridazin probes (Xan 14-Xan 15) [31,32] displayed a faster response rate than the alkyl-substituted (Xan 13) [30] or phenyl substituted hydrazide xanthene (Xan 11-Xan 12).The response was usually specific, without interferences from a lot of metal ions and other reactive oxygen and nitrogen species.[28][29][30][31][32] Peroxynitrite generated from different cells, such as HeLa, RAW264.7,HepG2, HSC-2, and Cal-27, could be detected by hydrazide xanthenes.Meanwhile, these probes could detect peroxynitrite in zebrafish and mouse models.These outstanding performances made hydrazide xanthenes capable of revealing the important roles of peroxynitrite in many kinds of diseases, such as respiratory infectious diseases and inflammation in the future.

Oxidative Cleavage of the Recognition Groups to Release Xanthene Probes
Utilizing the oxidative ability of peroxynitrite, the recognition groups at the 2-or 6-hydroxyl or amino group of xanthenes derivative could be cleaved to release xanthenes with high fluorescence (Figure 2).

Oxidative Cleavage of the Recognition Groups to Release Xanthene Probes
Utilizing the oxidative ability of peroxynitrite, the recognition groups at the 2-or hydroxyl or amino group of xanthenes derivative could be cleaved to release xanthen with high fluorescence.(Figure 2) Yang et al. developed the HKGreen series of rhodamine probes (Xan 16 [33] and X 24 [41]) for detecting peroxynitrite with the employment of the trifluoromethyl ketone the recognition group, which involved dioxirane rearrangement and oxidative O-or diarylation.The response was highly selective and sensitive, with high-fold fluoresce enhancement, though the response time was relatively long (>15 min).
While using the benzyl boronates moiety as a recognition group, Xan 17-19 [33-3 exhibited quite different responsive behaviors upon reactive species.Xan 17 [33] react not only to peroxynitrite but also with hypochlorite and hydrogen peroxide, though w different second-order rate constants.However, Xan 18 [34] and Xan 19 [35] respond exclusively to peroxynitrite, even when hypochlorite and hydrogen peroxide were much higher concentrations.Nevertheless, all of them displayed obvious fluorescent e hancement and low detection limits, and thus they were all further employed for fluor cent imaging in biosystems involving diseases such as drug-introduced liver injury.Yang et al. developed the HKGreen series of rhodamine probes (Xan 16 [33] and Xan 24 [41]) for detecting peroxynitrite with the employment of the trifluoromethyl ketone as the recognition group, which involved dioxirane rearrangement and oxidative O-or N-diarylation.The response was highly selective and sensitive, with high-fold fluorescent enhancement, though the response time was relatively long (>15 min).
While using the benzyl boronates moiety as a recognition group, Xan 17-19 [33][34][35] exhibited quite different responsive behaviors upon reactive species.Xan 17 [33] reacted not only to peroxynitrite but also with hypochlorite and hydrogen peroxide, though with different second-order rate constants.However, Xan 18 [34] and Xan 19 [35] responded exclusively to peroxynitrite, even when hypochlorite and hydrogen peroxide were at much higher concentrations.Nevertheless, all of them displayed obvious fluorescent enhancement and low detection limits, and thus they were all further employed for fluorescent imaging in biosystems involving diseases such as drug-introduced liver injury.
The 1-methylindoline-2,3-dione group can also be employed as the recognition moiety for the specific detection of peroxynitrite (Xan 20 [36] and Xan 21 [37]).The mechanism involving intramolecular cyclization of peroxynitrite with indoline-2,3-dione, rearrangement, and 1,6-elimination was proposed [36].Leveraging the probes, the two-photon (TP) in vivo NIR imaging technique was applied to observe the peroxynitrite level in a mouse tumor, a tumor onset on the second day, a kidney injury of zebrafish, and the microvessels of mouse brains with strokes [37].

Oxidation of Pyrylium
Yuan et al. discovered an aminophenyl-substituted pyrylium as a highly sensitive and selective scaffold towards peroxynitrite after the screening of nineteen dyes and then further modified it to a FRET probe (Xan 29) [46] with TP absorption.After the response, the pyrylium emission band at 651 nm disappeared, and a coumarin characteristic emission band at 473 nm was enhanced.Detailed response mechanisms involving nucleophilic addition, oxidation, elimination, and hydrolysis reactions on chromenylium fluorophore were proposed and verified by MS spectra.Although the destroyed-type response led to the decrease of the emission wavelength, the combination technique of the ratiometric measure and TP imaging made it possible to specifically and rapidly visualize the peroxynitrite in an inflamed mouse model.Furthermore, the detection limit was as low as 11.3 nM, which was at a super level among the peroxynitrite probes.Subsequently, similar structures were synthesized for different applications.Gong et al. reported esterified Xan 30 [47] with better membrane penetrability and mitochondria targeting ability, which could image the peroxynitrite in the acute liver injury model in living cells.Li et al. introduced a piperazine ring to respond to the pH and finally realized the fluorescent imaging of the cellular peroxynitrite level as well as the mitophagy behavior [48].
Yuan et al. performed an original structure-activity relationship study of the substituents at the recognition site.(Figure 3) They discovered that pyrylium involving aryl substituents with strong electron-withdrawing groups could improve the sensitivity; meanwhile, pyrylium involving aryl substituents with strong electron-donating groups could improve the selectivity.Hence, they designed a coumarin, which was a not strong electronwithdrawing and -donating group, substituted pyrylium (Xan 32) [49] to satisfy the high requirements of both selectivity and sensitivity, and the results showed that an outstanding detection sensitivity of 4.1 nM of the detection limit as well as a high 130-fold ratiometric emission signal were realized.Employing the probe, the changing content of peroxynitrite in the diseases model involving nonalcoholic fatty liver and drug-induced liver injury was successfully visualized to unfold the functionality of a related enzyme.Zhou et al. introduced a naphthimide fluorophore in the xanthene carboxylic position.After the response, both coumarin and naphthimide fluorescence were produced to output a multicolor signal.The probe Xan 33 was applied for the early detection and evaluation of arthritis [50].
However, a similar structure-activity relationship study conducted by Tang et al. produced totally different results and response mechanisms, in which electron-withdrawing groups were installed in the 6-position of coumarin moiety.They found that because of the installation of the electron-withdrawing groups in the 6-position of coumarin, Xan 34 However, a similar structure-activity relationship study conducted by Tang produced totally different results and response mechanisms, in which electron-withd ing groups were installed in the 6-position of coumarin moiety.They found that bec of the installation of the electron-withdrawing groups in the 6-position of coumarin, 34 produced 4-(2-carboxylphenyl)-7-diethylaminocoumarin (λem = 520 nm) and 3 droxy-6-bromocoumarin (non-fluorescent) as products after the response [51]; neve less, Xan 32 and Xan 33 produced 3-carboxyl-7-diethylaminocoumarin (λem = ~468 and a ring-opening product of pyrylium (non-fluorescent).Furthermore, Xan 34 c also detect biothiols by the additional recognition site on coumarin.

Oxidation of Hydrogenated Xanthene
Peroxynitrite can oxidize t non-fluorescent hydrogenated xanthene to produce h fluorescent aromatic products.(Figure 4) Gong et al. developed 9,10-dihydroacridine 35 as the peroxynitrite detection probe.An over 100-fold fluorescence enhancement c be achieved after reacting with peroxynitrite.The probe was utilized to detect intracel peroxynitrite [52].Similar O-, Si-, and P-hydrogenated rhodamine systems were als ported.Xan 36-37 [53,54]   An over 100-fold fluorescence enhancement could be achieved after reacting with peroxynitrite.The probe was utilized to detect intracellular peroxynitrite [52].Similar O-, Si-, and P-hydrogenated rhodamine systems were also reported.Xan 36-37 [53,54] displayed a very fast response speed (<20 s); for Xan 38 [55], the relatively low response speed was probably due to the low reactivity caused by the presence of the electron-withdrawing phosphonic group.Nevertheless, Xan 35-38 [52][53][54][55] all exhibited very low detection limits at the nanomolar level, and they were all applied to fluorescent imaging of cell endogenous peroxynitrite.[56], which introduced 1 thylhydrazone as a peroxynitrite recognition group.(Figure 5) The probe was n rescent as a result of the rotational vibration of the C=N bond.Using the oxidativ  [56], which introduced 1,1-dimethylhydrazone as a peroxynitrite recognition group.(Figure 5) The probe was non-fluorescent as a result of the rotational vibration of the C=N bond.Using the oxidative ability of peroxynitrite, the hydrazine was oxidatively cleaved into the corresponding aldehyde with significant fluorescence.The response exhibited a low detection limit (57 nM) with a short response time (<60 s).The probe was applied in the fluorescent imaging of exogenous and endogenous peroxynitrite in living cells.
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW of peroxynitrite, the hydrazine was oxidatively cleaved into the correspondin with significant fluorescence.The response exhibited a low detection limit (57 short response time (<60 s).The probe was applied in the fluorescent imagin nous and endogenous peroxynitrite in living cells.Miao et al. reported Xan 40 [57] as a peroxynitrite off-on probe.The pro little fluorescence because of the photo-induced electron transfer (PeT) quen of the 3-dibenzylaminophenyl group.Upon reaction with peroxynitrite, a be was removed and formed an N-oxide product, and the fluorescent was turned Li et al. released the study on Xan 41 [58] as a peroxynitrite probe, in whi rescence was turned off by the intramolecular charge transfer (ICT) effect of th thiophenyl group.After the response with peroxynitrite, the thiol ether was t into sulfoxide and discontinued the ICT effect, thus recovering the fluorescen izing the detection of the peroxynitrite concentration.
Zhang et al. reported a novel rhodamine probe Xan 42 with the dibenzo pine core as the responsive moiety [59].Synthesized by the reaction of rhod hydroxylamine, the probe was of little fluorescence at 672 nm.However, afte ment with peroxynitrite, oxazines was generated with high fluorescence.The used to monitor the peroxynitrite level in living cells.

Dicyano-Based Compounds as Fluorophore Core
Dicyano-based compounds are characteristic of their donor-π-accepto which endows them with large Stokes shifts and excellent photostability as a r ICT process.In addition, this sort of chromophore was generally easily synth structurally modified.Thus, great attention has been attracted towards dic compounds to build probes with different functionalities [60].
The designing rule for dicyano-based peroxynitrite probes was a consen was described in Figure 6.In general, the responsive groups, such as diphenylp benzyl boronates, and 4-hydroxyphenyl, were modified on the donor moiety ICT process.The response with peroxynitrite would break the links between moiety and the response groups and release the dicyano-based chromophores fluorescence.Miao et al. reported Xan 40 [57] as a peroxynitrite off-on probe.The probe showed little fluorescence because of the photo-induced electron transfer (PeT) quenching effect of the 3-dibenzylaminophenyl group.Upon reaction with peroxynitrite, a benzyl group was removed and formed an N-oxide product, and the fluorescent was turned on.
Li et al. released the study on Xan 41 [58] as a peroxynitrite probe, in which the fluorescence was turned off by the intramolecular charge transfer (ICT) effect of the 4methylthiophenyl group.After the response with peroxynitrite, the thiol ether was transformed into sulfoxide and discontinued the ICT effect, thus recovering the fluorescence and realizing the detection of the peroxynitrite concentration.
Zhang et al. reported a novel rhodamine probe Xan 42 with the dibenzo [1,4]oxazepine core as the responsive moiety [59].Synthesized by the reaction of rhodamine with hydroxylamine, the probe was of little fluorescence at 672 nm.However, after the treatment with peroxynitrite, oxazines was generated with high fluorescence.The probe was used to monitor the peroxynitrite level in living cells.

Dicyano-Based Compounds as Fluorophore Core
Dicyano-based compounds are characteristic of their donor-π-acceptor structure, which endows them with large Stokes shifts and excellent photostability as a result of the ICT process.In addition, this sort of chromophore was generally easily synthesized and structurally modified.Thus, great attention has been attracted towards dicyano-based compounds to build probes with different functionalities [60].
The designing rule for dicyano-based peroxynitrite probes was a consensus, which was described in Figure 6.In general, the responsive groups, such as diphenylphosphonyl, benzyl boronates, and 4-hydroxyphenyl, were modified on the donor moiety to stop the ICT process.The response with peroxynitrite would break the links between the donor moiety and the response groups and release the dicyano-based chromophores with strong fluorescence.
Dic 5 [65] with the 4-nitrophenyl oxoacetyl group as the responsive unit showed a much faster response rate (<2 s) than Dic 1-4, but its detection limit was at a similar level (81 nM).The probe was used for fluorescence imaging of the endogenous peroxynitrite in zebrafish and mice.
All of the benzyl boronates derived dicyano-based probes Dic 6-10 showed analogous response times to each other.Interestingly, Dic 6 [66] and Dic 10 [70] only displayed greenchannel fluorescence, although they both have an extra conjugate phenyl ring compared Dic 7 [67] and Dic 9 [69], respectively.The boronate group of Dic 10-11 was oxidized to the hydroxyl group in situ by peroxynitrite, and the transformation generated the donor, thus forming the ICT process and producing fluorescence.As summarized in Table 1, Dic 1-4 [61][62][63][64], with diphenylphosphonyl as a rec tion group, took more than 10 min to respond, which was relatively longer than tho Dic 5-14 [65][66][67][68][69][70][71][72][73][74].This was probably due to their high intrinsic structural stability.H ever, their detection selectivity and sensitivity were not reduced.Thus, they were ployed for fluorescent imaging of the exogenous peroxynitrite in living cells.Am them, Dic 4 were further used to manifest the changing peroxynitrite concentration i rat epilepsy model with the aid of two-photon fluorescent technology [64].
Dic 5 [65] with the 4-nitrophenyl oxoacetyl group as the responsive unit show much faster response rate (<2 s) than Dic 1-4, but its detection limit was at a similar (81 nM).The probe was used for fluorescence imaging of the endogenous peroxynitr zebrafish and mice.
All of the benzyl boronates derived dicyano-based probes Dic 6-10 showed a gous response times to each other.Interestingly, Dic 6 [66]   The response rates of the probes Dic 12-14 [72][73][74], which employed 4-hydroxylphenyl as a masking group, were all found to be ultrafast, which were 5, 25, and 1 s, respectively.This phenomenon was in accordance with those of Xan 25-26 [42,43] and Xan 28 [45], suggesting the great advantage of this mask group.Possessing the superior detecting sensitivity and selectivity, the probes Dic 12-14 were applied to visualize the peroxynitrite in different diseases, including inflammation, acute liver injury, and Parkinson's disease [72][73][74].

Coumarin as Fluorophore Core
The research history of coumarin (also known as 1-benzopyran-2-one or 2H-chromen-2-one) was more than 200 years.Plenty of extensive investigations have been performed to modify the weak fluorescent parent coumarin to its derivatives with different desired photophysical properties, with a considerable amount of them now very active in the commercial market [75].
Inserting the electron-donors in the 7-position leads to a bathochromic shift to the emission wavelength; in addition, a donor-π-donor structure was formed, which facilitates the use of itself to design the ICT type probes by further introducing an electron-acceptor recognition group.(Figure 7) Xie et al. adopted this strategy and synthesized Cou 1 [76].The electron effect of the substituents of the 3-position of 7-dialkylaminocoumarin derivatives decided their emission properties.The existence of an electron-acceptor can cause a strong ICT effect and fluorescence, and the stronger the electron-withdrawing ability the group owned, the longer the emission wavelength and stronger fluorescence the probes owned.If the electron-withdrawing ability changed, the fluorescent property would change accordingly.For example, the formyl group is a medium-ability electron-withdrawing group.If it was transformed into stronger electron-withdrawing groups, the emission wavelength of the product, Cou 4, would increase [79].In reverse, after the response with peroxynitrite, the C=C bond of Cou 4 broke and generated the aldehyde product.The response was completed in a very short time with high selectivity and sensitivity to peroxynitrite.If the aldehyde group was reacted with hydrazine, the hydrazone product Cou 5 would emit only little fluorescence.However, after the reaction with peroxynitrite, the fluorescence would recover [80].

Coumarin as Fluorophore Core
The research history of coumarin (also known as 1-benzopyran-2-one or chromen-2-one) was more than 200 years.Plenty of extensive investigations have performed to modify the weak fluorescent parent coumarin to its derivatives with d ent desired photophysical properties, with a considerable amount of them now very a in the commercial market [75].
Inserting the electron-donors in the 7-position leads to a bathochromic shift to emission wavelength; in addition, a donor-π-donor structure was formed, which f tates the use of itself to design the ICT type probes by further introducing an elec acceptor recognition group.(Figure 7) Xie et al. adopted this strategy and synthesized 1 [76]  The electron effect of the substituents of the 3-position of 7-dialkylaminocoum derivatives decided their emission properties.The existence of an electron-acceptor cause a strong ICT effect and fluorescence, and the stronger the electron-withdraw ability the group owned, the longer the emission wavelength and stronger fluoresc the probes owned.If the electron-withdrawing ability changed, the fluorescent prop would change accordingly.For example, the formyl group is a medium-ability elect withdrawing group.If it was transformed into stronger electron-withdrawing groups emission wavelength of the product, Cou 4, would increase [79].In reverse, after th The ICT process is very strong in the quaternized pyridinium probe Cou 6 [81].After the addition of peroxynitrite, the diphenyl phosphinate was eliminated, and the product owned a very weak ICT process.The fluorescence undergoes a hypochromatic shift from 643 nm to 538 nm, and the emission ration displays a 153-fold increase.The probe was applied to detect the peroxynitrite in living cells.
Parthiban et al. reported a coumarin-chalcone hybrid peroxynitrite probe Cou 7 containing a tetrahydroquinoxaline ring [82].The probe displayed a large Stokes shift of 149 nm.The aryl boronate group was employed as the recognition group for peroxynitrite.The probe exhibited exceptional speed and sensitivity in detecting peroxynitrite.Palanisamy described another coumarin probe Cou 8 with a 7-position aryl boronate group as the response moiety, and the probe was applied to fluorescence imaging of peroxynitrite in a high-fat diet-induced obese mouse model [83].
Wang et al. reported a coumarin probe Cou 9-based 7-position benzyl borate as a recognition group.The probe exhibited weak ICT and weak fluorescence at 421 nm [84].After the reaction with peroxnitrie, a strong ICT and FRET process was turned on and led to an incredible 1200-fold enhancement of the fluorescence.The probe was used for fluorescent imaging of the content of peroxnitrie in cancer cells.The hydroxyl group was also converted from the borate group.The probe NCou 4 exhibited high speed and sensitivity in detecting peroxynitrite [88].The detection limit of the probe for peroxynitrite was 0.83 nM.
applied to detect the peroxynitrite in living cells.
Parthiban et al. reported a coumarin-chalcone hybrid peroxynitrite probe Co taining a tetrahydroquinoxaline ring [82].The probe displayed a large Stokes shi nm.The aryl boronate group was employed as the recognition group for perox The probe exhibited exceptional speed and sensitivity in detecting peroxynitr anisamy described another coumarin probe Cou 8 with a 7-position aryl boronat as the response moiety, and the probe was applied to fluorescence imaging of pe trite in a high-fat diet-induced obese mouse model [83].
Wang et al. reported a coumarin probe Cou 9-based 7-position benzyl bor recognition group.The probe exhibited weak ICT and weak fluorescence at 421 After the reaction with peroxnitrie, a strong ICT and FRET process was turned on to an incredible 1200-fold enhancement of the fluorescence.The probe was used rescent imaging of the content of peroxnitrie in cancer cells.The hydroxyl group was also converted from the borate group.The probe exhibited high speed and sensitivity in detecting peroxynitrite [88].The detection the probe for peroxynitrite was 0.83 nM.

1,8-Naphthalimide as Fluorophore Core
1,8-Naphthalimide and its derivatives have been employed in a variety of detecting applications owing to their good chemical stability and outstanding pho ical properties [89].(Figure 9) The switch to control the off-and-on state of the fluor was usually installed on the 4-or 5-position of the hydroxy or amino group at naphthalimide.Through protection with a recognition group on the hydroxy o group, the ICT process stopped.After the reaction with peroxynitrite, the reco group was removed, the ICT process was restored, and the fluorescence was enha 2.5.1,8-Naphthalimide as Fluorophore Core 1,8-Naphthalimide and its derivatives have been employed in a variety of analytedetecting applications owing to their good chemical stability and outstanding photophysical properties [89].(Figure 9) The switch to control the off-and-on state of the fluorescence was usually installed on the 4-or 5-position of the hydroxy or amino group at the 1,8naphthalimide.Through protection with a recognition group on the hydroxy or amino group, the ICT process stopped.After the reaction with peroxynitrite, the recognition group was removed, the ICT process was restored, and the fluorescence was enhanced.
Wang et al. reported a 4-hydroxyl-1,8-naphthalimide derivative probe Nap 1 targeting lysosomes with benzyl borate as the response group for the detection of peroxynitrite.After the addition of peroxynitrite, the fluorescence at 550 nm was greatly increased [90].The response finished in a very short period (<70 s), without interference by a lot of common metal ions and ROS, and the detection limit was only 130 nM.The probe was used for the vi-sualization of the changing levels of peroxynitrite in three types of acute liver injury mouse models.Sun et al. described a similar probe, Nap 2, with a p-toluenesulfonamide group used as the endoplasmic reticulum (ER)-targeted group [91].With the aid of ratiometric two-photon fluorescent technology, they revealed the increased exogenous peroxynitrite level at ER in the hippocampus of the depressive mouse.The aminophenol group could also be used to prevent the ICT pro droxy-1,8-naphthalimide and quench its fluorescence.Meanwhile, it wa and de-arylated.Thus, it was very suitable to be employed as the reco the peroxynitrite probe.Nap 3 [92] and Nap 4 [93] probes were built on th Both of them exhibited good sensitivity and specificity over peroxynitri used for the fluorescent imaging of the exogenous and endogenous pe living cells and zebrafish or C. elegans.
To enclose the ROS level during the ferroptosis process in the mit al. built a photocontrol peroxynitrite probe Nap 5 [94].The fluorescence on only when the probe was simultaneously exposed to peroxynitrite The aminophenol group could also be used to prevent the ICT process of the 5-hydroxy-1,8-naphthalimide and quench its fluorescence.Meanwhile, it was easily oxidized and de-arylated.Thus, it was very suitable to be employed as the recognition group for the peroxynitrite probe.Nap 3 [92] and Nap 4 [93] probes were built on the above strategy.Both of them exhibited good sensitivity and specificity over peroxynitrite, and they were used for the fluorescent imaging of the exogenous and endogenous peroxynitrite of the living cells and zebrafish or C. elegans.
To enclose the ROS level during the ferroptosis process in the mitochondria, Xie et al. built a photocontrol peroxynitrite probe Nap 5 [94].The fluorescence could be turned on only when the probe was simultaneously exposed to peroxynitrite and light irradiation.This avoided the false fluorescent signal generated outside the mitochondria.The ability to target mitochondria was endowed by the lipophilic cation group.Based on the solid evidence, the authors revealed the changing peroxynitrite level and its possible biological source during ferroptosis and suggested that the mitochondrial peroxynitrite was closely related to ferroptotic progression.
The N-methyl-D-aspartate (NMDA) receptor acted as a significant role in memoryrelated molecular biology.Lee et al. developed a 1,8-naphthalimide-based probe, Nap 6, to visualize peroxynitrite near the NMDA receptor in neuronal cells and hippocampal tissues [95].The oxidation of the boric acid by peroxynitrite led to the generation of a hydroxy group at the 5-position of 1,8-naphthalimide.The fluorescence was increased after the response.The cytotoxicity of Nap 6 was negligible, and its sensitivity and selectivity to peroxynitrite upon other ROS and RNS were extremely high.Thus, it could be used to investigate the cellular functions related to peroxynitrite near NMDA receptors.Xie et al. described an oxindole derivative probe Nap 7 for the detection of peroxynitrite [96].The probe could specifically and quickly respond to peroxynitrite.In addition, it was able to cross the blood-brain barrier.Therefore, it was used to visualize the peroxynitrite level in live animals to disclose the cerebral peroxynitrite stress state in the 4-month-old Alzheimer's disease (AD) mouse model.
Zeng et al. discovered that peroxynitrite could oxidize 4-akylamino-1,8-naphthalimide Nap 8 and cause a reduction in fluorescence [97].The ratiometric behavior could be used to detect the concentration of peroxynitrite.The recognition was highly selective and sensitive and can be used to sense the peroxynitrite in living cells and zebrafish.

Cyanines as Fluorophore Core
Cyanines have of long research history and are widely used in photo diagnostic and therapy applications due to their excellent optical properties as well as their facile structural modification.Meanwhile, cyanines have remarkable biocompatibility; thus, they are often employed in fluorescent imaging-related clinical trials in which Indocyanine Green (ICG) has been approved by the FDA [98,99].Cyanines are readily accessed by traditional pyridine or cycloalkyl ketone-initiated procedures or by furfurals derivative started protocols which were recently developed by Mo et al. [100].
When exposed to oxidants or nucleophiles, the polymethine bridge of the cyanines could be broken, or form adducts [101].(Figure 10) Additionally, the longer the bridge is, the more fragile it will be, and the more likely the destructive reactions will happen [102].Based on this phenomenon, Jia et al. developed Cyanine 3 and Cyanine 5 covalent small-molecule Cy 1 as the FRET-based ratiometric probe for the detection of peroxynitrite [103].As the probe response to peroxynitrite, the Cyanine 5 fluorescence band at 660 nm decreased, while the Cyanine 3 band at 560 nm was enhanced.The fluorescent intensity ratio between the two bands realized a 324-fold increase.The detection limit was as low as 0.65 nM, which was an incredible value among those produced from the reported peroxynitrite probes.The probe was used to semiquantitatively detect the peroxynitrite in living cells [103].In comparison, the probes Cy 2 [104] and Cy 3 [105] contained only one cyanine dye.Consequently, their reaction with peroxynitrite resulted in the observation of a relatively smaller wavelength fluorescent signal generated from a cleaved aldehyde fragment.
The conjugated system of phenol-ether center Cyanine 7 was divided in half, which was not capable of emitting typical Cyanine 7 fluorescence.However, when the phenolether was fused and turned into the quinone form, the molecule became a heptathine cyanine conjugate system and produced Cyanine 7 fluorescence.Compared to traditional Cy 7anine, quinone Cyanine 7 displayed a generally large Stokes shift of more than 100 nm.In Cy 4 [106] and Cy 5 [107], a benzyl boronate and a 1-methylindoline-2,3-dione group were installed in the phenol-ether, which could be fused by peroxynitrite and thus turn the fluorescence on.This fluorescent response was sensitive, exhibiting 55.9 and 25.5 nM of the detection limits, respectively.The probes were applied to visualize peroxynitrite in the mouse model of hepatotoxicity and stroke [106,107].
Huang et al. reported an anisole C4-substituted Cyanine 7 as a peroxynitrite probe Cy 6 [108].The probe's fluorescence was efficiently quenched by the 1,1,1-trifluoro-4-(4oxyphenyl)butan-2-one group, which produced a clean fluorescent background.After the treatment with peroxynitrite, a dienone product was formed and produced fluorescence at 630 nm.The detection limit was only 9.2 nM.Using the probe, the changing concentration of peroxynitrite in zebrafish and mice under several hypoxic conditions was evaluated, proving that the peroxynitrite produced from hypoxic stress could oxidatively damage cells and tissues.
between the two bands realized a 324-fold increase.The detection limit was as low as nM, which was an incredible value among those produced from the reported peroxyn probes.The probe was used to semiquantitatively detect the peroxynitrite in living [103].In comparison, the probes Cy 2 [104] and Cy 3 [105] contained only one cyanine Consequently, their reaction with peroxynitrite resulted in the observation of a relat smaller wavelength fluorescent signal generated from a cleaved aldehyde fragment.The conjugated system of phenol-ether center Cyanine 7 was divided in half, w was not capable of emitting typical Cyanine 7 fluorescence.However, when the phe ether was fused and turned into the quinone form, the molecule became a heptat   11) Although the hydroxyl or amine group is usually used to regulate the optical performance, the C=C bond was a reliable recognition site when the HDs were used to detect peroxynitrite.By the oxidative cleavage of the C=C bond in HDs, an aldehyde product with a lower fluorescent wavelength was generated [110][111][112].The response was fast and was generally finished in a few minutes.With the Cy 10 probe, the fluorescence intensity ratio achieved a 1728-fold enhancement after the reaction with peroxynitrite [113].The aldehyde product was well characterized by MS and NMR, proving the response mechanism.With the use of the probe, the authors realized the ratiometric image visualization of the peroxynitrite in living cells.Very similar spectral behaviors were obtained with similar structure HDs Cy 11-15 [114][115][116][117][118], regardless of their difference in the quaternized heterocycles and aryl substituents.
With the Cy 10 probe, the fluorescence intensity ratio achieved a 1728-fold enhancem after the reaction with peroxynitrite [113].The aldehyde product was well character by MS and NMR, proving the response mechanism.With the use of the probe, the aut realized the ratiometric image visualization of the peroxynitrite in living cells.Very s lar spectral behaviors were obtained with similar structure HDs Cy 11-15 [114-118 gardless of their difference in the quaternized heterocycles and aryl substituents.The hemicyanine type of peroxynitrite probes could also be constructed via the densation of the quaternized heterocycles with various aryl aldehydes.(Figure 12) aryl group in aryl aldehydes included EDG-substituted naphthalene (Cy 12 [115] and 13 [116]) and dihydronaphthalene (Cy 14 [117]), coumarin (Cy 15-20 [118][119][120][121][122][123]), porphrin (Cy 21 [124]), and rhodamine (Cy 22 [125])).The porphrin and rhodamine groups were relatively less electron-donating than others, leading to longer response times (90 min and 40 min, respectively).However, their detection sensitivity was not reduced (56 and 13 nM, respectively).Depending on the size of the conjugate system, the fluorescence wavelength ranged from 477 to 680 nm.All of these probes were capable of detecting exogenous and endogenous peroxynitrite from living cells.The C=C bonds of the above half-cyanine probes were fused after the reaction with peroxynitrite.(Figure 13) However, when there was another responsive unit in the halfcyanine structure (Cy 23-29 [126][127][128][129][130][131]), the C=C bond would be maintained, which avoids the hypochromatic shift of the fluorescent wavelength.In these probes, EWG responsive units, including benzyl boronates 1,1,1-trifluoro-4-(4-oxyphenyl)butan-2-one diphe- The C=C bonds of the above half-cyanine probes were fused after the reaction with peroxynitrite.(Figure 13) However, when there was another responsive unit in the halfcyanine structure (Cy 23-29 [126][127][128][129][130][131]), the C=C bond would be maintained, which avoids the hypochromatic shift of the fluorescent wavelength.In these probes, EWG responsive units, including benzyl boronates 1,1,1-trifluoro-4-(4-oxyphenyl)butan-2-one diphenylphosphonyl, were employed to form the ICT process and quench the fluorescence of the probes.The Cy 23 probe developed by Sonawane et al. displayed good water solubility as a result of the incorporation of a sulfonate group [126].A remarkable 32-fold fluorescent enhancement was achieved after the response with peroxynitrite.The probe was found to have a mitochondria-targeting ability, and it was applied to investigate peroxynitrite in the zebrafish inflammatory model.The probe Cy 25 exhibited a wide pH application range of pH 3-9 for the detection of peroxynitrite [128], which was utilized for the fluorescent imaging of peroxynitrite in living cells and thus diagnosing drug-induced liver injury.The probe Cy 26 could respond to peroxynitrite at a very fast rate with very good selectivity and sensitivity [129].The authors used the probe to detect the changing concentration of the cell endogenous peroxynitrite and proved that H

Summary and Outlook
In conclusion, we systemically reviewed over 100 peroxynitrite-responsive fluorescent probes based on their fluorophore core.The response pathways, in vivo peroxynitrite response data, bio-system peroxynitrite produce mode, and fluorescent bioimaging objects of the probes were summarized in detail.Based on the overall experimental results, specific and sensitive detection of peroxynitrite could be achieved for most of the reported probes.Many of the probes have been applied to reveal the important role of peroxynitrite in a great diversity of disease processes.
Although the number of articles concerning peroxynitrite responsive fluorescent probes has appeared to have had an explosive increase in the last 6 years and remarkable progress has been achieved, the design and application of a new class of fluorophore core, new responsive moiety, new application mode, and probes with higher potential in clinical translation are still challenging and greatly required.

Figure 2 .
Figure 2. Chemical structures of the oxidative cleavage xanthene probes.
displayed a very fast response speed (<20 s); for Xan 38 [55 relatively low response speed was probably due to the low reactivity caused by the ence of the electron-withdrawing phosphonic group.Nevertheless, Xan 35-38 [52-5 exhibited very low detection limits at the nanomolar level, and they were all appli fluorescent imaging of cell endogenous peroxynitrite.

Figure 3 .
Figure 3.Chemical structures of the pyrylium oxidative xanthene probes.2.1.4.Oxidation of Hydrogenated Xanthene Peroxynitrite can oxidize t non-fluorescent hydrogenated xanthene to produce highly fluorescent aromatic products.(Figure 4) Gong et al. developed 9,10-dihydroacridine Xan 35 as the peroxynitrite detection probe.An over 100-fold fluorescence enhancement could be achieved after reacting with peroxynitrite.The probe was utilized to detect intracellular peroxynitrite[52].Similar O-, Si-, and P-hydrogenated rhodamine systems were also reported.Xan 36-37[53,54] displayed a very fast response speed (<20 s); for Xan 38[55], the relatively low response speed was probably due to the low reactivity caused by the presence of the electron-withdrawing phosphonic group.Nevertheless, Xan 35-38[52][53][54][55] all exhibited very low detection limits at the nanomolar level, and they were all applied to fluorescent imaging of cell endogenous peroxynitrite.

Figure 4 .
Figure 4.Chemical structures of the oxidative hydrogenated xanthene probes.2.1.5.Others Wu et al. described a Rhodol-based probe, Xan 39[56], which introduced 1,1-dimethylhydrazone as a peroxynitrite recognition group.(Figure5) The probe was non-fluorescent as a result of the rotational vibration of the C=N bond.Using the oxidative ability of peroxynitrite, the hydrazine was oxidatively cleaved into the corresponding aldehyde with

Figure 5 .
Figure 5.Chemical structures of the other xanthene probes.

Figure 5 .
Figure 5.Chemical structures of the other xanthene probes.
and Dic 10 [70] only displ green-channel fluorescence, although they both have an extra conjugate phenyl ring
The 4-nitrophenyl oxoacetyl recognition group reacted with peroxynitrite rapidly and produced the deprotected product Cou 2 [77].They used Cou 1, together with the twophoton fluorescent imaging technology, to visualize the peroxynitrite produced in the mitochondria in an anthracycline-induced cardiotoxicity mouse model.However, Li et al. reported that the deprotected product, Cou 2, also further reacted with peroxynitrite in 5 s in the concentration range of 0.064-0.64µM, and the resulting nitration products were confirmed by ESI-MS analysis.The 3-position of coumarin could also be introduced with electron-donors to generate the donor-π-donor structure.Wei et al. developed Cou 3 as the peroxynitrite probe using the 4-nitrophenyl oxoacetyl group as a recognition moiety [78].The fluorescence of Cou 3 was quenched but could be quickly recovered with eight-fold enhancement after the response with peroxynitrite.The probe was used to image exogenous peroxynitrite formation in living cells in a biosystem.
. The 4-nitrophenyl oxoacetyl recognition group reacted with peroxynitrite rap and produced the deprotected product Cou 2 [77].They used Cou 1, together with two-photon fluorescent imaging technology, to visualize the peroxynitrite produce the mitochondria in an anthracycline-induced cardiotoxicity mouse model.Howeve et al. reported that the deprotected product, Cou 2, also further reacted with peroxyn in 5 s in the concentration range of 0.064-0.64µM, and the resulting nitration prod were confirmed by ESI-MS analysis.The 3-position of coumarin could also be introd with electron-donors to generate the donor-π-donor structure.Wei et al. developed 3 as the peroxynitrite probe using the 4-nitrophenyl oxoacetyl group as a recognition ety [78].The fluorescence of Cou 3 was quenched but could be quickly recovered eight-fold enhancement after the response with peroxynitrite.The probe was used to age exogenous peroxynitrite formation in living cells in a biosystem.

Figure 7 .
Figure 7.Chemical structures of the coumarin probes.

Figure 7 .
Figure 7.Chemical structures of the coumarin probes.

Figure 8 .
Figure 8.Chemical structures of the N-substituted coumarin probes and their responsiv nism.

Figure 8 .
Figure 8.Chemical structures of the N-substituted coumarin probes and their responsive mechanism.

Figure 10 .
Figure 10.Chemical structures of the cyanine probes.

Figure 10 .
Figure 10.Chemical structures of the cyanine probes.

2. 7 .
Half-Cyanines as Fluorophore Core As a milestone event, Yuan et al. accidentally obtained a new category of hydroxyl hemicyanine (also known as HDs) by the treatment of chloro-substituted Cyanine 7 with resorcin [109].The HDs produced NIR range fluorescence, offering an outstanding platform for the establishment of off-on probes.(Figure
2 S was able to scavenge the peroxynitrite produced in living cells.Zhang et al. reported the use of Cy 27 for the real-time fluorescent and photoacoustic dual-modal imaging of peroxynitrite in the mice tumor, achieving, respectively, 2.1-and 5.3-fold higher signals than the background [130].Xu et al. developed a dual-responsive probe, Cy 28, for the detection of viscosity and peroxynitrite [131].The fluorescent signals were at 740 nm and 580 nm, respectively.The probe showed low cytotoxicity, very good sensitivity, and high selectivity over a variety of oxidizing species as well as metal, halide, and sulfite ions.The authors employed the probe to realize the fluorescent imaging of peroxynitrite in living HepG2 cells.Int.J. Mol.Sci.2023, 24, x FOR PEER REVIEW 15 of 3 selectivity and sensitivity [129].The authors used the probe to detect the changing con centration of the cell endogenous peroxynitrite and proved that H2S was able to scaveng the peroxynitrite produced in living cells.Zhang et al. reported the use of Cy 27 for th real-time fluorescent and photoacoustic dual-modal imaging of peroxynitrite in the mic tumor, achieving, respectively, 2.1-and 5.3-fold higher signals than the background [130] Xu et al. developed a dual-responsive probe, Cy 28, for the detection of viscosity and per oxynitrite [131].The fluorescent signals were at 740 nm and 580 nm, respectively.Th probe showed low cytotoxicity, very good sensitivity, and high selectivity over a variety of oxidizing species as well as metal, halide, and sulfite ions.The authors employed th probe to realize the fluorescent imaging of peroxynitrite in living HepG2 cells.

Table 1 .
The response behavior of probes.