Research Progress on Naphthalimide Fluorescent Probes

Naphthalimide derivatives are attracting increasing attention as fluorescent probes due to their excellent modifiability and photophysical properties. Their large, rigid coplanar structures and conjugated systems enable easy generation of fluorescence. The imide units and bromonaphthalene functionality offer facile and concise modification pathways in preparing amenable probes for different metal cations, anions, small molecules, and organelles. Therefore, naphthalimide probes have great potential for application in research fields, such as analytical chemistry, cell biology, and clinical diagnosis.


Introduction
The selective detection of small chemical species by fluorescent probes facilitates the recognition of intracellular events and other analytes.[3][4] In light of this, many chemical probes have been developed for different purposes. [5][8] The scaffold is widely used as fluorescent labeling material and fluorescent dye due to its good photochemical and thermal stability, high fluorescence quantum yield, excellent chemical modifiability.Naphthalimide core is commonly constructed via the condensation reaction of 4−bromonaphthalene anhydride with primary amines.

DOI: 10.1002/adsr.202300032
The resulting conjugated cores can be excited by light of particular wavelengths, and thus possess the capability to achieve the transfer of an electron between photoexcited and ground-state molecules, [9] which is defined as photoinduced electron transfer (PET) process.The fluorophore could be further modified with a proper acceptor at the 4-position [10] to deliver a D-A  system, which would allow the charge to be redistributed within the molecule in excited state (the intramolecular charge transfer process, ICT).Additionally, naphthalimide scaffold could also be modified based on FRET (fluorescence resonance energy transfer) mechanism when donor-acceptor fluorophore pair was installed in proximity of each other.The donor in excited state could transfer its electronic energy to the vicinal acceptor in the ground state.It is worthy noting that these processes could be interrupted as off when naphthalimide probes coordinate with particular ions and molecules.Based on these colorimetric phenomena, diverse functional naphthalimide fluorescent probes that respond to different chemical species with simplicity and high sensitivity (Figure 1) have been rationally designed, synthesized, and have been found to possess the following characteristics: 1) The luminescent properties of naphthalimide probes largely depend on the substituent group R 2 .Commonly, strong fluorescence is obtained when R 2 is an electron−donating group, whereas when R 2 is an electron-withdrawing group, there is weak or no fluorescence.
2) The conjugated system with minimized bond rotations and/or vibrations is responsible for the observed high quantum yield of the naphathalimide fluorophore.3) Naphthalimide probes often have an imide group within their core skeleton and a strong electron-donating group at 4-position of the naphthalimide ring.Therefore, the naphthalimide-based systems with ICT effect can be easily constructed.4) The substituent groups R 1 and R 2 can be fine-tuned for different chemical species with excellent selectivity.
At present, naphthalimide fluorescent probes have been successfully used to detect various metal ions such as Cu 2+ , Zn 2+ , Al 3+ , Hg 2+ , Fe 3+ , and Pb 2+ , as well as anions such as F − , CN − and HSO 3 − .They have been further used in bioimaging with small molecules, and some biological organelles as targets.These works on fluorescent naphthalimides before 2019 were comprehensively reviewed by Lin et al. [11] In the current review, we focus mostly on the development of naphthalimide-based fluorescent probes after 2020, and present the most recent progress and perspectives.

Fluorescent Probes for Cu 2+ Detection
Copper ion is the third most plentiful transition element in human body and supports important functions for human health.However, an excessive intake of Cu 2+ may not only have adverse effects on the liver/kidney and gastrointestinal tract, but may also result in Wilson diseases and some neurodegenerative syndromes including Alzheimer's and Menkes. [12]o detect Cu 2+ , Ni's group designed a new 1,8-naphthalimidebased Schiff base 1 (Figure 2). [13]Probe 1, which was prepared by a simple condensation reaction between 2-butyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-carbaldehyde and 2-aminophenol, exhibited high sensitivity and selectivity for Cu 2+ over a wide pH range in H 2 O/Tetrahydrofuran (THF) (v/v = 7/3).The fluorescence intensity of free probe 1 is extremely weak.However, after binding with Cu 2+ , the fluorescence intensity is significantly enhanced and visibly changes from light blue to dark cyan.The limit of detection (LOD) of probe 1 is as low as 0.48 × 10 −6 M. The proposed binding mode of probe 1 and Cu 2+ was confirmed by 1 H NMR, Job's plot and TOF-MS experiments.Moreover, probe 1 has been used successfully to detect Cu 2+ in HeLa cells with great biocompatibility and low toxicity.
In 2021, Yang's group fabricated a new water-soluble naphthalimide-based chemical sensor 2 in dimethylsulfoxide (DMSO)/4-(2-hydroxyethyl)−1-piperazineethanesulfonic acid (HEPES) (v/v = 1/9) (Figure 2). [14]Probe 2 detects Cu 2+ within 2 min with unique selectivity and sensitivity via fluorescent quenching.The LOD of probe 2 for Cu 2+ is 45.5 nM.The stoichiometry of the 2-Cu 2+ complex is 2:1 according to Job's plot analysis and the Benesi-Hildebrand method.Probe 2 is complexed with Cu 2+ through the phenolic hydroxyl oxygen on naphthalimide and through the imine group on quinoline.This binding mechanism has also been verified by density functional theory (DFT) studies.In addition, the findings show that probe 2 is highly effective in detecting Cu 2+ in vivo and in vitro.Duygu Aydin's group has developed a new 1,8-naphthalimidebased fluorescent probe (probe 3) for nanomolar Cu 2+ sensing (Figure 2). [15]Probe 3 demonstrates a ratiometric fluorescence response toward Cu 2+ with a LOD (17 nM).We presume that the ratiometric fluorescence caused by Cu 2+ owes to the intraligand charge transfer signals, which is more important than Cu 2+ metal-ligand charge transfer.The binding stoichiometry of probe 3 and Cu 2+ is 2:1 according to a Job's plot and TOF-MS.Furthermore, probe 3 has been proven to be effective in Cu 2+ detection in real food samples, and to be an excellent chemosensor for the onsite detection of Cu 2+ without the need for sophisticated equipment.
The same group also came up with the fast responding colorimetric and ratiometric fluorescent probe 4 for Cu 2+ based on 1,8naphthalimide moiety (Figure 2). [16]Probe 4 exhibits high selectivity toward Cu 2+ and has ultralow LOD (9.53 nM).Upon binding with Cu 2+ , probe 4 displays a color change from bright yellow to blue that can be observed with naked eye.The stoichiometric ratio between probe 4 and Cu 2+ was calculated to be 1:1 based on Job's plot and MALDI TOF-MS.Furthermore, the prepared probe is not only used to detect Cu 2+ in bottled drinking water but also used for vegetables.
The new fluorescent probe 5, which contains 1,8naphthalimide and picolinate moiety, was prepared for detecting Cu 2+ by Zhu's team (Figure 2). [17]Probe 5 shows a fluorescence quenching response to Cu 2+ with a LOD (52 nM).Furthermore, upon binding with Cu 2+ , an obvious color change in the sample solution from colorless to pale yellow can be observed.In addition, the weakly fluorescent N-ethyl-4-hydroxyphenyl-1,8naphthalimide is formed by hydrolysis reaction in the presence of complex 5-Cu 2+ in the polar ethanol-HEPES buffer, which is the reason for the fluorescence quenching of 5 in the presence of Cu 2+ .Moreover, probe 5 has potential practical applications in environmental samples.
Probe 6 was synthesized by simple condensation reaction betwwen naphthalimide aldehyde and 2-picolinyl hydrazide (Figure 2). [18]Probe 6 exhibits a turn-on fluorescence response toward Cu 2+ under acidic conditions.The reason for its color change, which is readily distinguished by the naked eye under 365 nm ultraviolet (UV) lamp, is further explained by the hydrolysate of probe 6 under the assistance of Cu 2+ .The LOD is 19.4 nM for Cu 2+ .Furthermore, probe 6 can be used to image lysosomal Cu 2+ in HepG2 cells, as is demonstrated in Figure 2.

Fluorescent Probes for Zn 2+ Detection
As an essential metallic element, zinc takes part in a series of significant physiological processes in human growth and de-velopment.The imbalance of zinc ions would cause serious health problems. [19]Although several probes have been developed for Zn 2+ detection, it is still a great challenge to design and develop Zn 2+ fluorescent probes that have excellent sensing performance. [20,21]Since Zn 2+ and Cd 2+ belong to the same main group and have similar optical properties, it is challenging to design chemosensor for rapid detection of Zn 2+ without interference from Cd 2+ . [22,23]He's group developed the naphthalimide-based fluorescence sensor 7 (Figure 3) [24] and probe 8 (Figure 3) [25] Probe 7 uses monoiminoethoxy acetic acid as an acceptor, coordinates with Zn 2+ to form the distorted four-coordination geometry through the N and O atoms of iminoethoxy acetic acid arm and methoxy group.Probe 7 exhibits a turn-on response to Zn 2+ based on the mechanism of PET process.Probe 8 uses iminoacetic acid and iminoethoxy acetic acid as acceptors, and coordinates with Zn 2+ using the O atoms of the arm of iminoacetic acid and iminoethoxy acetic acid and the N atom of amidine.Both probe 7 and probe 8 can also be used to image Zn 2+ successfully in living cells.
Shanmugaraju's group reported a novel 4-mino-1,8naphthalimide hydrazine-based turn-on fluorescent probe 9 for Zn 2+ detection (Figure 3). [26]The fluorescence emission intensity of probe 9 was remarkably enhanced upon binding with Zn 2+ in THF solution and was not disturbed by coexistent metal ions.Moreover, the fluorescence titration profile shows the formation of 1:2 complex between probe 9 and Zn 2+ .Computational calculations were performed to validate the experimental observations.

Fluorescent Probes for Al 3+ Detection
Al 3+ is the third most abundant heavy metal ion in the earth crust, and it has been widely used in human activities.However, Al 3+ is a non-essential element that may have harmful effects on biological systems and environment.Furthermore, its detection seems to be more difficult over other metal ions, because high charge to radius ratio makes Al 3+ hydrophilic and hard in character. [27,28]To address this challenge, the new reversible naphthalimide-based fluorescent probe (probe 10) (Figure 4) was prepared by Li's team. [29]After probe 10 coordinates with Al 3+ , the color of the fluorescence changes from green to blue in DMSO/tris (v/v = 2:8), and the fluorescence emission shows a large Stokes shift.This experimental phenomenon effectively prevents fluorescence detection error and automatic quenching.Probe 10 exhibits excellent selectivity and sensitivity for Al 3+ and has LOD of 290 nM.Probe 10 shows cell permeability and low toxicity and thus it can be used to successfully detect aluminum ions in zebrafish and in HeLa cells.
In 2021, our team developed a new Schiff base fluorescent probe 11 (Figure 4). [30]Probe 11 has high sensitivity and selectivity for Al 3+ , undergoes a visible color change, and shows significant fluorescence enhancement and extremely low LOD (80 nM).In addition, the coordination compound that was prepared in situ using 11 with Al 3+ can be used to detect F − , indicating that probe 11 shows repeatability in alternative detection of Al 3+ and then F − .Probe 11 has good biocompatibility and thus it has been successfully applied to biosensor of aluminum ions in living HeLa cells.In the same year, our team also used 1,8-naphthalimide carbaldehyde and isoquinoline-1-carbohydrazide as the raw materials to prepare Schiff base 12 for selectively detecting Al 3+ (Figure 4). [31]When Al 3+ was added to the solution of probe 12, there was a significant color change and fluorescence enhancement, which may be due to the inhibition of light-induced electron transfer and chelation-enhanced fluorescence after probe 12 and Al 3+ were coordinated.The coordination ratio between probe 12 and Al 3+ is 1:1.The coordination mechanism of probe 12 and Al 3+ was verified using DFT calculations.The LOD of probe 12 was 52 nM, which is far lower than the World Health Organization (WHO) standard (0.9 mg L −1 ). [32]Moreover, probe 12 has been successfully used to monitor trace Al 3+ in living cells and real water samples.
Recently, Sahoo's team fabricated the luminescent chemosensor 13 for the selective detection of Al 3+ based on 2-hydroxy naphtahaldehyde and naphthalimide framework (Figure 4). [33]he chemosensor 13 shows high selectivity and sensitivity to Al 3+ with a remarkable color change from non-fluorescence to bright blue fluorescence.The LOD of probe 13 is 33 nM.In addition, probe 13 can be successfully used to detect and qualify the amount of aluminum in drugs and supplements.

Fluorescent Probes for Hg 2+ Detection
Mercury is a highly toxic heavy metal, and mercury species exist widely in nature and various human activities. [34,35]As studies have shown, even trace amounts of mercury ions (Hg 2+ ) can cause a serious hazard to human beings and environment.Therefore, it is extremely important to develop a simple analytical method for Hg 2+ detection that is highly selective, sensitive, and has a short response time. [36,37]he novel water-soluble fluorescent probe 14 based on naphthalimide was developed for Hg 2+ detection by Zhu's team (Figure 5). [38]Probe 14 exhibits high selectivity for Hg 2+ without interference from other metal ions by forming a specific imide-Hg-imide complex.Moreover, the modification of the structure of naphthalimide with morpholine gives probe 14 good water solubility, which means it can be directly to real water samples.When probe 14 binds with Hg 2+ , it emits a remarkable yellow fluorescence under UV lamp.It is worth noting that probe 14 has certain potential for imaging Hg 2+ in living cells and zebrafish.
Manivannan's team designed and synthesized the new probe (probe 15), which bears a 1,8-naphthalimide group appended with a hydrazinecarbothioamide moiety for the detection of Hg 2+ and Ag + (Figure 6). [39]Probe 15 exhibits a dual colorimetric and fluorometric response to Hg 2+ and Ag + upon excitation with 410 nm light.The LODs of probe 15 for Hg 2+ and Ag + were calculated as 20 and 40 nM, respectively.What's more, the 15-Hg 2+ complex can be reversed by EDTA but this is not true for probe 15-Ag + complex.Probe 15 was also used for the intracellular detection of Hg 2+ and Ag + ions in MDA-MB-231 and HDF cells at a concentration of 5 μM.

Fluorescent Probe for Fe 3+ Detection
As the fourth most abundant metallic element on earth, iron, especially Fe 3+ , has unique chemical properties and plays a vital role in many physiological functions. [40,41]The destruction of Fe 3+ homeostasis may lead to potential health hazards.At present, the common use of iron tools in our daily life may cause excessive Fe 3+ accumulation in the environment, which poses a toxic threat to the ecosystem by way of food chain. [42]Hence, it would be meaningful to develop rapid and highly sensitive analytical methods for detecting excess Fe 3+ in environmental and biological fields.
Lyer's team designed the on-off-on colorimetric fluorescent probe 16 (Figure 7). [43]The isomerization of the C = N of probe 16 weakens the ligand-to-ligand charge transfer, which results in a weak ICT absorption band.After the N of the imine and phenol hydroxyl groups on the probe is complexed with Fe 3+ , the isomerization at C = N is suppressed, which results in strong ICT   transformation and absorption.Furthermore, the fluorescence emission of probe 16-Fe 3+ complex returns the original state on addition of EDTA, which indicates Fe 3+ detection by probe 16 is chemically reversible.Probe 16 can be used as potential detector for Fe 3+ recognition in W138 cells.

Fluorescent Probes for Pb 2+ Detection
Rapid and quantifiable detection of lead ions (Pb 2+ ) in biological and environmental systems is crucial.Lead ion, one of the most lethal heavy metals, has severe effects on human health even at low levels. [44]Moreover, Pb 2+ can permanently exist in the environment, having harmful effects on plants and animals. [45]To analyze Pb 2+ in the ecosystem, Ye's team designed probe 17 with methylthio imide as the recognition site and naphthalimide as the fluorophore (Figure 8). [46]It has good selectivity and sensitivity to Pb 2+ in CH 3 CN/H 2 O (v/v = 1:1) solution.Under UV light, the fluorescence of the sample solution changes from light green to yellow and is not disturbed by other ions.The LOD of Pb 2+ is 4.7 nM.Probe 17 is also used for fluorescence imaging of Pb 2+ in human stromal cell lysosomes.

Detection of Anionic Fluorescent Probes Based on Naphthalimide
Anions play an important role in biology, chemistry, medicine, and environmental science.They promote blood circulation in the human body and regulate the nervous system.47∓50]

Fluorescent Probes for F− Detection
The fluoride ion is the smallest anion of the many anions, and possesses a relatively high charge density.As an essential element in the human body, F − exerts beneficial effects on human dental and bone health.The fluoride ions are easily ingested from foodstuffs and groundwater. [51]However, studies have shown that if abnormal amounts of F − are absorbed into human body, this could result in a series of health problems. [52]Hence, considerable attention has been paid to develop sensors for fluoride ion detection in real-time.
Ragini's research group developed a colorimetric and "off" probe 18 (Figure 9). [53]The probe is complexed with F − in a stoichiometric ratio of 1:3.In DMSO/H 2 O (v/v = 9:1), this probe undergoes a dual-mode color reaction with F − and shows a significant color change from orange to blue.
Guo's research group designed the selective naphthalimide−based colorimetric and fluorescent probe 19 for the "naked-eye" detection of F − via fluorescent quenching (Figure 9). [54]Probe 19 features a rapid-response color change for F − detection through the interaction between F − and N-H of 9-anthraldehyde hydrazone.The coordination ratio of probe 19 with F − measured using Job's plot is 1:1, and the LOD for F − measured using fluorescence titration is 806 nM.

Fluorescent Probes for CN− Detection
Cyanide has more attracted attention than F − because of its extremely high toxicity to environment and human health. [55]he lethal dose of cyanide ions for humans is 1.5 mg k −1 g of body weight.The maximum allowable cyanide in drinking water is 1.9 μM. [56,57]Therefore, developing effective detection methods for selectively monitoring of cyanide ions is crucially important. [58,59]Considering the above facts, Choi's team developed the turn-on fluorescent probe 20 for the detection of cyanide ions based on the naphthalimide-benzothiazole group (Figure 10). [60]Probe 20 exhibited high selectivity toward CN − in THF, with a LOD (3.35 × 10 −8 M).Furthermore, probe 20 displays two-mode aggregation-induced emission in THF/H 2 O mixed solvent system.The different water content of THF/H 2 O mixed solvent system induces the prepared probe to generate blue or cyan two-mode fluorescence emissions.

Fluorescent Probes for HSO 3 − Detection
Bisulfite has been widely used in foodstuffs, drinks and pharmaceutical products as a preservative. [61]However, excessive levels of HSO 3 − can lead to health problems.The permissible levels of HSO 3 − in food are strictly limited in many countries: the maximum safety limitation is no >0.1 g kg −1 in sugar. [62]In 2020, Figure 10.Structure of probe 20 and proposed response mechanism for CN − .Reproduced with permission. [59]Copyright 2021, Elsevier.
Qian's group developed the ratiometric probe 21 for the selective detection of HSO 3 − via 1,4-Michael addition at the nanomolar level (Figure 11). [63]Probe 21 possesses a naphthalimide fluorophore as donor and a hemocyanin as acceptor and recognition site for HSO

Naphthalimide Fluorescent Probes to Detect Small Molecules and Organelles
Naphthalimide derivatives were further developed for detecting small molecules in living cells and some organelles.Their permeability through cell membrance and strong fluorescence in living cells were well proved. [64]For instance, they demonstrate some promising results for dynamically tracking the concentration of small, potentially harmful endogenous metabolites within cells.Some organelles may also be selectively stained by naphthalimide-based fluorescent probes, such as the endoplasmic reticulum (ER).These findings greatly expand their use in cell biology and related areas.

Fluorescent Probes for H 2 S Detection
H 2 S plays a significant role in various biological processes.The fluctuation of H 2 S concentration may be involved in various human diseases, including tumors. [65]Moreover, the leakage of H 2 S into the environment during industrial activities could be fatal to humans. [66]Thus, development of feasible and rapid-response sensors for H 2 S detection is greatly desired.
In 2020, Zhang et al constructed a water-soluble naphthalimide-based luminescent probe (probe 22) bearing hydroxyl group for enhanced water solubility, and azide groups for recognizing H 2 S (Figure 12). [67]This probe successfully monitors H 2 S in HepG-2 cells and hydrogen sulfide release in laboratories or chemical plants without the use of organic solvents or surfactants.The color change is apparent with LOD as low as 50.8 nM.A logic gate circuit and the corresponding truth table were then constructed from three input systems H 2 S, HSO 3 − /SO 3 2− and other ions, which showed that the luminescent probe had excellent selectivity for H 2 S.
Sun and co-workers developed the colorimetric and turn-on fluorescent probe 23 for H 2 S detection (Figure 12). [68]Probe 23 was prepared using a naphthalimde fluorophore as the color and fluorescence signal group, a dinitrobenzenesulfonyl moiety as the H 2 S-selective cleavable group and a polyethylene glycol chain to give the prepared probe good water solubility.Probe 23 can be used as a selective chemosensor to monitor H 2 S in aqueous solutions and H 2 S gas in air with color changes visible by naked-eye.In 2022, Lee and co-workers developed an azide-containing naphthalimide derivative probe 24, which can detect H 2 S in aqueous solution (Figure 12). [69]This fluorescent turn−on probe can be selectively reduced by H 2 S, and shows the colorimetric change from colorless to pale yellow.The probe had a fluorescent peak at 550 nm and a low LOD of 2.29 μM for H 2 S.This redox mechanism for H 2 S's sensing was verified by characterizing the expected product (E)−6-amino-2-((5-(diethylamino)−2-hydroxybenzylidene)amino)−1Hbenzo[de]isoquinoline-1,3(2H)-dione. 1 H NMR, the ESI-mass diagram and the absorption and emission spectra are consistent with the proposed detection mechanism.
Recently, Lee's team further functionalized an azidecontaining naphthalimide with biotin to fabricate the biocompatible probe 25 (Figure 12). [70]Probe 25 has fluorescent peak at 545 nm and can be utilized to perform the in vivo real-time analysis of H 2 S. It showed good selectivity for H 2 S independent of the presence of other biological reductants or under biological relevant pH effects.In the presence of various H 2 S inducers and inhibitors, the functionalized probe often provides effective fluorescence response.Moreover, this biotincoupled probe can penetrate cell membranes.No cytotoxicity was demonstrated in A549 cells.

Fluorescent Probes for HClO Detection
HClO is one of the important reactive oxygen species (ROS) produced within cells.HClO is generated via the Golgi apparatus (GA) and has strong oxidation ability. [71]The excessive generation of HClO may lead to organelles dysfunction, thus, causing diseases like Alzheimer's and Parkinson's diseases. [72]n 2020, Ni et al synthesized the naphthalimide derivative probe NAP-OH (26) for monitoring the dynamic balance of HClO/ClO -(Figure 13). [73]Probe 26 has an on-off-on fluorescence response to ClO -/ascorbic acid (AA).NAP-OH can be oxidized by HClO/ClO -into NAP-O containing 2,2,6,6-tetramethyl-1-oxo-piperidinium segments.NAP-O can be then reduced using AA to restore fluorescence.This probe demonstrates a response time of >8 s and a LOD of 10.3 nM with good cycle stability.
In 2022, Wang et al developed the GA localized probe 27 to detect hypochlorous acid (HOCl), which is a product of the excess ROS of GA (Figure 13). [74]Probe 27 was constructed from naphthalimide derivatives with dimethylthiocarbamate as the recognition unit.HClO can oxidize and remove dimethythiocarbamate to trigger the ICT process, thereby resulting in a strong green fluorescence.The LOD of this probe for HOCl is 5.7 × 10 −8 M. The probe has good biocompatibility and is used to image Golgi HOCl in HeLa cells.aging and subsequently give rise to aging-related diseases. [75,76]n 2022, Qian et al developed a novel MGO-Naph-A probe (probe 28) with a naphthalimide unit as the fluorophore (Figure 14). [77]robe 28 demonstrates high sensitivity to MGO.In this probe, ophenylenediamine recognition group is used to recognize MGO.To use this probe in living cells, the targeting group morpholine for lysosome is installed so that the probe can accumulate near the lysosome and monitor the MGO concentration in the lysosomes.The fluorescent peak of this probe is close to 532 nm, and has a LOD of 1.36 μM.

Fluorescent Probes for
Phosgene is a poisonous chemical that is widely used in chemical industries.Phosgene cause lung damage in humans within 2 min even at a low level of 20 ppm and it results in death at a concentration of 90 ppm for 30 min. [78,79]In 2022, Liu et al synthesized a 1,8-naphthalimide-based fluorescent probe (probe 29) to detect phosgene (Figure 14). [80]Probe 29 itself with odiaminobenzene as recognition unit has no fluorescence.However, after rapid cycled reactions with phosgene, a large fluorescence intensity increase with low LOD of 0.23 nM was generated.Probe-loaded test paper was also examined for selectivity against other interference analytes.It is noteworthy that this probe had extremely high sensitivity for concentration below 1 ppm in the gas phase.
As a key component of the ROS, hydrogen peroxide (H 2 O 2 ) plays an essential role in the physiological processes of many organisms. [81]However, the abnormal production and accumulation of H 2 O 2 would give rise to a series of diseases and even death.Therefore, it is crucial to develop an efficient assay for the detection of hydrogen peroxide in biological fields. [82]In 2021, Peng's team developed a novel naphthalimide−based fluorescent probe (probe 30) for the ratiometric detection of H 2 O 2 in vitro and in vivo (Figure 14). [83]Probe 30 displayed excellent sensing of H 2 O 2 by virtue of its high selectivity, low cytotoxicity, low LOD (12.8 nM), and rapid response (< 40 s). the probe 30 demonstrates a large Stokes shift and dual well-resolved channels (425 nm and 550 nm) after installation of the well-known benzyl boric acid ester as a H 2 O 2 responsive moiety.Owing to the nature of weakly basic, the modification of the pyridine group makes probe 30 a lysosome-targetable chemosensor for H 2 O 2 .Furthermore, probe 30 has shown satisfactory results for H 2 O 2 detection in inflamed tissues.

Fluorescent Probes for the Detection of Lipid Droplets and Endoplasmic Reticulum, Nucleoside Polyphosphates and Esterase
Organelles, as fundamental subunits in eukaryotic cells, play a variety of indispensable roles in complex biological systems. [84]n 2022, Yu et al introduced a 1,8-naphthalimide-based fluorescent probe (probe 31) for simultaneous and discriminative imaging of lipid droplets (LDs) and ER through a dual-targeting group strategy (Figure 15). [85]Tosyl sulfoamide moiety and dimethylamine group were introduced on the naphthalimide fluorophore as ER targeting group and the LD targeting groups, respectively.Owing to its high sensitivity to slight variations in polarity, probe 31 could discriminately visualize the ER and LDs with different fluorescence.Moreover, probe 31 demonstrated a dynamic balance of LDs and ER when the living environment changed.
In 2023, Singh et al synthesized a novel hydrophilic naphthalimide-based fluorescent probe (probe 32) for simultaneous detection of adenosine triphosphate (ATP) and cytosine triphosphate (CTP) (Figure 15). [86]Probe 32 demonstrates a selective turn-on concentration-based response to ATP (5 mM) and CTP (400 mM) in DMSO/HEPES buffer solution at an emission wavelength at 440 nM.It successfully bio-images the nucleoside triphosphates in MCF-7 tumor cells, which provide an in vivo solution for detecting these nucleoside triphosphates.
In 2023, Zhang et al developed a ratiometric fluorescent probe (probe 33) that specifically targeted the ER (Figure 15). [87]The ER-specific probe 33 comprises an acetyl ester group and a sulfonamide group.Initially, the hydroxy group of 4-hydroxy-1,8naphthimide was protected by an acetoxyl group (an electronwithdrawing group) to form an acceptor--acceptor (A--A) electronic structure, and probe 33 emitted blue fluorescence.Then, the A--A structure was then converted to an acceptor-−donor (A--D) framework via hydrolysis using esterase, and the final product emitted green fluorescence and red-shifted emission from 435 to 560 nm.The attachment of a p-toluene sulfonamide group to the structure of napthalimide endows probe 33 with ERspecificity.Probe 33 has low LOD of 1.8 × 10 −4 U L −1 for esterase activity.Copyright 2023, Elsevier.

Conclusion and Prospects
Compared with other complex chemical molecules, naphthalimide derivatives have simplicity, high selectivity and high sensitivity and have broad research prospects.Moreover, naphthalimide probes have become well developed recently.In terms of detection, they can recognize various ions and some can even recognize small molecules and organelles with high selectivity.Different fluorescent mechanisms, such as PET, excited state proton transfer (ESPT) [88] and chelation enhanced fluorescence (CHEF), [89] were developed based on the naphthalimide framework.This greatly expands the application of naphthalimide in chemical sensing, materials, biological imaging, and other fields.However, from the perspective of application, there are several problems to be solved: 1) probes for detection in aqueous media have been designed, but most chemical sensing is still carried out in a mixed system of organic solvents and water.In biochemical research (especially in cell imaging research), the use of  [85] Copyright 2022, Royal Society of Chemistry.(b) Reproduced with permission. [86]Copyright 2023, Royal Society of Chemistry.organic solvents usually destroys the normal function of biological molecules.Thus, the application scope of existing naphthalimide probes may be limited.2) Biological imaging has greats prospect in medicine, and depends on the permeability of cell membrane.To obtain probes with enhanced light transmittance through the cell membrane, naphthalimide probes with excitation and emission wavelengths in the red and near-infrared region should be prior to be designed and developed.3) Most of the probes can only detect single ions, and multifunctional probes are few.Multifunctional fluorescent probes can interact with multiple analytes through the same or different action sites, which not only improves the detection efficiency but also reduces the cost.Overall, new probes with excellent performance, concise synthesis, low cost, good water solubility, high sensitivity, and low LOD are greatly needed.Such probes would be superior to existing probes and play a greater role in environmental and biological research systems.

Figure 5 .
Figure 5. Structure of probe 14 and proposed response mechanism for Hg 2+ .

Figure 6 .
Figure 6.Structure of probe 15 and proposed response mechanism for Hg 2+ or Ag + .

Figure 7 .
Figure 7. Structure of probe 16 and proposed response mechanism for Fe 3+ .

Figure 8 .
Figure 8. Structure of probe 17 and proposed response mechanism for Pb 2+ .

Figure 9 .
Figure 9. Structures of probes 18−19 and proposed response mechanisms for F − .

3 − 3 −
. Probe 21 demonstrates high specificity to HSO with short response time of 150 s and low LOD (98.1 nM).The low toxicity of probe 21 enables it to image exogenous HSO 3 − in living cells.

Figure 11 .
Figure 11.Structure of probe 21 and proposed response mechanism for HSO 3 − .

Figure 12 .
Figure 12.Structures of probes 22-25 and their proposed response mechanisms.

Ying− Guo
Liu acquired his M.S. degree in State Key Laboratory of Natural and Biomimetic Drugs from Peking University, in 2012, and his Ph.D. Degree in School of Physical and Mathematical Sciences from Nanyang Technological University, in 2020.He currently worked at Zhengzhou University as an associate professor.His research interests lie in synthetic chemistry, asymmetric synthesis and bioprobe for anti−aging process.Wen Sun received his Ph.D in 2017 from Max−Planck−Institute for Polymer Research (Germany).He joined Dalian University of Technology as an associate professor from 2018, where he became a full professor in 2020.His research focuses on developing functional dyes for biomedical applications including bioimaging, fluorescence diagnosis, and phototherapy.Until recently, he has authored more than 50 papers in international journals with a H−index of 40.He served as an associate editor of Frontiers in Chemistry from 2021.