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Construction and Application of 1,8⁃Naphthalimide⁃Based Multi⁃Analyte Fluorescent Probes

Yanqin Lai , Zhenda Xie , Manlin Fu , Xuan Chen , Qi Zhou , Jin⁃Feng Hu

Progress in Chemistry ›› 2022, Vol. 34 ›› Issue (9) : 2024 -2034.

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Progress in Chemistry ›› 2022, Vol. 34 ›› Issue (9) : 2024-2034 DOI: 10.7536/PC211117
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Construction and Application of 1,8⁃Naphthalimide⁃Based Multi⁃Analyte Fluorescent Probes
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Abstract

Life are governed by cellular complex metabolic processes. One of the on⁃going endeavours in the scientific community is to study and understand these dynamic biochemical reactions and the roles of biomolecules in sustaining life. Fluorescent probes have been widely used in the visualization of analytes in physiological and pathological processes, due to their advantages such as easy operation, low cost, high sensitivity, capability of multi⁃channel and real⁃time visualization in vivo. However, most fluorescent probes can only respond to single analyte, which is not suitable to mornitor multiple analytes in complex biological system. In recent five years, significantly more multi⁃analyte fluorescent probes based on 1,8⁃naphthimide fluorophore have been designed in biological or environmental field. Categorized by the fluorescent mechanisms, such as single fluorescent mechanism (e.g. photoinduced electron transfer (PET), intramolecular charge transfer (ICT) and fluorescence resonance energy transfer (FRET)), dual fluorescent mechanisms (e.g. PET⁃ICT, PET⁃FRET, and ICT⁃FRET) and so on, this review highlights the recent progress in designing strategies, corresponding recognition process, optical property and cell imaging of 1,8⁃naphthalimide⁃based multi⁃analyte fluorescent probes. Finally, the development trend and possible challenges of such fluorescent probes are prospected.

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1,8⁃naphthalimide / fluorescent probes / multi⁃analyte detection / fluorescent mechanisms

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Yanqin Lai, Zhenda Xie, Manlin Fu, Xuan Chen, Qi Zhou, Jin⁃Feng Hu. Construction and Application of 1,8⁃Naphthalimide⁃Based Multi⁃Analyte Fluorescent Probes. Progress in Chemistry, 2022, 34(9): 2024-2034 DOI:10.7536/PC211117

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Contents

1 Introduction
2 Multi⁃analyte fluorescent probes based on single fluorescent mechanism
2.1 Multi⁃analyte fluorescent probes based on PET
2.2 Multi⁃analyte fluorescent probes based on ICT
2.3 Multi⁃analyte fluorescent probes based on FRET
3 Multi⁃analyte fluorescent probes based on dual fluorescent mechanisms
3.1 Multi⁃analyte fluorescent probes based on PET and ICT
3.2 Multi⁃analyte fluorescent probes based on PET and FRET
3.3 Multi⁃analyte fluorescent probes based on ICT and FRET
3.4 Multi⁃analyte fluorescent probes based on ICT and TICT
4 Other multi⁃analyte fluorescent probes
5 Conclusion and outlook

1 Introduction

Since the concept of fluorescence was proposed by Irish scientist Stokes in 1852, a large number of organic fluorescent dyes have been developed as chemical and biological indicators. Up to now, with the continuous development of fluorescence spectrometer technology and confocal fluorescence microscopy, fluorescent probes have the advantages of good selectivity, high sensitivity, multi-color detection and good cytocompatibility.It is widely used in the detection and visualization of analytes such as internal environment, metal ions, reactive oxygen molecules and enzymes in cells/living bodies, among which the development of single-analyte fluorescent probes is the most common[1⇓⇓⇓⇓⇓~7]. However, there are several shortcomings in the use of single-analyte fluorescent probes: (1) There is a biological association between analytes in cells/living bodies, so in theory, multiple single-analyte probes can be used to measure multiple analytes at the same time, but different probes have different cell uptake and localization, which will lead to inaccurate detection results[8⇓~10]; (2) The occurrence of most pathological processes will induce changes in the content of multiple analytes, and using the content of a single analyte as the basis for measurement, on the one hand, is not conducive to the study of the mechanism of disease, on the other hand, it is easy to cause misjudgment of the disease; (3) Single-analyte fluorescent probes have only one fluorescence quenching group, and their detection signal-to-noise ratio is often lower than that of multi-analyte fluorescent probes. The above shortcomings have led to the narrowing of the application scope and the reduction of the reliability of single-analyte fluorescent probes in physiological and pathological processes, which has become one of the bottlenecks restricting the development of fluorescence analysis technology. In order to eliminate the problems caused by single-analyte fluorescent probes, scientists have devoted themselves to developing new multi-analyte fluorescent probes.
Fluorescent probes based on 1,8-naphthalimide as a fluorescent reporter group have the advantages of large Stokes shift and good photostability, based on photoinduced electron transfer, intramolecular charge transfer,ICT, twisted intramolecular charge transfer (TICT) and fluorescence resonance energy transfer (FRET) have been used in life science, medicine, environmental science and other fields[11,12][13⇓⇓~16][17][18⇓~20][21⇓⇓⇓⇓⇓~27]. We have also done some positive work in the design, synthesis and bioimaging of 1,8-naphthalimide multi-analyte fluorescent probes, especially in the construction of pH formaldehyde and pH palladium dual-analyte fluorescent probes based on PET ICT mechanism and the construction of viscosity hydrogen sulfide dual-analyte fluorescent probes based on TICT ICT[28,29][30]. It is gratifying that in recent years, a group of scholars at home and abroad have explored the multi-analyte fluorescent probes based on 1,8-naphthalimide, and realized the visualization of multi-analyte in biological systems. In view of this, according to the classification of fluorescence luminescence mechanism, it can be divided into single luminescence mechanism (PET, ICT and FRET), dual luminescence mechanism (PET ICT, PET FRET, ICT FRET and ICT TICT) and other ways. The research progress of detection mechanism, fluorescence response performance and imaging application of this kind of probe is summarized, and the possible challenges and development trends are put forward.

2 Multi-analyte fluorescent probe based on single luminescence mechanism

2.1 Multi-analyte fluorescent probe based on PET mechanism

Zinc ion (Zn2+) is the second most abundant transition metal ion in the human body, and during oxidative stress, Zn2+ can rapidly accumulate to lysosomes and lead to lysosomal membrane permeabilization[31]. Therefore, it is of great significance to monitor the changes of Zn2+ concentration in lysosomes. In 2016, Cho AND Ahn et al. Developed a two-photon and logic gate fluorescent probe 1 (Fig. 1) for detecting Zn2+ in lysosomes, which was prepared by introducing morpholine group and N, N di (2 pyridylmethyl) ethylenediamine (DPEN) ligand on two-photon 1,8 naphthalimide molecule[32]. The research data showed that the fluorescence emitted by probe 1 was almost negligible due to the PET effect of morpholine group and DPEN ligand. At pH 5.0, the addition of Zn2+ inhibited the PET process of the probe, resulting in fluorescence enhancement, and at pH 7.4, probe 1 showed only negligible fluorescence even after binding to Zn2+, indicating that the probe can specifically recognize Zn2+ in lysosomes. Probe 1 has strong binding affinity (binding constant Limit of detection), high selectivity (Cu2+, Co2+ and Cd2+ ions do not interfere with the fluorescence signal) and good sensitivity, and its Limit of detection (LOD) is as low as 0.18 μmol/L. Probe 1 was successfully used to determine low concentrations of Zn2+ in lysosomes of NIH 3T3 cells and in mouse brain tissue under light excitation at 900 nm. Therefore, this study provides an effective monitoring tool for the visualization of Zn2+ in lysosomes in various biological processes.
Intracellular redox cycling is a complex process that affects many important biological processes[33,34]. In 2018, Lv and Guo et al. Designed and synthesized a mitochondria-targeted HClO/GSH reversible fluorescent probe 2 by introducing selenomorpholine group and triphenylphosphine group (Scheme 2)[35]. Selenium can quench the fluorescence of 1,8-naphthalimide molecules through the PET mechanism, and is easily oxidized to selenoxide by HOCl/ClO-, so it can be selected as an ideal recognition site for HOCl/ClO-. After treatment with HOCl/ClO-, selenium was converted to selenoxide, the PET mechanism disappeared, and the fluorescence turned on. Upon addition of glutathione, a reduction reaction occurs and the fluorescence is quenched. In PBS, the fluorescence intensity of probe 2 at 540 nm increased about 50-fold with the addition of HOCl/ClO- and was linear with the concentration of ClO- (0 – 6.5 μmol/L). Its LOD is as low as 4.8 nmol/L, its response time is short (less than 2 s), and its specificity is strong. Finally, cell imaging studies confirmed that probe 2 was specifically localized to the mitochondria of living cells, not only to monitor the endogenous/exogenous HClO/ClO- concentration in RAW264.7 cells, but also to further study the relevant biological processes of redox biology in biological systems.

2.2 Multi-analyte fluorescent probe based on ICT mechanism

Phosgene and its derivatives (diphosgene, triphosgene), as a classical and indispensable intermediate of chemical products, are widely used in organic synthesis fields such as polymer materials, pesticides, spices, pharmaceuticals and so on. However, phosgene and its derivatives can also be used as a highly toxic and irritating chemical weapon, which can seriously threaten public safety[36]. In 2019, Song et al. Constructed a new fluorescent probe 3 (Fig. 3) with 1,8-naphthyl imide as the fluorophore and 2-naphthyl (2-naphthylaminophenyl) imidazole as the recognition site, which realized the differential detection of phosgene and triphosgene for the first time[37]. Due to the significant difference in nucleophilicity between chlorocarbonyl and trichloromethoxycarbonyl, the ICT effect of the reaction products of probe 3 with phosgene or triphosgene is different. It was found that the probe reacted with phosgene twice to form a cyclic product with green fluorescence, while the probe reacted with triphosgene only once to form an acyclic product with blue fluorescence. The sensor exhibited high sensitivity (LOD of 3.2 nmol/L for phosgene and 1.9 nmol/L for triphosgene) and selectivity. In addition, a simple test paper containing probe 3 was prepared by electrospinning technology, which can selectively detect phosgene with a LOD of 42 ppb (lower than the concentration of phosgene that causes clinical symptoms and signs in humans) and a short response time (≤ 10 s), and is expected to become a practical phosgene detection tool.
Similarly, taking advantage of the ICT effect difference of the products after the reaction between the probe and different analytes, in 2021, Li et al. First synthesized a ratiometric fluorescent probe 4 (Fig. 4) for the differential detection of bisulfite ion ( HSO 3 -) and ClO- through the ligation of 1,8 naphthalimide and indolesulfonate[38]. During the test, probe 4 itself has red fluorescence (λEm=620 nm), which disappears with the addition of ClO- or HSO 3 -.The corresponding products emit fluorescence of the respective emission channels (ClO-:λEm=515 nm, HSO 3 - :λEm=548 nm), thus achieving the effect of a ratiometric measurement. Further experiments showed that the probe had good water solubility (DMF ∶ PBS = 1 ∶ 99, V/V), large Stokes shift (ClO-:115 nm, HSO 3 -: 88 nm) and low LOD(ClO-:40 nmol/L ( HSO 3 -: 71.9 nmol/L). Nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS) explained the ClO- or HSO 3 - reaction mechanism of the probe, which further indicated that the specific detection of the probe was based on the ICT mechanism. Cytocompatibility and imaging experiments showed that the probe could effectively detect ClO- and HSO 3 - in cells. Finally, the probe was applied to the detection of ClO- and HSO 3 - in plasma environment, and the identification of tumor cells was achieved by using the different content of endogenous ClO- in normal cells and tumor cells.
In the study of reversible monitoring of HClO/GSH redox cycle, Ye et al. Designed and synthesized a lysosome-targeted two-photon reversible fluorescent probe 5 (Figure 5)[39]. Similar to the selenium atom of probe 2, the sulfur atom also has similar redox properties. The thioether of probe 5 can be oxidized to sulfoxide by HOCl, and then the sulfoxide can be reduced to thioether by reducing agent. Different from the detection mechanism of probe 2, the structural change of probe 5 after thioether redox changes the electron donating ability of the substituent at the 4-position of the dye, thus changing the degree of ICT effect. After the addition of 10-fold equivalent of OCl-, the absorption peak of the probe at 405 nm disappeared accompanied by the appearance of a new absorption peak at 350 nm, and in addition, the fluorescence at 505 nm was quenched. Fluorescence titration experiments showed that the probe was linear with the concentration of ClO- (3 ~ 150 μmol/L), and the LOD was 0.674 μmol/L. Competitive experiments showed that reactive oxygen species, reactive nitrogen species and anions and cations did not interfere with the fluorescence signal of the probe. Fortunately, when GSH was added to the mixture of probe 5 and ClO-, the fluorescence intensity almost returned to the original level. However, the addition of other reducing agents could not regenerate the fluorescence of the probe. These results indicate that probe 5 can continuously monitor the redox cycling process between ClO- and GSH. Finally, the colocalization experiment of Lyso Tracker Red DND-99 and probe 5 showed that the probe could well detect the level of Lyso Tracker Red DND in lysosomes.
Similarly, Wang et al. Constructed a responsive fluorescent probe 6 for HOCl/GSH detection based on the ICT mechanism using thioether as the reaction site (Scheme 6)[40]. This probe increases the probe quantum yield (Φ = 0.93) by introducing a benzothiazole group. At the same time, the probe has the advantages of high selectivity, fast response (less than 2 min), large Stokes shift (120 nm) and low LOD (0. 237 μmol/L) in the detection of HOCl, and has good application prospects. In addition, GSH can restore the fluorescence of the probe oxidized by HOCl, and the fluorescence recovery rate can reach 95% within 1 min. Other antioxidants including cysteine, Fe2+, vitamin C, vitamin E, histamine, etc. Have no response ability. Redox cycling experiments with HOCl and GSH showed that the redox cycle could be repeated at least 3 times. The successful implementation of this probe will shed light on the construction of more reversible probes.
Real-time dynamic imaging of DNA damage in tumor cells can provide important information for early diagnosis and pathogenesis of cancer[41]. Based on the fact that γ-glutamyl transpeptidase (GGT) is overexpressed in tumor cells and N-acetyltransferase (NAT) is overexpressed in DNA damage areas, Zhang and Guo et al. Chose the two enzymes as the recognition targets of DNA damage in tumor cells, and constructed a novel two-photon fluorescent probe 7 based on the sequence ICT process (Figure 7)[42]. Firstly, GGT can make the glutamic acid group hydrolyze and leave, the ICT effect is enhanced, and the probe fluorescence changes from blue to green; Then the 5 '-amino group of the probe is acetylated by NAT, the ICT effect is weakened, and the probe returns from green to blue. In the cell imaging experiment, it was found that the excellent hydrophilicity caused the probe to be located on the cell membrane surface and show blue fluorescence at the beginning (30 s), and the overexpression of GGT outside the tumor cell membrane and the targeting performance of the probe caused the probe to enter the nucleus and show green fluorescence after a period of time (250 s). Under the action of NAT, the fluorescence of the probe returned to blue at the end (2 H).
Interestingly, both naked-eye (365 nm UV lamp assisted) and two-photon (800 nm) fluorescence microscopy can observe distinct, high signal-to-noise ratio fluorescence from blue to green and back to blue in animals. To sum up, the sequence ICT process fluorescent probe is expected to become a visualization tool for real-time dynamic imaging of DNA damage.

2.3 Multi-analyte fluorescent probe based on FRET mechanism

Lysosomes have been implicated in the metabolism of formaldehyde (FA)[43]. In order to specifically detect FA levels in lysosomes, Wang AND Tang et al. Reported a lysosome-targeted acidic pH-activated FA-responsive two-photon and fluorescent probe 8 (Figure 8) in 2017[44]. The probe uses the FA recognition site homoallylicamine as a link bridge between coumarin and morpholinostilbene naphthalimide to achieve the FRET mechanism. Unlike the traditional lysosome-targeted FA probe, this probe is responsive to FA only under acidic conditions (pH = 4 to 6). At pH 5. 0, with the increase of FA concentration, the probe underwent a series of reactions such as aza-cope rearrangement and hydrolysis, and the coumarin fluorophore and naphthalimide fluorophore were separated, the fluorescence intensity of the probe increased, and the maximum emission wavelength blue-shifted from 566 nm to 506 nm. There was a good linear correlation between the fluorescence intensity at 506 nm and the concentration of FA in the range of 10 ~ 250 μmol/L, and the detection limit was 3. 0 μmol/L. In addition, the probe has significant specificity for FA and good lysosomal targeting ability. Finally, two-photon imaging experiments showed that the probe could detect FA levels in living cells (PC _ 12 cells and HepG2 cells) and tissues, and proved that acetylcysteine was a potential drug for the treatment of FA-related diseases.
Studies have shown that there is a close relationship between FA and sulfur dioxide (SO2) in cell signal transduction pathways, but the mechanism of their interaction is still unclear[45,46]. In the past two years, Lin et al. And Zhou et al. Have reported three cases of reversible fluorescent probes 9 to 11 that can detect SO2 and FA (Fig. 9)[47⇓~49]. Based on the fact that the emission wavelength of 1,8-naphthimide overlaps with the absorption wavelength of benzopyran, 1,8-naphthimide was selected as the energy donor and benzopyran as the energy acceptor to form FRET system probes. At the same time, benzopyran is susceptible to Michael addition reaction and reversible, which can be used as the reaction site of SO2/FA. The experiment proved that the probe initially showed obvious far-infrared fluorescence, while the addition of NaHSO3 led to the destruction of benzopyran conjugation and the blockade of FRET process, accompanied by a gradual decrease in fluorescence intensity and an increase in green fluorescence intensity. The addition of FA leads to the occurrence of retro-Michael addition reaction, the recovery of benzopyran conjugation, and the disappearance of green fluorescence accompanied by the recovery of far-infrared fluorescence. Further experiments showed that the response time of the probe was short, and similar fluorescence changes could be observed after the alternate addition of NaHSO3 and FA, which proved that the probe could track the dynamic interaction between SO2 and FA in living cells. Finally, all three probes were successfully used to monitor SO2 and FA levels in living cells and mice. Unlike probes 9 and 10, probe 11 further utilized the two-photon property of 1,8 naphthalimide to achieve deep tissue imaging, and the imaging results showed that its maximum penetration depth exceeded 155 μm.

3 Multi-analyte fluorescent probe based on dual-luminescence mechanism

3.1 Multi-analyte fluorescent probe based on PET-ICT mechanism

Under certain physiological conditions, there is a correlation between pH and FA concentration[50]. In 2018, Zhu et al. Reported the first dual-analyte fluorescent probe 12 (Figure 10) with 1,8-naphthyl imide as the fluorescent group and homoallylamino as the recognition group, which can detect pH and FA respectively[28]. The detection process is coordinated by two fluorescence mechanisms, PET and ICT. Initially, probe 12 has almost no fluorescence, and its homoallylamino group can specifically undergo condensation with FA, aza ￡ Cope rearrangement, hydrolysis and β ￡ elimination reaction, followed by the release of 4 ￡ hydroxy ￡ 1,8 ￡ naphthalimide, and the ICT mechanism becomes stronger, realizing the opening of green channel fluorescence (λEm=555 nm). On the other hand, the nitrogen atom of the homoallylamino group makes probe 12 pH-sensitive, with the PET mechanism disappearing under acidic conditions, achieving turn-on of the blue channel fluorescence (λEm=455 nm). Probe 12 has good response performance (FA:LOD=10μmol/L;pH:pKa=6.3) and anti-interference ability to FA and pH, and is less toxic to cells. Finally, based on the fact that the lysosomal pH value is about 4.5 to 5.5, the confocal imaging results show that the blue channel fluorescence generated by probe 12 overlaps well with the Red channel fluorescence of the commercial Lyso Tracker Red (Pearson correlation coefficient is 0.88); In addition, probe 12 was successfully applied to confocal imaging of endogenous/exogenous FA in living cells, and the above results show that it has broad application prospects in the field of dual analyte detection.
On the basis of the previous work, Zhu and Wang et al. Constructed the first dual-analyte fluorescent probe 13 (Fig. 11) that can detect pH and zero-valent palladium (Palladium(0),Pd0) separately, which is similar to the fluorescence emission mechanism of probe 12[29]. Unlike probe 12, the dual analyte recognition groups of probe 13 are located at the imide and naphthalene ring moieties, respectively. The morpholine group in the imide is a typical lysosomal targeting and pH-sensitive group; The allyl carbamate group on the naphthalene ring is the traditional Pd-responsive site. The results showed that with the decrease of pH value, the morpholine of probe 13 was protonated and the PET effect was inhibited, the existence of blue channel fluorescence turn-on (λEm=485 nm);Pd0 led to the occurrence of Tsuji Trost reaction, the ICT effect was enhanced, and the yellow channel fluorescence turned on (λEm=545 nm). The 120 nm,pKa of Stokes shift is 5.95 when the probe is used to detect pH. The Stokes shift and LOD of Pd0 were 100 nm and 9 nmol/L, respectively, indicating good detection performance. Finally, the low toxicity of the probe to HeLa cells was verified by XTT experiment, and the changes of pH value and Pd0 concentration in lysosomes of HeLa cells were successfully detected by confocal imaging technology. In addition, zebrafish in vivo experimental results confirmed the effectiveness of this dual-analyte probe in a living system.
In 2019, Wang, Guo, AND Zhu et al. Constructed a fluorescent probe 14 (Fig. 12) based on the AND logic gate property to specifically detect HSO 3 - in lysosomes[51]. The probe has two recognition sites, pH and HSO 3 -, which together lead to dye fluorescence quenching by PET and ICT mechanisms. Only when both HSO 3 - and H+ are present, the morpholine group protonation leads to the disappearance of the PET effect, and the destruction of the carbon-carbon double bond between thiophene and hemicyanine by HSO 3 - inhibits the ICT process, which is accompanied by the strong fluorescence emission of probe 14 at 524 nm. That is, the fluorescence signal will be "output" only when SO2 and H+ are "input" at the same time. The results show that the probe not only has the characteristics of high selectivity, fast response (less than 200 s) and low detection limit (LOD = 20. 7 nmol/L), but also has good biocompatibility. Based on the above excellent characteristics, the probe was used for the specific detection of HSO 3 - in lysosomes of living cells.
Hypoxia and acidic environment are the main characteristics of tumor cells[52,53]. In order to better achieve selective imaging of tumor cells, Cheng et al. Reported a bifunctional ratiometric fluorescent probe 15 for nitroreductase (NTR) and acidity detection (Figure 13)[54]. The probe introduces a morpholine group and a p-nitrobenzyl group at the No.4 position of the 1,8-naphthimide as the recognition sites of pH and NTR, respectively. The experimental data show that the probe has almost no fluorescence in neutral solution due to PET and weak ICT effects. In acidic environment, the protonation of morpholine group inhibits the PET process, showing weak blue fluorescence. In non-acidic environment, the nitro-group is reduced by NTR to release quinone, and the CO2,ICT effect is enhanced, showing weak green fluorescence. Under the simultaneous action of acidity and NTR, the inhibition of PET process and the enhancement of ICT effect occurred simultaneously, showing strong green fluorescence. Probe 15 has a rapid response to NTR and high sensitivity (LOD is 0.92μg·mL-1 at pH = 5), and can effectively detect the acidity and NTR of A549 cells by confocal fluorescence microscopy, which has potential application value in the field of precise tumor imaging.
It is important to monitor hydrogen sulfide (H2S) and hypobromous acid (HOBr) concentrations in real time and explore their complex relationships in various physiological processes[55]. Here, Wu and Zhang et al. Designed and constructed a sequence-type fluorescent probe 16 for detecting H2S and HOBr in organelles (Fig. 14)[56]. Due to the electron-withdrawing ability of the azide group, the probe itself only has weak fluorescence, and the fluorescence intensity of the probe increases about 45 times with the addition of H2S(60μmol/L), and then the fluorescence decreases with the addition of HOBr (70 μmol/L), which proves that the probe has the ability of sequence detection. The probe showed high sensitivity and selectivity for H2S(LOD=23.5 nmol/L) and HOBr (LOD = 254 nmol/L). In addition, fluorescent probe 16 has good lysosomal targeting ability, which can realize the visualization of exogenous/endogenous H2S and HOBr in RAW 264.7, HeLa and HepG2 cells. Based on the fluorescence response of the sequence, the probe successfully confirmed that acetylcysteine mainly depends on its metabolite H2S to scavenge excessive HOBr and thus play a role in cell regulation and protection.

3.2 Multi-analyte fluorescent probe based on PET-FRET mechanism

H2S and quinine oxidoreductase 1 (hNQO1) are potential biomarkers for some cancers, and Li, Yi, and Xi et al. Developed a dual-response fluorescent probe 17 for detection of hNQO1 and H2S (Fig. 15)[57]. Probe 17 has a double quenching effect, that is, the PET effect of recognizing the trimethylquinone (TLQ) moiety of hNQO1 and the FRET effect of naphthalimide and the 7 nitro 1,2,3 benzoxadiazole (NBD) moiety that can recognize H2S. When probe 13 encounters both hNQO1 and H2S, TLQ and NBD leave the probe, the fluorescence quenching effect disappears, and the naphthalimide fluorescence turns on at 535 nm (up to 400-fold intensity change). In addition, cell visualization studies showed that endogenous H2S concentration and hNQO1 activity were the highest in HT29 and HepG2 compared with HeLa, HCT116, and FHC cell lines. In addition, the exogenous H2O2 induction model, probe 17 showed significant green fluorescence, indicating that H2S and hNQO1 were spontaneously up-regulated during oxidative stress. Therefore, this kind of dual-response fluorescence system can visually display the synergistic antioxidant response of H2S and hNQO1 under oxidative stress, highlighting its application value in cancer biology research.

3.3 Multi-analyte fluorescent probe based on ICT-FRET mechanism

It was found that there was an appropriate spectral overlap between the ultraviolet absorption of 1,8 naphthalimide (acceptor) and the fluorescence emission of coumarin (donor), and the FRET process could be achieved through a suitable linker[58,59]. Based on this principle, in 2019, Wu and Bhuniya et al. Used two fluorophores, coumarin and naphthalimide, to prepare a multicolor fluorescent probe 18 that can detect hydrogen peroxide (H2O2) and H2S (Fig. 16)[60]. The probe selects the borate ester group as the recognition group for H2O2 and the azide group as the recognition group for H2S (ICT mechanism). Upon addition of H2O2, probe 18 exhibited blue fluorescence at 460 nm (LOD=12.1×10-5μmol/L); After the addition of H2S, it showed green fluorescence at 550 nm (LOD=52.3×10-5μmol/L); Its signal strength is not interfered by other substances. Finally, the probe can detect exogenous/endogenous H2O2/H2S in tumor cells through a multicolor channel and realize multicolor fluorescence visualization in zebrafish.
Similar to the construction mechanism of the above probe, Wang and Chen et al. Constructed the first dual-reaction site FRET fluorescent probe 19 for simultaneous detection of HClO and H2O2 in 2020 (Fig. 17)[61]. The probe selects N, N dimethylthiocarbamate as the recognition group of HClO and borate as the recognition group of H2O2 (ICT mechanism)[62]. After the addition of HClO, the fluorescence of probe 19 at 452 nm was enhanced by nearly 213-fold (λEx=400 nm,LOD=28.2 nmol/L); After the addition of H2O2, the fluorescence of the probe at 550 nm was enhanced by about 260 times. (λEx=450 nm,LOD=64.6 nmol/L);HClO and H2O2 co-incubation, the probe showed fluorescence enhancement at 550 nm (λEx=400 nm) due to the FRET process. Probe 19 has the advantages of low toxicity and good tissue penetration ability, and can simultaneously detect HClO and H2O2 in cells and tissues.

3.4 Multi-analyte fluorescent probe based on ICT-TICT mechanism

Many studies have shown that the concentration and viscosity of small molecules change in many physiological or pathological processes[63,64]. In 2018, Yang and Zhang et al. Constructed a viscosity and peroxynitrite (ONOO-) -sensitive dual-response fluorescent probe 20 (Fig. 18) by introducing a hemicyanine group to a 1,8-naphthyl imide dye[65]. The probe provides two fluorescence signals: sensitivity to ONOO- on green fluorescence (λEm=510 nm) based on the ICT mechanism and sensitivity to viscosity on red fluorescence (λEm=610 nm) based on the TICT mechanism.
In addition, the introduction of the morpholine group realizes the localization of subcellular lysosomes. The experimental results show that when the viscosity increases from 1.0 cP to 1410 cP, the fluorescence of fluorescent probe 20 at 610 nm increases by 50 times. In addition, with the addition of ONOO-, the fluorescence of the probe at 510 nm increased by 3.5-fold (LOD = 0.24 μmol/L) in a short time. Finally, the changes of viscosity and ONOO- in lysosomes of RAW 264.7 cells were successfully monitored by fluorescent probe 20 using confocal imaging technology.
Parkinson's disease (PD) is a common neurodegenerative disease in middle-aged and elderly people, and its pathological condition is closely related to the changes of Parkinson's disease and viscosity levels.Development of dual-analyte analysis tools that can monitor Parkinson's disease and viscosity in PD model will help to clarify the pathogenesis of PD. Therefore, Hu, Fu, and Zhu et al. Prepared a viscosity and H2S dual-analyte fluorescent probe 21 with 1,8-naphthalimide dicyanoisopherone as a fluorophore (Fig. 19)[30]. Due to the TICT and ICT effects, the fluorescence of the probe itself is negligible. After the H2S treatment, the sulfonyl group reacts with the H2S to generate a hydroxyl group, and the red channel fluorescence is turned on; At the same time, the high viscosity medium can inhibit the rotation of the single bond, and the yellow channel fluorescence is turned on. In the detection process, the response performance of the probe to viscosity and H2S is higher than that of most reported probes (H2S:LOD=79 nmol/L; Viscosity: fluorescence enhanced by about 50 times). The research proves that the probe 21 can not only be used for the detection of viscosity and H2S in HeLa cells under different fluorescence channels, but also be used for the visual research of rotenone-induced cell and zebrafish PD models, proving that the occurrence of PD can lead to the simultaneous increase of H2S and viscosity levels.

4 Other multi-analyte fluorescent probes

Hypoxia is a common feature of solid tumors, but the changes in intracellular adenosine triphosphate (ATP) levels during hypoxia are not known. Therefore, the study of cellular hypoxia by monitoring intracellular NTR and ATP will help to better reveal the nature of cancer cells. In 2018, Ma et al. Designed and prepared fluorescent probe 22 (Figure 20) by combining rhodamine and 1,8-naphthalimide dyes[66]. NTR reduced the nitro group of 1,8-naphthyl imide to amino group, and significant fluorescence was observed at 520 nm (LOD = 0.12 μg/mL). Correspondingly, the interaction between ATP and the diethylenetriamine of the probe led to the opening of the rhodamine spiro ring and induced the emission of fluorescence at 580 nm (LOD = 0.05 mmol/L). Fortunately, in the presence of ATP and NTR, the signals of the two fluorescence channels hardly interfere with each other. Finally, the probe was used to monitor NTR and ATP levels in hypoxic cells by dual-channel cell imaging, and demonstrated that ATP concentration decreased in hypoxic tumor cells, contributing to the search for new cellular hypoxia markers.
Real-time monitoring of the dynamic changes of H2O2 and ATP levels in mitochondria is of great research significance[67]. Therefore, in 2020, Tian et al. Developed a two-photon excited fluorescence lifetime probe 23 (Fig. 21) that can simultaneously, selectively, and in real time detect changes in H2O2 and ATP in mitochondria[68]. The design concept of the probe is similar to that of probe 22: the naphthalimide fluorescence is quenched by the pyridine cation, and the rhodamine fluorescence is quenched by the spirolactam ring; In addition, pyridine cations can also promote the accumulation of the probe in mitochondria. After H2O2 treatment, the benzilic acid ester bond was broken, which showed a strong blue fluorescence signal. ATP binds to multiple amino groups of the probe, giving an intense red fluorescence. Interestingly, these two emission peaks do not interfere with each other in the presence of both H2O2 and ATP. Thus, probe 22 has the ability to detect both H2O2 and ATP. Finally, the changes of H2O2 and ATP content in mitochondria under different stimulation conditions were detected by fluorescence lifetime imaging of the probe. The successful implementation of this work revealed the correlation of H2O2 and ATP within cellular mitochondria induced by oxidative stress.
H2S and cysteine (Cys) are ubiquitous biological thiols in living systems, and their abnormal concentration levels are associated with many diseases[69,70]. Based on this, Han and Dong et al. Constructed a novel fluorescent probe 24 (Fig. 22), which introduced triethylene glycol to increase water solubility and introduced 7-chlorphenoxazole 4-sulfonimide (CBD) as a recognition group to achieve specific detection of Cys and H2S under dual fluorescence channels[71]. The study of fluorescence response and reaction mechanism showed that, on the one hand, the nucleophilicity of HS- was enough to replace chloride ion and lead to the cleavage of sulfonamide bond, which turned on the fluorescence of 1,8-naphthyl imide at 535 nm with LOD of 11.5 nmol/L; On the other hand, the substitution of the amino group of Cys for the sulfur atom after the substitution of RS- for chloride ion promoted intramolecular rearrangement, which turned on the fluorescence of benzofurazole at 571 nm with a LOD of 11.6 nmol/L. The probe has wide application pH range, can distinguish Cys and H2S from other biological thiols (GSH and Hcy), has low cytotoxicity, and can detect endogenous/exogenous H2S and Cys in MCF 7 cells through dual emission signals.

5 Conclusion and prospect

To sum up, in recent years, multi-analyte fluorescent probes with 1,8-naphthimide as the fluorescent reporter group have become one of the key research directions in the field of fluorescent probes. In this paper, the recognition mechanism, molecular design and spectral changes of different fluorescent probes are introduced, and the visualization applications of these fluorescent probes in various physiological and pathological processes are discussed. It can be seen that based on the characteristics of 1,8-naphthalimide fluorophore, most fluorescent probes have the advantages of large Stokes shift, high selectivity, and high sensitivity.
However, the molecular structure of 1,8-naphthalimide multi-analyte fluorescent probes is complex, and the design and preparation are difficult, so the efficiency of scientific research output is not as good as that of single-analyte fluorescent probes. In addition, the design and preparation of some reported 1,8-naphthalimide multi-analyte fluorescent probes still need to be improved: (1) most probes can only be used for qualitative detection of multi-analytes; (2) there is still a lack of multi-analyte fluorescent probes with organelle targeting properties; (3) Some multi-analyte fluorescent probes have poor water solubility, which is not conducive to their imaging studies in cellular environments. Therefore, the follow-up research still needs the unremitting exploration of the vast number of scientific researchers. It is also hoped that the summary of the existing work can provide new research ideas for future work.

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Funding

Taizhou Science and Technology Project(2003gy14)

Project of Taizhou University(2019PY021)

National College Students Innovation and Entrepreneurship Training Program(202010350033)

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