A Naphthalimide-Based Fluorescence “Off-on-Off” Chemosensor for Relay Detection of Al3+ and ClO−

A novel Al3+ chemosensor NPA was designed and synthesized on basis of the mechanism of ICT and CHEF. Upon addition of Al3+, the probe NPA displayed a bright green fluorescence under UV radiation and visual color change from yellow to colorless. Spectrum titrations showed that NPA could be recognized as a fluorescent turn-on probe with 10−8 M detection level. The probe was successfully applied in real water sample and test paper. More important, NPA-Al3+ complex were used as a fluorescent turn-off probe for the detection of ClO− with the detection as low as 2.34 × 10−8 M. The performance of NPA to Al3+ and NPA-Al3+ complex to ClO− demonstrated that NPA could be served as a sensitive probe and exhibit INHIBIT logic gate behavior with Al3+ and ClO− as inputs.


INTRODUCTION
Aluminum, the third most abundant metal element in the Earth's crust, is widely used in human's daily life including pharmaceuticals, textile, kitchen utensils, and paper industries Kaur and Kaur, 2017). Moreover, Al 3+ ion widely exists in the environment, normally in natural waters and many plants, which can enter the human body through foods and water. However, excess aluminum can damage the human nervous system, and has tightly relation to many diseases such as Alzheimer's disease, Parkinson's disease, anemia, dementia, encephalopathy, and gastrointestinal diseases (Helal et al., 2013;Liu et al., 2014Liu et al., , 2017Jiang et al., 2015;Zeng et al., 2018). In addition, hypochlorous acid (HOCl), known as an important reactive oxygen species (ROS) in many living organisms, plays a vital role in many biological processes (Zhu et al., 2014). Hypochlorite is a key microbicide that is used for natural defense because it behaves as a strong nucleophilic non-radical oxidant (Shi et al., 2010). However, it also results in many pathological diseases, especially is implicated in inflammation-associated injury including hepatic ischemia-reperfusion injury, lung injury, rheumatoid arthritis, and atherosclerosis. Moreover, excessive or misplaced of hypochlorite can also cause detrimental effect on tissues (Lei et al., 2017;Lin et al., 2017;Shen et al., 2017). Therefore, the determination of Al 3+ and ClO − in biological samples is of great importance.
In the last few decades, the designing a molecular system, which displayed significant changes in electronic, magnetic, or optical signals even at low concentration during the detection for a specific guest species, was a hot topic for many researchers Liu et al., 2014;Lim et al., 2017). Among them, small-molecule optical probes, which exhibited many merits including high selectivity and sensitivity, tunability, simple manipulation, and direct visualization, were employed as a powerful tool in the facet of trace analysis and rapid detection for various analytes (Qin and Yang, 2015;Wang et al., 2015;Simon et al., 2016;Liu et al., 2017;Murugan et al., 2017). Up to now, lots of papers that one probe only for the detection of one special analyte such as Al 3+ ion Gupta and Kumar, 2016;Xie et al., 2017) or Hypochlorous acid  had been reported, but the design idea, one fluorescent probe for successively recognition of two different analytes, had gained increasing attention in considering its high efficiency and potential cost reduction Ye et al., 2014;Zhao et al., 2015;Wen and Fan, 2016;Xie et al., 2016;Zhai et al., 2016;Zhu et al., 2016;Wu et al., 2017). Many excellent probes had been reported for relay recognition of two different ions through fluorescent "off-on-off " (Borasea et al., 2015;He et al., 2015;Zhao et al., 2015;Rai et al., 2016;Bhattacharyya et al., 2017;Das et al., 2017;Jo et al., 2017;Dwivedi et al., 2018;Feng et al., 2018;Lim et al., 2018) or "on-offon" (Diao et al., 2016;Zhao et al., 2016;Sarkar et al., 2017) mode. Moreover, 1, 8-naphthalimide fluorophore, holding many excellent photophysics properties, such as high photostability, visible absorption and fluorescence emission, large Stokes' shift and high fluorescence quantum yield (Dimov et al., 2014;Yu and Zhang, 2014;Li et al., 2018;Fu et al., 2019), was successfully applied in designing fluorescent probes toward various analytes.
Taking above statements into consideration, in this paper, we prepared and characterized a 1, 8-naphthalimide-based fluorescent probe NPA for the detection of Al 3+ embodied in colorimetric and fluorescent turn-on was ascribed to the co-contribution of intramolecular charge transfer (ICT) SCHEME 1 | Synthesis of probe NPA. and chelation enhanced fluorescence (CHEF). The binding stoichiometry between the NPA and Al 3+ had been clarified according to various spectroscopic measurements and data analysis. Moreover, the performance of in situ formed NPA-Al 3+ complex for the detection of ClO − was investigated. The NPA-Al 3+ complex exhibited a fluorescent turn-off response in the detection of ClO − . Inspiringly, to the best of our knowledge, a single probe for sequential detection of Al 3+ and ClO − through fluorescence "off-on-off " mode is scarcely documented.

Reagents and Instrument
All the solvents and reagents (analytical or spectroscopic grade) were purchased commercially and used as received. O] were obtained from commercial suppliers, and used as received without further purification. 1 H NMR and 13 C NMR spectra were recorded on a AV-600 spectrometer in DMSO-d 6 solution. The chemical shifts (δ) are reported in ppm and coupling constants (J) in Hz relative to TMS (0.00) for 1 H NMR and 13 C NMR. Mass spectra were measured on a Waters Xevo UPLC/G2-SQ Tof MS spectrometer. Absorption spectra were recorded using a Pgeneral TU-1901 UV-vis spectrophotometer. Fluorescence measurements were performed on a Perkin Elmer LS55 fluorescence spectrometer at room temperature.

Synthesis of the Probe NPA
NPA was prepared in four steps from naphthalimide as the starting material, as shown in Scheme 1. Compound 1-4 were synthesized using the literature method Li et al., 2018).

Preparation of Solutions for Spectral Detection
All stock solutions (10 mM) including metal cations and the anions were prepared with distilled water, while the stock solution of compound NPA (0.1 mM) was prepared in CH 3 OH (100 mL). One milliliter of NPA solution (0.1 mM) was diluted in 9 mL CH 3 OH to make the test solutions (10 µM). For fluorescence measurements, excitation was set at 400 nm, and the excitation and emission slit widths were 10 and 10 nm, respectively. All spectroscopic measurements were performed in CH 3 OH at room temperature.
Stock solution of NPA (0.1 mM) was prepared by NPA (0.01 mmoL) was dissolved in CH 3 OH (100 mL), the test solutions of NPA (10 µM) was prepared by adding 1 mL of NPA stock solution (0.1 mM) was diluted in 9 mL CH 3 OH in CH 3 OH.
Stock solutions (10 mM) including the metal cations and the anions were prepared with ultrapure water, respectively. For spectrum measurement, the test solutions were prepared by adding certain amount of stock solution using a pipette into NPA stock solution. For fluorescence measurements, excitation was set at 400 nm, and the excitation and emission slit widths were 10 and 10 nm, respectively.

Determination of Binding Constant and Detection Limit
According to the fluorescence intensity data, the binding constant of NPA with Al 3+ was calculated based on the modified Benesi-Hildebrand equation  as followed. Where, F max, F and F min are the fluorescence intensities of NPA in the presence of Al 3+ at saturation, at an intermediate Al 3+ concentration, and absence of Al 3+ , respectively. K is the stability constant.
The limit of detection (LOD) of Al 3+ was calculated on the basis of 3δ/K according to the fluorescence changes, δ is the standard deviation of the blank solution, and K is slope of calibration curve (Borasea et al., 2015;Zeng et al., 2018).

RESULTS AND DISCUSSION
The UV-Vis Spectra Responses of Probe NPA Various metal ions: K + , Na + , Ca 2+ , Mg 2+ , Ba 2+ , Pb 2+ , Cu 2+ , Co 2+ , Ni 2+ , Fe 2+ , Cd 2+ , Hg 2+ , Mn 2+ , Ag + , Zn 2+ , and Al 3+ were used to observe the selectivity of probe NPA (10 µM) and their UV-vis spectra were measured in CH 3 OH. As shown in Figure 1, NPA exhibited a characteristic absorbance band at 400 nm in the presence of tested metal ions (K + , Na + , Ag + , Ca 2+ , Mg 2+ , Ba 2+ , Pb 2+ , Cu 2+ , Co 2+ , Ni 2+ , Fe 2+ , Cd 2+ , Hg 2+ , Mn 2+ , Zn 2+ ) and there was almost no difference compared with that of NPA itself. However, upon the addition of Al 3+ , the maximum absorbance peak of NPA was blue-shifted with 21 nm from 399 to 378 nm, and the solution color was changed from yellow to colorless. This result might attribute to the decrease of conjugated degree resulting from the decrease of electron donating ability of piperazine ring to the naphthalimide after the complexation with Al 3+ (De Silva et al., 1997;Urano et al., 2015;. In addition, the quantitative sensing of Al 3+ ion was elucidated by UV-vis titration of probe NPA in CH 3 OH solution (Figure 2). NPA alone showed a major absorbance band at 399 nm, a new band at 378 nm appeared upon addition of Al 3+ and the intensity increased gradually and then kept constant until the amount added of Al 3+ was more than 20 µM. The good relationship was found between the ratio of absorbance (A 378 /A 399 ) vs. the concentration of Al 3+ (Supplementary Figure 4), and the limit of detection (LOD) for Al 3+ was calculated as 3.61 × 10 −8 M on the basis of 3 δ/K (where δ is deviation of the blank signal and K is slope of calibration curve; Borasea et al., 2015;Zeng et al., 2018). These results indicated that NPA could be used as an absorbance-ratiometric probe for the detection of Al 3+ .

Fluorescence Spectral Responses of NPA
The fluorescence properties of NPA were investigated in the presence of a variety of metal ions in CH 3 OH (Figure 3). NPA itself exhibited almost no fluorescence emission upon excitation at 400 nm. However, the addition of Al 3+ (5 equiv.) into NPA induced an obvious fluorescence enhancement at 505 nm, which might be attributed to the CHEF effect through the formation of a rigid system after binding with Al 3+ Zeng et al., 2019). In contrast, few fluorescence changes were observed in the presence of other tested metal ions, demonstrating that NPA was highly selective for Al 3+ over competing metal ions.
To further validate the utility of probe NPA, the fluorescence titrations of NPA were performed by gradually increasing  the concentration of Al 3+ (Figure 4). The emission intensity of NPA at 505 nm progressively increased upon addition of Al 3+ , and the emission intensity remained constant when the quantity of Al 3+ added was over 15 µM (Figure 4, Insert). Moreover, the fluorescence enhancement of sensor NPA depending on the concentration of Al 3+ was in a linear Frontiers in Chemistry | www.frontiersin.org   Figure 5). The detection limit (LOD) for Al 3+ ion according to fluorescence changes was measured to be 2.03 × 10 −8 M on the basis of 3 δ/K (Borasea et al., 2015;Zeng et al., 2018). The result clearly demonstrated that the probe NPA was highly efficient in sensing Al 3+ at nanomolar level.

manner (Supplementary
Further, tolerance of fluorescence intensity of NPA-Al 3+ complex in presence of other metal ions was tested (Figure 5).  All competitive metal ions had no obvious interference on the Al 3+ detection, indicated that NPA-Al 3+ complex was hardly affected by these coexistent metal ions. Accordingly, NPA can be used as selective fluorescent probe for Al 3+ determination without disturbance of other competing metal ions.

Response Time Studies
As for an excellent fluorescent probe, fluorescent stability and response time are two crucial factors. Hence, the changes of fluorescent intensity of NPA and the response time of NPA to Al 3+ were investigated (Supplementary Figure 6). The results demonstrated that the fluorescence signal of NPA remained stable for a long time in the absence of Al 3+ , implying the NPA had good fluorescence stability. Moreover, after addition of Al 3+ , the fluorescence intensity of NPA at the 505 nm reached the maximum within 10 s and maintained constant more than 3 min, indicating the unique feature of high complexation ability of NPA with Al 3+ .

Binding Stoichiometry and Sensing Mechanism
The total concentration of Al 3+ and ligand was 50 µM with the molar ratio of Al 3+ changed from 0.1 to 0.9. The fluorescence mission was measured for each sample in CH 3 OH with the excitation wavelength at 400 nm. The maximum point appeared at a mole fraction of 0.5 (Figure 6), indicating a 1:1 stoichiometry of the binding mode between NPA and Al 3+ , and which was further clarified by a peak at m/z 594.1575, which was assignable to [NPA -2H + + K + + Al 3+ + NO − 3 ] + (calcd. m/z 594.1547) in the ESI mass spectrum (Figure 7).
According to the above results, the association constant was calculated as 7.06 × 10 4 M −1 according to the fluorescence titration data (Supplementary Figure 7), basing on the Benesi-Hildebrand plot .
To better evaluate the interaction of NPA with Al 3+ , 1 H NMR spectra of NPA were constructed in the absence and presence of different equivalent Al 3+ in DMSO (Figure 8). The protons of H 1 and H 2 of NPA at around 2.80 and 3.27 ppm were shifted downfield to 3.42 upon the addition of Al 3+ . In addition, the peak of NPA at around 3.10 ppm was also shifted downfield to 3.42 ppm, indicated that the two nitrogen atoms of piperazine ring might coordinate the Al 3+ . Moreover, the proton of amide was disappeared supporting the occurrence of deprotonation upon the interaction of amide with the Al 3+ . According to the above results, the two nitrogen atoms of piperazine ring and the amide nitrogen atom might coordinate with Al 3+ (Scheme 2).

Reversibility of NPA Toward Al 3+
The fluorescence intensity enhancement of NPA on interaction with Al 3+ was found to be reversible by using EDTA as the recovering reagent. When the strong metal ion chelating agent EDTA was added gradually to a mixture of NPA-Al 3+ , the UV-vis absorbance spectral and fluorescence spectral of the solution of NPA-Al 3+ almost recovered to the original condition in the absence of Al 3+ (Supplementary Figures 8, 9). These results indicating that recognition process can be made reversible merely by treatment with EDTA. Besides, as for an excellent chemosensor, reversibility and regeneration are crucial for the fabrication of apparatus to sense Al 3+ .

Application of NPA for Al 3+ Analysis in Test Paper
Interestingly, the noticeable colorimetric changes of the system and qualitative recognition of Al 3+ in solution were confirmed by the simple test strips. The required test strips were immersed in CH 3 OH (10 mL) including Al 3+ and then dried in air. The color changes in various concentrations of Al 3+ under sunlight and under 365 nm UV light were illustrated in Supplementary Figure 10. Visual color changed from yellow to colorless and bright-green fluorescence increased gradually with FIGURE 8 | 1 H NMR spectra of NPA with Al 3+ in DMSO d 6 .
Frontiers in Chemistry | www.frontiersin.org  increasing amounts of Al 3+ were clearly observed. This result showed that NPA might be used as a portable detector for Al 3+ .

Application of NPA for Al 3+ Analysis in Water Samples
In order to verify the practical application of NPA, ultrapure water and tap water samples were analyzed by the proposed fluorimetric method. The results were summarized in Supplementary Table 1 with satisfactory recovery of Al 3+ , indicating that the present fluorescent probe seem to be applicable for the determination of Al 3+ in environmental analysis.

Fluorescent "On-Off" Sensing of ClO − by NPA-Al 3+ Complex
In order to further explore the performance of NPA-Al 3+ complex to different anionic, the fluorescence spectra of NPA-Al 3+ complex were investigated in presence of various 100 µM FIGURE 12 | Fluorescence spectrum (A) and UV-vis absorbance spectrum (B) of NPA in CH 3 OH while adding Al 3+ (3 eq.) and ClO − (10 eq.) to the solution. SCHEME 2 | Proposed mechanism for the fluorescent sensing of NPA and its in situ complex with Al 3+ and ClO − .  (Figure 9). The result showed that fluorescence intensity of NPA-Al 3+ complex was affected to tolerable degree by I − , H 2 O 2, and BF − 4 except for ClO − , which completely quenched the fluorescence of NPA-Al 3+ complex. This result suggested that the NPA-Al 3+ complex could distinguish ClO − anion over other ROO − , H 2 O 2 , OH − , NO − , Cl − , Br − , NO − 3 , BF − 4 , ClO − 4 by fluorescence "on-off " mode. Furthermore, competition experiments were carried out by addition of various anions (100 µM) to the solution of NPA-Al 3+ in the presence of 100 µM of ClO − . As shown in Figure 10, competitive anion had no prominent interference with the determination of ClO − , which meant that NPA-Al 3+ could perform as a highly selective probe for ClO − via a fluorescence "turn-off " mechanism.
To further investigate the binding ability and limit of detection of the NPA-Al 3+ complex with ClO − . The complex sensing capability was studied in detail analysis with fluorescence titration (Figure 11). Upon addition of ClO − to the solution containing NPA-Al 3+ complex, fluorescent intensity at 505 nm gradually diminishes, and 40 µM of ClO − could lead to complete fluorescent quenching (Figure 11, inset). Obviously, the fluorescence intensity of NPA-Al 3+ complex depending on the concentration of ClO − was in a linear manner (Supplementary Figure 11). According to the fluorescence titration data, the detection limit of NPA-Al 3+ complex was determined and be found to 2.34 × 10 −8 M (Supplementary Table 3). Furthermore, fluorescence quenching in situ also indicated that the occurrence of dissociation of NPA-Al 3+ complex, and the NPA was regenerated upon the addition of ClO − , which further supported by the verified experiments as followed. Firstly, the peak at m/z 469.2438 appeared in ESI-MS spectrum of NPA-Al 3+ complex upon addition of ClO − in CH 3 OH, which was assignable to [NPA+H + ] + (calcd m/z 469.2451; Supplementary Figure 12) compared with the ESI-MS spectrum of NPA itself (Supplementary Figure 13). Moreover, the UV-vis absorbance and fluorescence spectrum of NPA-Al 3+ complex in the absence and presence of ClO − were measured (Figure 12), respectively. The result showed that both of them were almost the same as that of NPA itself. Lastly, the titration of 1 H NMR (Supplementary Figure 14) and 13 C NMR (Supplementary Figure 15) were also investigated to clarify the sensing mechanism. The result displayed that NPA was regenerated to some extent upon the addition of ClO − to the NPA-Al 3+ complex. The above result implies that the NPA-Al 3+ complex could act as a fluorescent turn-off probe for ClO − recognition. According to above results, the "off-on-off " mechanism was achieved with sequence specificity (Al 3+ and ClO − ) in CH 3 OH (Scheme 2). In addition, the results of the comparison between NPA and those reported sensors (one fluorescent probe for successively recognition of two different analytes) were summarized in Supplementary Table 2.

Application as Logic Gate Function
NPA alone displayed very weak fluorescence emission. Maximum emission at 505 nm appeared after coordination of NPA with the Al 3+ . Moreover, when ClO − was added to the above solution, the emission intensity at 505 nm was quenched. In addition, the fluorescence "off-on-off " response for Al 3+ and ClO − were carried out (Supplementary Figure 16), and the result showed that cycle times was more than 5 according to NPA fluorescent signal upon the alternate addition of Al 3+ and ClO − . Due to the remarkable fluorescence changes of probe NPA in the presence Al 3+ and ClO − , it would be able to construct a two output combinatorial logic circuit with two signal inputs that are input 1 (Al 3+ ) and input 2 (ClO − ; Figure 13A). Input 1 caused fluorescence enhancement, equivalent to a YES operation, while input 2 resulted in fluorescence quenching, thereby enforcement of the NOT gate. In the presence of both inputs, the quenching (by Input 2) has precedence over the fluorescence enhancement by Input 1, which was in accordance with the truth table illustrated in Figure 13B. Hence, by monitoring the emission maxima at 505 nm by two inputs (Al 3+ and ClO − ), a monomolecular circuit displaying an INHIBIT logic function could be achieved.

CONCLUSION
In conclusion, we had developed a naphthalimide-based sensor NPA which exhibited a turn-on fluorescence response toward Al 3+ with a bright green fluorescence under UV radiation and visual color change from yellow to colorless detected by nakedeye. The binding stoichiometry of NPA with Al 3+ was 1:1 confirmed by job's plot, HRMS and 1 H NMR and 13 C NMR titration. The application of NPA for detection and assessing the existence of Al 3+ in real sample was also successfully achieved. Moreover, NPA-Al 3+ complex were further used as a fluorescent turn-off probe for the detection of ClO − with the detection as low as 2.34 × 10 −8 M. The INHIBIT molecular logic gate was effectively constructed by the performance of NPA to Al 3+ and NPA-Al 3+ complex to ClO − . The development of multifunctional chemosensor for the detection of biological-related analyst in pure water is our future work.

DATA AVAILABILITY
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

AUTHOR CONTRIBUTIONS
Z-YX constructed the workflow and completed the paper. N-NL synthesized and purified the compound. X-JS and T-TL measured and interpreted the data the UV/Vis and fluorescence experiments. HF and FY performed the real sample analysis.

ACKNOWLEDGMENTS
We would like to thank our team leader Professor Ying Fu (Northeast Agricultural University) for financial supporting in paper publishing.