An Acylhydrazone-Based Fluorescent Sensor for Sequential Recognition of Al3+ and H2PO4−

A novel acylhydrazone-based fluorescent sensor NATB was designed and synthesized for consecutive sensing of Al3+ and H2PO4−. NATB displayed fluorometric sensing to Al3+ and could sequentially detect H2PO4− by fluorescence quenching. The limits of detection for Al3+ and H2PO4− were determined to be 0.83 and 1.7 μM, respectively. The binding ratios of NATB to Al3+ and NATB-Al3+ to H2PO4− were found to be 1:1. The sequential recognition of Al3+ and H2PO4− by NATB could be repeated consecutively. In addition, the practicality of NATB was confirmed with the application of test strips. The sensing mechanisms of Al3+ and H2PO4− by NATB were investigated through fluorescence and UV–Visible spectroscopy, Job plot, ESI-MS, 1H NMR titration, and DFT calculations.


Introduction
Al 3+ , the third most abundant metallic element in nature [1,2], is broadly employed in daily life in packaging materials, pharmaceuticals, food additives, machinery, clinical medicines, and water purification [3,4]. Owing to its widespread usage, Al 3+ could be readily accumulated in the body, which leads to the development of diverse diseases such as Parkinson's and Alzheimer's disease [5,6]. Dihydrogen phosphate (H 2 PO 4 − ) is an important component related to many intercellular activities, such as signaling mediation, protein phosphorylation, enzymatic reactions, ion-channel regulation, and so on [7][8][9]. However, excessive agricultural use of phosphate causes eutrophication or massive algal growth, leading to a deficiency in oxygen levels [10][11][12]. For these reasons, there has been a strong demand for the development of sensing and detection methods for Al 3+ and H 2 PO 4 − . The traditional analytical methods reported for the analysis of cations and anions, such as ICP-AES, AAS, and electrochemical methods, have been largely restricted due to their expensive instruments, complicated procedures, and the need for highly trained operators [13][14][15]. In contrast, fluorescence methods have shown the advantages of costeffectiveness, simplicity, easy operation, and high sensitivity [16][17][18]. While numerous fluorescent chemosensors for a single analyte have been reported, fluorescent chemosensors that allow the sequential sensing of multiple analytes with great selectivity and sensitivity are still needed [19][20][21] because they are more cost-effective, recyclable and practical [22][23][24]. Several fluorescent sensors have been addressed for consecutive sensing of Al 3+ and several anions [25][26][27][28] [32]. The practical importance of sequential sensing may have potential applications such as logic gates and molecular switches. Nevertheless, a sequential fluorescent sensor that can exclusively detect Al 3+ and H 2 PO 4 − has not been reported to date.
As Al 3+ is a hard cation, chemosensors containing hard base units, such as nitrogen or oxygen atoms, prefer to coordinate with Al 3+ [33][34][35]. In this regard, acylhydrazone derivatives, having oxygen and nitrogen atoms, are expected to be a suitable functional group to design an Al 3+ chemosensor [36][37][38]. Naphthalene moieties have been widely applied for the design of fluorescent sensors because of their excellent photophysical properties as a fluorophore [39][40][41]. Hence, we expected that a compound including both acylhydrazone and naphthalene may operate as a fluorescence chemosensor for Al 3+ .
In the current study, we designed an acylhydrazone-based fluorescent sensor, NATB, which showed green fluorescence emissions with Al 3+ and could sequentially detect H 2 PO 4 − through fluorescence quenching with high sensitivity and selectivity. A sensing mechanism of NATB to Al 3+ and H 2 PO 4 − was illustrated by fluorescence and UV-Vis spectroscopy, Job plot, ESI-MS, 1 H NMR titration, and calculations.

Materials and Equipment
All solvents and reagents were commercially obtained from TCI (TCI, Nihonbashi-Honcho, Tokyo, Japan) and Sigma-Aldrich (MilliporeSigma, Burlington, MA, USA). NMR experiments were conducted using DMSO-d 6 as the solvent, and the data were recorded on a Varian spectrometer (Varian, Palo Alto, CA, USA). Fluorescence and UV-Visible spectra were measured with Perkin Elmer machines (Perkin Elmer, Waltham, MA, USA). The quantum yields of NATB and NATB-Al 3+ were relatively determined with quinine (Φ = 0.54 in 1 × 10 −1 M H 2 SO 4 ) as a reference. ESI-MS data were recorded on a Thermo Finnigan machine (Thermo Finnigan LLC, San Jose, CA, USA).

Competitive Experiments
For Al 3+ , 6 µL (10 mM) of an NATB stock in DMSO was mixed into MeOH (2 mL) to make 30 µM. A total of 4.5 µL of various cations (20 mM) in DMF was diluted in NATB to make 45 µM. Finally, 4.5 µL (20 mM) of an Al 3+ stock in DMF was mixed into each solution to produce 45 µM, and their fluorescent spectra were measured.
For H 2 PO 4 − , 6 µL (10 mM) of an NATB stock in DMSO and 4.5 µL (20 mM) of an Al 3+ stock in DMF were diluted into MeOH (2 mL) to produce 30 µM of NATB-Al 3+ . We added 4.5 µL of various anions (20 mM) in H 2 O to NATB-Al 3+ to produce 45 µM. A total of 4.5 µL (20 mM) of an H 2 PO 4 − stock was diluted into each solution to produce 45 µM. Their fluorescent spectra were measured.

Determination of Association Constant (K)
The association constant (K) was calculated using Li's method [43]. If the ligand (L) and the analyte (M) form an m-n complex, M m L n , the equilibrium constant of the corresponding complex, K, can be expressed by the following equation: Calculations were achieved with the Gaussian 16 program [44]. Optimal geometries of NATB and NATB-Al 3+ were provided with the DFT method [45,46]. B3LYP was selected as the hybrid functional basis set. The 6-31G(d,p) basis set was implemented to all atoms except Al 3+ [47,48], and the LANL2DZ basis set was employed for applying ECP to Al 3+ [49][50][51]. No imaginary frequency was found in the optimized states of NATB or NATB-Al 3+ , indicating their local minima. The solvent effect of MeOH was considered with IEFPCM [52]. Based on the energy-optimized structures of NATB and NATB-Al 3+ , the plausible UV-Vis transition states were calculated by the TD-DFT method with 20 lowest singlet states.

Spectroscopic Examination of NATB to Al 3+
To confirm the fluorescence selectivity of NATB, the fluorescence emission was studied with a variety of cations in MeOH ( Figure 1). As a result, NATB exhibited notable fluorescence emission at 526 nm with Al 3+ , while NATB and NATB with other cations showed negligible or no fluorescence emission (λex = 358 nm). These outcomes demon-Scheme 1. Synthesis of NATB.

Spectroscopic Examination of NATB to Al 3+
To confirm the fluorescence selectivity of NATB, the fluorescence emission was studied with a variety of cations in MeOH ( Figure 1). As a result, NATB exhibited notable fluorescence emission at 526 nm with Al 3+ , while NATB and NATB with other cations showed negligible or no fluorescence emission (λ ex = 358 nm). These outcomes demonstrated that NATB could be utilized as a fluorescent probe for the selective sensing of Al 3+ . On the other hand, NATB was soluble in aqueous media, but it did not show any selectivity to Al 3+ . In addition, the fluorescence emission of NATB was examined with various anions including dihydrogen phosphate. NATB had no selectivity for the anions.

Spectroscopic Examination of NATB to Al 3+
To confirm the fluorescence selectivity of NATB, the fluorescence emission was studied with a variety of cations in MeOH ( Figure 1). As a result, NATB exhibited notable fluorescence emission at 526 nm with Al 3+ , while NATB and NATB with other cations showed negligible or no fluorescence emission (λex = 358 nm). These outcomes demonstrated that NATB could be utilized as a fluorescent probe for the selective sensing of Al 3+ . On the other hand, NATB was soluble in aqueous media, but it did not show any selectivity to Al 3+ . In addition, the fluorescence emission of NATB was examined with various anions including dihydrogen phosphate. NATB had no selectivity for the anions.  To check the concentration-dependent properties of NATB to Al 3+ , fluorescence titration was carried out (Figure 2). NATB exhibited little fluorescence with a tiny quantum yield (Φ = 0.008). However, the continuous increase in Al 3+ up to 1.5 equiv significantly enhanced the green fluorescence emission at 526 nm (Φ = 0.162). UV-Vis spectrometry was also conducted with Al 3+ to examine its photophysical characteristics ( Figure 3). Upon the addition of Al 3+ , the absorption of 310 nm clearly decreased, while a new absorption of 325 nm constantly increased up to 1.5 equiv. An explicit isosbestic point was observed at 315 nm, verifying that the coordination of NATB with Al 3+ produced a stable complex.
The 1 H NMR titrations were achieved to investigate the binding mechanism of NATB toward Al 3+ (Figure 4). Upon the addition of Al 3+ to NATB, the proton H 14 continually disappeared and the protons H 5 and H 6 were deshielded. These results indicate that the deprotonated oxygen on the tert-butylphenol group and the oxygen and nitrogen on the acylhydrazone group may be coordinated to Al 3+ (Scheme 2).
was also conducted with Al 3+ to examine its photophysical characteristics ( Figure 3) the addition of Al 3+ , the absorption of 310 nm clearly decreased, while a new abso of 325 nm constantly increased up to 1.5 equiv. An explicit isosbestic point was ob at 315 nm, verifying that the coordination of NATB with Al 3+ produced a stable co   the addition of Al 3+ , the absorption of 310 nm clearly decreased, while a new absor of 325 nm constantly increased up to 1.5 equiv. An explicit isosbestic point was obse at 315 nm, verifying that the coordination of NATB with Al 3+ produced a stable com   Al 3+ was confirmed to be 3.6 × 10 4 M −1 ( Figure S5) based on Li's method [43]. The detection limit of NATB toward Al 3+ was 0.83 μM, based on 3σ/slope ( Figure S6).
The 1 H NMR titrations were achieved to investigate the binding mechanism of NATB toward Al 3+ (Figure 4). Upon the addition of Al 3+ to NATB, the proton H14 continually disappeared and the protons H5 and H6 were deshielded. These results indicate that the deprotonated oxygen on the tert-butylphenol group and the oxygen and nitrogen on the acylhydrazone group may be coordinated to Al 3+ (Scheme 2). To verify the practicability of NATB as a probe for Al 3+ , an interference experiment was conducted ( Figure S7). NATB could detect Al 3+ with other cations without significant interferences, except for In 3+ , Fe 3+ and Cu 2+ . These three cations bound more tightly to NATB than Al 3+ . For the practical application of NATB, test kits were prepared by im- limit of NATB toward Al 3+ was 0.83 μM, based on 3σ/slope ( Figure S6).
The 1 H NMR titrations were achieved to investigate the binding mechanism of NATB toward Al 3+ (Figure 4). Upon the addition of Al 3+ to NATB, the proton H14 continually disappeared and the protons H5 and H6 were deshielded. These results indicate that the deprotonated oxygen on the tert-butylphenol group and the oxygen and nitrogen on the acylhydrazone group may be coordinated to Al 3+ (Scheme 2). To verify the practicability of NATB as a probe for Al 3+ , an interference experiment was conducted ( Figure S7). NATB could detect Al 3+ with other cations without significant interferences, except for In 3+ , Fe 3+ and Cu 2+ . These three cations bound more tightly to NATB than Al 3+ . For the practical application of NATB, test kits were prepared by im-Scheme 2. Sequential recognition mechanism of Al 3+ and H 2 PO 4 − by NATB.
To verify the practicability of NATB as a probe for Al 3+ , an interference experiment was conducted ( Figure S7). NATB could detect Al 3+ with other cations without significant interferences, except for In 3+ , Fe 3+ and Cu 2+ . These three cations bound more tightly to NATB than Al 3+ . For the practical application of NATB, test kits were prepared by immersing filter paper strips in the NATB solution. When NATB-coated test kits were immersed in a range of concentrations of Al 3+ solutions, the obvious green fluorescence emission showed up above 2 mM of Al 3+ under UV light (Figure 5a). However, the fluorescence was not displayed when those strips were applied to the same concentration of other cations (Figure 5b). These results indicate the potential applications of NATB in easily recognizing Al 3+ without any complicated tools. mersing filter paper strips in the NATB solution. When NATB-coated test kits were im-mersed in a range of concentrations of Al 3+ solutions, the obvious green fluorescence emission showed up above 2 mM of Al 3+ under UV light (Figure 5a). However, the fluorescence was not displayed when those strips were applied to the same concentration of other cations (Figure 5b). These results indicate the potential applications of NATB in easily recognizing Al 3+ without any complicated tools.

Calculations
To comprehend the Al 3+ -sensing property of NATB, DFT calculations were performed with the Gaussian 16 program ( Figure 6). As the Job plot, ESI-MS, and 1 H NMR titration implied a 1:1 stoichiometric coordination of NATB with Al 3+ , all calculations were conducted with 1:1 stoichiometry. NATB showed a dihedral angle of 0.013° (1O, 2C, 3N, and 4C) with a planar structure (Figure 6a). The coordination of NATB with Al 3+ distorted its structure, showing a dihedral angle of 98.875° (Figure 6b).

Calculations
To comprehend the Al 3+ -sensing property of NATB, DFT calculations were performed with the Gaussian 16 program ( Figure 6). As the Job plot, ESI-MS, and 1 H NMR titration implied a 1:1 stoichiometric coordination of NATB with Al 3+ , all calculations were conducted with 1:1 stoichiometry. NATB showed a dihedral angle of 0.013 • (1O, 2C, 3N, and 4C) with a planar structure (Figure 6a). The coordination of NATB with Al 3+ distorted its structure, showing a dihedral angle of 98.875 • (Figure 6b).
Based on the energy-minimized structures of NATB and NATB-Al 3+ , TD-DFT calculations were conducted to inspect the transition energies and molecular orbitals. NATB featured the main absorption induced from the HOMO → LUMO (347.28 nm), showing intra-charge transfer (ICT) transition from the tert-butylphenol to the naphthol ( Figure S8). The major absorption of NATB-Al 3+ derived from the HOMO-1 → LUMO transition (412.27 nm) also showed a similar ICT transition (Figures S9 and S10). The reduction in the energy gap was consistent with the red-shift of the experimental absorption. These outcomes led us to conclude that the fluorescence turn-on mechanism of NATB to Al 3+ may be a chelation-enhanced fluorescence (CHEF) effect [53]. Based on experimental data and theoretical calculations, an appropriate binding structure of NATB-Al 3+ is proposed in Scheme 2.  (Figures S9 and S10). The reduction in the energy gap was consistent with the red-shift of the experimental absorption. These outcomes led us to conclude that the fluorescence turn-on mechanism of NATB to Al 3+ may be a chelation-enhanced fluorescence (CHEF) effect [53]. Based on experimental data and theoretical calculations, an appropriate binding structure of NATB-Al 3+ is proposed in Scheme 2.  The fluorescence titration experiments were conducted to verify the fluorescence quenching ability of H 2 PO 4 − toward NATB-Al 3+ (Figure 8). The fluorescence of NATB-Al 3+ consistently diminished with the addition of H 2 PO 4 − up to 1.5 equiv (Φ = 0.005). UV-Vis spectroscopy showed that the continuous addition of H 2 PO 4 − increased the absorbance at 310 nm, while those at 270 and 325 nm decreased with the explicit isosbestic points at 253 and 315 nm (Figure 9). The UV-Vis spectrum of H 2 PO 4 − with NATB-Al 3+ is analogous to that of free NATB, implying that the addition of H 2 PO 4 − released Al 3+ from the NATB-Al 3+ complex ( Figure S11). The fluorescence titration experiments were conducted to verify the fluorescence quenching ability of H2PO4 − toward NATB-Al 3+ (Figure 8). The fluorescence of NATB-Al 3+ consistently diminished with the addition of H2PO4 − up to 1.5 equiv (Φ = 0.005). UV-Vis spectroscopy showed that the continuous addition of H2PO4 − increased the absorbance at 310 nm, while those at 270 and 325 nm decreased with the explicit isosbestic points at 253 and 315 nm (Figure 9). The UV-Vis spectrum of H2PO4 − with NATB-Al 3+ is analogous to that of free NATB, implying that the addition of H2PO4 − released Al 3+ from the NATB-Al 3+ complex ( Figure S11).   The stoichiometry of H 2 PO 4 − toward NATB-Al 3+ was determined by the Job plot experiment ( Figure S12), which exhibited a 1:1 stoichiometry. The mass spectral analysis displayed a peak of 395.06 (m/z), which demonstrated the regeneration of NATB ([NATB + H + + MeOH] + ; calcd. 395.20) (Figure S13). These outcomes supported the mechanism that the addition of H 2 PO 4 − released Al 3+ from NATB-Al 3+ , which resulted in the loss of fluorescence. Based on Li's method [43], the association constant (K) for H 2 PO 4 − with NATB-Al 3+ was calculated as 1.2 × 10 4 M −1 ( Figure S14). The detection limit of NATB-Al 3+ toward H 2 PO 4 − was determined as 1.7 µM, based on 3σ/slope ( Figure S15). Importantly, NATB is the first fluorescent sensor for the consecutive sensing of Al 3+ and H 2 PO 4 − (Table S1). On the other hand, NATB showed higher detection limits for Al 3+ and H 2 PO 4 − compared to Kumar's work [32], but it could solely detect H 2 PO 4 − without the interference of HSO 4 − . The reversibility in the response of NATB was verified through the alternative additions of Al 3+ and H 2 PO 4 − (Figure 10). The fluorescence emission of NATB repeated its enhancing and quenching processes several times without fluorescence efficiency loss. To verify that NATB-Al 3+ is an effective fluorescence probe for H 2 PO 4 − , the interference of other anions was tested ( Figure S16

Conclusions
An acylhydrazone-based chemosensor NATB was developed and its sequential recognition of Al 3+ and H2PO4 − was studied. NATB showed a strong fluorescence increase with Al 3+ , and its complex NATB-Al 3+ sequentially detected H2PO4 − by releasing Al 3+ with turn-off fluorescence. Importantly, NATB is the first sequential fluorescent probe for se-

Conclusions
An acylhydrazone-based chemosensor NATB was developed and its sequential recognition of Al 3+ and H 2 PO 4 − was studied. NATB showed a strong fluorescence increase with Al 3+ , and its complex NATB-Al 3+

Conflicts of Interest:
The authors declare no conflict of interest.