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

Abstract

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.

1. Introduction

Al³⁺, 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, Al³⁺ 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 (H₂PO₄⁻) 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 Al³⁺ and H₂PO₄⁻.

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 cost-effectiveness, 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 Al³⁺ and several anions [25,26,27,28] or several cations and H₂PO₄⁻ [29,30,31]. In addition, Kumar et al. reported a fluorescent sensor for sequential sensing of Al³⁺ and H₂PO₄⁻/HSO₄⁻ [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 Al³⁺ and H₂PO₄⁻ has not been reported to date.

As Al³⁺ is a hard cation, chemosensors containing hard base units, such as nitrogen or oxygen atoms, prefer to coordinate with Al³⁺ [33,34,35]. In this regard, acylhydrazone derivatives, having oxygen and nitrogen atoms, are expected to be a suitable functional group to design an Al³⁺ 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 Al³⁺.

In the current study, we designed an acylhydrazone-based fluorescent sensor, NATB, which showed green fluorescence emissions with Al³⁺ and could sequentially detect H₂PO₄⁻ through fluorescence quenching with high sensitivity and selectivity. A sensing mechanism of NATB to Al³⁺ and H₂PO₄⁻ was illustrated by fluorescence and UV–Vis spectroscopy, Job plot, ESI-MS, ¹H NMR titration, and calculations.

2. Experimental Section

2.1. 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-d₆ 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-Al³⁺ were relatively determined with quinine (Φ = 0.54 in 1 × 10⁻¹ M H₂SO₄) as a reference. ESI-MS data were recorded on a Thermo Finnigan machine (Thermo Finnigan LLC, San Jose, CA, USA).

2.2. Synthesis of N′-[(E)-(3-tert-butyl-2-hydroxyphenyl)methylidene]-3-hydroxynaphthalene-2-carbohydrazide (NATB)

The intermediate compound, 3-hydroxy-2-naphthohydrazide (2), was synthesized following a previously reported method [42]. The excess amounts of 3-(tert-butyl)-2-hydroxybenzaldehyde (1, 1.8 mmol) and 3-hydroxy-2-naphthohydrazide (2, 0.3 mmol) were mixed in absolute EtOH (10 mL) with a catalytic amount of HCl and stirred at room temperature for 1 day. A yellow precipitate was filtered, rinsed with cold absolute EtOH, and dried (77.2 mg, 70.1%); ¹H NMR in DMSO-d₆: δ 12.42 (s, 1H), 12.24 (s, 1H), 11.19 (s, 1H), 8.63 (s, 1H), 8.45 (s, 1H), 7.93 (d, 1H), 7.77 (d, 1H), 7.53 (t, 1H), 7.38 (t, 1H), 7.35 (s, 1H), 7.32 (d, 1H), 7.30 (d, 1H), 6.91 (t, 1H), 1.43 (s, 9H). ¹³C NMR in DMSO-d6: δ 163.23 (1C), 156.90 (1C), 153.75 (1C), 151.45 (1C), 136.36 (1C), 135.84 (1C), 130.31 (1C), 129.53 (1C), 128.59 (1C), 128.54 (1C), 128.20 (1C), 126.69 (1C), 125.75 (1C), 123.75 (1C), 119.86 (1C), 118.70 (1C), 117.54 (1C), 110.54 (1C), 34.39 (1C), 29.16 (3C). ESI-MS (m/z): [NATB + H⁺]⁺ calcd 363.17, found 363.04.

2.3. Preparation of Spectroscopic Experiments

An NATB stock (10 mM) was prepared in DMSO. The stock solutions (20 mM) of varied cations were prepared using their nitrate salts (Al³⁺, Na⁺, Cr³⁺, Fe²⁺, Ca²⁺, Cd²⁺, Zn²⁺, Pb²⁺, Co²⁺, Cu²⁺, In³⁺, Mn²⁺, Ga³⁺, Ni²⁺, Mg²⁺, Ag⁺, Hg²⁺ and K⁺) or perchlorate salt (Fe³⁺). The concentrated solutions (20 mM) of varied anions were prepared using their tetrabutylammonium salts (H₂PO₄⁻, SCN⁻, BzO⁻, N₃⁻, OAc⁻ and NO₂⁻), tetraethylammonium salts (F⁻, Cl⁻, Br⁻, I⁻ and CN⁻), sodium salts (S²⁻ and ClO⁻), or potassium salts (HPO₄²⁻, PO₄³⁻, HSO₄⁻ and P₂O₇⁴⁻(PPi)). All spectroscopic experiments were conducted in MeOH.

2.4. Competitive Experiments

For Al³⁺, 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 Al³⁺ stock in DMF was mixed into each solution to produce 45 μM, and their fluorescent spectra were measured.

For H₂PO₄⁻, 6 μL (10 mM) of an NATB stock in DMSO and 4.5 μL (20 mM) of an Al³⁺ stock in DMF were diluted into MeOH (2 mL) to produce 30 μM of NATB-Al³⁺. We added 4.5 μL of various anions (20 mM) in H₂O to NATB-Al³⁺ to produce 45 μM. A total of 4.5 μL (20 mM) of an H₂PO₄⁻ stock was diluted into each solution to produce 45 μM. Their fluorescent spectra were measured.

2.5. 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_mL_n, the equilibrium constant of the corresponding complex, K, can be expressed by the following equation:

$${\left[M\right]}^m=\frac{1}{nK}\frac{1}{{\left[L\right]}_T^{n-1}}\frac{1-\alpha }{{\alpha }^n}$$

where,

$$\begin{gathered} \left[M\right]=the concentration of analyte \\ {\left[L\right\}}_T=the total concentration of ligand \end{gathered}$$

and α could be described as:

$$\alpha =\frac{I-I_{max}}{I_{min}-I_{max}}$$

where,

$$I=the fluorescence intensity of complex$$

2.6. Calculations

Calculations were achieved with the Gaussian 16 program [44]. Optimal geometries of NATB and NATB-Al³⁺ 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 Al³⁺ [47,48], and the LANL2DZ basis set was employed for applying ECP to Al³⁺ [49,50,51]. No imaginary frequency was found in the optimized states of NATB or NATB-Al³⁺, indicating their local minima. The solvent effect of MeOH was considered with IEFPCM [52]. Based on the energy-optimized structures of NATB and NATB-Al³⁺, the plausible UV–Vis transition states were calculated by the TD-DFT method with 20 lowest singlet states.

3. Results and Discussion

The synthesis of NATB was conducted as depicted in Scheme 1. The condensation reaction of 3-(tert-butyl)-2-hydroxybenzaldehyde (1) and 3-hydroxy-2-naphthohydrazide (2) produced the desired product, N’-[(E)-(3-tert-butyl-2-hydroxyphenyl)methylidene]-3-hydroxynaphthalene-2-carbohydrazide (NATB), which was verified with ¹H NMR, ¹³C NMR (Figures S1 and S2), and ESI-MS.

3.1. Spectroscopic Examination of NATB to Al³⁺

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 Al³⁺, 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 Al³⁺. On the other hand, NATB was soluble in aqueous media, but it did not show any selectivity to Al³⁺. 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 Al³⁺, fluorescence titration was carried out (Figure 2). NATB exhibited little fluorescence with a tiny quantum yield (Φ = 0.008). However, the continuous increase in Al³⁺ up to 1.5 equiv significantly enhanced the green fluorescence emission at 526 nm (Φ = 0.162). UV–Vis spectrometry was also conducted with Al³⁺ to examine its photophysical characteristics (Figure 3). Upon the addition of Al³⁺, 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 Al³⁺ produced a stable complex.

A 1:1 stoichiometric coordination between NATB and Al³⁺ was suggested by the Job plot experiment (Figure S3), which was explicitly supported by ESI-MS analysis (Figure S4). The positive ion mass displayed a large peak of 596.16 (m/z), which was correlated to [NATB(-H⁺) + Al³⁺ + 2 DMF + NO₃⁻]⁺ (calcd. 596.23). The association constant (K) of NATB-Al³⁺ was confirmed to be 3.6 × 10⁴ M⁻¹ (Figure S5) based on Li’s method [43]. The detection limit of NATB toward Al³⁺ was 0.83 μM, based on 3σ/slope (Figure S6).

The ¹H NMR titrations were achieved to investigate the binding mechanism of NATB toward Al³⁺ (Figure 4). Upon the addition of Al³⁺ 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 Al³⁺ (Scheme 2).

To verify the practicability of NATB as a probe for Al³⁺, an interference experiment was conducted (Figure S7). NATB could detect Al³⁺ with other cations without significant interferences, except for In³⁺, Fe³⁺ and Cu²⁺. These three cations bound more tightly to NATB than Al³⁺. 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 Al³⁺ solutions, the obvious green fluorescence emission showed up above 2 mM of Al³⁺ 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 Al³⁺ without any complicated tools.

3.2. Calculations

To comprehend the Al³⁺-sensing property of NATB, DFT calculations were performed with the Gaussian 16 program (Figure 6). As the Job plot, ESI-MS, and ¹H NMR titration implied a 1:1 stoichiometric coordination of NATB with Al³⁺, 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 Al³⁺ distorted its structure, showing a dihedral angle of 98.875° (Figure 6b).

Based on the energy-minimized structures of NATB and NATB-Al³⁺, 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-Al³⁺ 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 Al³⁺ may be a chelation-enhanced fluorescence (CHEF) effect [53]. Based on experimental data and theoretical calculations, an appropriate binding structure of NATB-Al³⁺ is proposed in Scheme 2.

3.3. Spectroscopic Examination of NATB-Al³⁺ to H₂PO₄⁻

We studied the fluorescence selectivity of NATB-Al³⁺ to a range of anions such as H₂PO₄⁻, Cl⁻, CN⁻, OAc⁻, F⁻, ClO⁻, I⁻, N₃⁻, BzO⁻, SCN⁻, Br⁻, NO₂⁻, S²⁻, HPO₄²⁻, PO₄³⁻, HSO₄⁻, and PPi in MeOH (Figure 7). Most of the anions did not affect the fluorescence emission of NATB-Al³⁺, while the addition of H₂PO₄⁻ toward NATB-Al³⁺ resulted in significant fluorescence quenching (λ_ex = 358 nm). The result demonstrated that NATB-Al³⁺ could be used as a chemosensor for H₂PO₄⁻ with fluorescence quenching.

The fluorescence titration experiments were conducted to verify the fluorescence quenching ability of H₂PO₄⁻ toward NATB-Al³⁺ (Figure 8). The fluorescence of NATB-Al³⁺ 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-Al³⁺ is analogous to that of free NATB, implying that the addition of H₂PO₄⁻ released Al³⁺ from the NATB-Al³⁺ complex (Figure S11).

The stoichiometry of H₂PO₄⁻ toward NATB-Al³⁺ 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 H₂PO₄⁻ released Al³⁺ from NATB-Al³⁺, which resulted in the loss of fluorescence. Based on Li’s method [43], the association constant (K) for H₂PO₄⁻ with NATB-Al³⁺ was calculated as 1.2 × 10⁴ M⁻¹ (Figure S14). The detection limit of NATB-Al³⁺ toward H₂PO₄⁻ was determined as 1.7 μM, based on 3σ/slope (Figure S15). Importantly, NATB is the first fluorescent sensor for the consecutive sensing of Al³⁺ and H₂PO₄⁻ (Table S1). On the other hand, NATB showed higher detection limits for Al³⁺ and H₂PO₄⁻ compared to Kumar’s work [32], but it could solely detect H₂PO₄⁻ without the interference of HSO₄⁻.

The reversibility in the response of NATB was verified through the alternative additions of Al³⁺ and H₂PO₄⁻ (Figure 10). The fluorescence emission of NATB repeated its enhancing and quenching processes several times without fluorescence efficiency loss. To verify that NATB-Al³⁺ is an effective fluorescence probe for H₂PO₄⁻, the interference of other anions was tested (Figure S16). The results indicated that the presence of other anions (1.5 equiv) did not interfere with the fluorescence quenching of NATB-Al³⁺ toward H₂PO₄⁻.

4. Conclusions

An acylhydrazone-based chemosensor NATB was developed and its sequential recognition of Al³⁺ and H₂PO₄⁻ was studied. NATB showed a strong fluorescence increase with Al³⁺, and its complex NATB-Al³⁺ sequentially detected H₂PO₄⁻ by releasing Al³⁺ with turn-off fluorescence. Importantly, NATB is the first sequential fluorescent probe for selective sensing of Al³⁺ and H₂PO₄⁻. Detection limits of NATB for Al³⁺ and H₂PO₄⁻ were calculated as 0.83 and 1.7 μM, respectively, based on 3σ/slope. NATB could repeat sequential recognition of Al³⁺ and H₂PO₄⁻ several times and could be applied to detect Al³⁺ in test strips. The sensing mechanism of NATB toward Al³⁺ and H₂PO₄⁻ was demonstrated with a Job plot, ESI-MS, ¹H NMR spectroscopy, and theoretical calculations. The detection mechanism of NATB toward Al³⁺ is suggested to be a CHEF effect through DFT calculations.