A dual-functional chemosensor based on acylhydrazone derivative for rapid detection of Zn(II) and Mg(II): Spectral properties, recognition mechanism and application studies

2.1 Materials and instruments

All of the reagents and chemicals used in this study were of analytical grade and used without further purification. Quinoline-8-carboxaldehyde was purchased from Energy Chemical (Shanghai, China), 6-hydroxy-2-naphthoic hydrazide was purchased from J&K Scientific ltd. (Beijing, China), and all other reagents were purchased from Sinopharm Chemical (Shanghai, China). The melting point was determined by a Beijing XT4-100x microscopic melting point apparatus (Beijing Electrooptical Instrument Factory, China). The ¹H NMR and ¹³C NMR spectra were measured by a Bruker AV-500 spectrometer (Bruker BioSpin Gmbh, Germany), the Fourier transform infrared (FT-IR) spectra were obtained on a Nicolet 6700 spectrometer (Thermo Fisher Scientific, USA), and the high-resolution mass spectra (HR-MS) spectra were obtained using a UPLC G2-XS mass spectrometer (Waters, USA). A TU-1901 UV–vis spectrophotometer (PGeneral Instrument Inc., China) and Cary Eclipse fluorescence spectrofluorometer (Agilent Technologies, USA) were used to measure absorption and fluorescent spectra, and the cell imaging experiments were observed by an Olympus FV3000 confocal laser-scanning microscope (Olympus FV3000, Japan).

A Bruker Smart CCD single-crystal diffractometer (Bruker AXS GmbH, Germany) was utilized to collect single crystal data. Measurement was performed at 173 K using a graphite monochromated Mo-Kα radiation (λ = 0.71073 Å). SHELXS-97 program was used to solve and refine the crystal structure by full-matrix least-squares techniques on F² (Sheldrick, 1997). The structure plots was used Diamond software (Brandenburg, 2012). Crystallographic data CCDC2192938 for the structure QN62 has been deposited with the Cambridge Crystallographic Data Centre and can be obtained free of charge via https://www.ccdc.cam.ac.uk.

2.2 Synthesis of compound QN62

The compound 6-hydroxy-N'-(quinolin-8-ylmethylene)naphthalene-2-carbohydrazide (QN62) was prepared as follows, quinoline-8-carboxaldehyde (250 mg, 1.60 mmol) was dissolved in 20 mL of 1,4-dioxane and then 6-hydroxy-2-naphthoic hydrazide (320 mg, 1.60 mmol) was added. After refluxed for 4 h, the solvent was removed under reduced pressure and the crude product was purified by column chromatography on silica gel (dichloromethane-methanol, 9:1, v/v). Crystallization from dichloromethane-methanol (4:1, v/v) gave fusiform yellow single crystals. Yield: 78 %. m.p.: 164.5–165.5 °C. IR (cm⁻¹) (Fig. S1): 3453 (–OH), 3200 (–NH), 3043 (HC = ), 1629 (C⚌O), 1578 (C⚌N), 1288 (C—N, amide III band), 1215(C—O). ¹H NMR (500 MHz, DMSO‑d₆, ppm) (Fig. S2): 12.20(1H, s, OH), 10.15(1H, s, NH), 9.80(1H, s,–CH = N), 9.01(1H, dd, J = 4.1, 1.7 Hz, ArH), 8.53(1H, s, ArH), 8.47(1H, m, ArH), 8.43(1H, d, J = 7.2 Hz, ArH), 8.10(1H, d, J = 8.0 Hz, ArH), 8.01–7.91(2H, m, ArH), 7.82(1H, d,J = 8.6 Hz, ArH), 7.74(1H, t, J = 7.7 Hz, ArH), 7.64(1H, dd, J = 8.3, 4.1 Hz, ArH), 7.25–7.17(2H, m, ArH). ¹³C NMR (125 MHz, DMSOd₆, ppm) (Fig. S3): 163.12, 157.08, 150.27, 145.27, 144.20, 136.62, 136.27, 131.25, 130.69, 129.87, 128.11, 127.98, 127.14, 126.52, 126.08, 125.53, 124.53, 121.83, 119.47, 108.59. ESI-Mass (Fig. S4): m/z calcd. for [QN62 + H]⁺, 342.1198; found, 342.1242.

2.3 Spectroscopic measurements

The QN62 stock solution (1.0 mM) was prepared in THF. Various solutions of metal ions (5.0 mM) were prepared by dissolving their nitrate or chloride salts in double distilled water. Test solutions were prepared with QN62 stock solution (100 μL) and a certain volume of metal ions solution, and the mixture was diluted to 5 mL by DMSO-H₂O (4:1, v/v) or ethanol-H₂O (9:1, v/v). Different pH of solutions was adjusted with 1.0 mM NaOH or HCl. All experiments and test procedures were performed at room temperature.

2.4 Binding constant determination

The binding constant K of complexes was determined based on the modified Benesi-Hildebrand in Eq. (1) (Zhang, et al., 2018).

2.5 Limit of detection (LOD) and limit of quantification (LOD) calculations

The limit of detection (LOD) and limit of quantification (LOQ) were calculated using the Eq. (2) and Eq. (3) (Goncalves, et al., 2022).

2.6 Quantum yield measurement

The fluorescence quantum yield was estimated using anthracene (Φ_R = 0.27 in ethanol) (Rurack, 2008) as reference and using the following Eq. (4) (Immanuel David, et al., 2022).

2.7 Cell imaging experiment

The MCF-7 cells were cultured with Dulbecco's modified Eagle's medium (DMEM) medium at 37˚C in a humidified incubator with 5 % CO₂. The cells were first incubated with 10 μM QN62 at 37 °C for 30 min and washed three times to remove the excess of QN62. Subsequently, the cells were exposed to 10 μM Zn²⁺/Mg²⁺ for 30 min under the same conditions. The fluorescence imaging of QN62 in MCF-7 cells was carried out on a confocal laser scanning microscope.

3.1 Characterization of Chemosensor QN62

The chemosensor QN62 was synthesized as shown in Scheme 1. The target compound QN62 was confirmed by FT-IR, ¹H NMR, ¹³C NMR, and HR-MS analyses, as well as by single crystal X-ray diffraction. The characteristic absorption band –CH = N- at 1578 cm⁻¹ appeared in the FT-IR spectrum of QN62 (Fig. S1). In the ¹H NMR and ¹³C NMR spectra of QN62, the proton and carbon signals of Schiff base CH = N resonated at their expected frequency ranges of 9.807 and 157.075 ppm, respectively (Fig. S2 and S3). In the HR-MS spectrum, the base peak was found at 342.1242 (calcd. 342.1198), corresponding to [QN62 + H]⁺ (Fig. S4). Moreover, the structure of compound QN62 was explicitly established by X-ray crystallography. As shown in Fig. 1a, the quinoline unit and naphthalene ring of QN62 were not in the same plane, presenting a dihedral angle of 13.87°. The lattice consisted of one QN62 unit, which incorporated one disordered dichloromethane solvent molecule (Fig. 1a), and the crystalline lattice contained intermolecular O—H⋅⋅⋅O and N—H⋅⋅⋅O hydrogen bonds (Fig. 1b). Centro-symmetrically related molecules were connected into ring R₂²(20) dimers by intermolecular N(3)-H(3)⋅⋅⋅O(2) hydrogen bonds and the dimers were further linked by intermolecular O(2)-H(2)⋅⋅⋅O(1) hydrogen bonds, which formed an undulating two-dimensional network (Fig. 1c). The intermolecular hydrogen bonds played a key role in the formation of crystalline solids (Shyamsivappan, et al., 2020). The QN62 compound for the crystallographic data, the selected bond lengths, and the bond angles were illustrated in Tables S1, S2, and S3, respectively.

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Scheme 1

Synthesis of compound QN62.

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Fig. 1

(a) Ortep view of QN62 drawn in 30 % probability thermal-displacement ellipsoids with the atom numbering scheme. (b) Part of the crystal structure of QN62 showing the intermolecular hydrogen bonds (dotted lines). (c) two-dimensional undulating structure of QN62. H-bonds are shown by dotted lines.

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3.2 Spectroscopic recognition of Zn²⁺ and Mg²⁺

The fluorescent properties of chemosensor QN62 were investigated in a series of solvents, including acetonitrile, ethanol, DMF, and DMSO. In the above solvents, QN62 exhibited very weak fluorescence emissions (Fig. S5). In the designed QN62 molecule, the combination of 6-position phenolic hydroxyl and 2-position acylhydrazone of naphthalene synergistically created a push–pull system, which possessed ICT features (Kim and Kim, 2021) and could be visualized by density functional theory (DFT) calculations, as presented later. Simultaneously, the isomerization of the -C⚌N- unit in the molecule easily underwent nonradiative deactivation of the excited states (Sivakumar and Lee, 2021). Thus, weak fluorescence was possibly caused by the synergistic contribution of C⚌N isomerization and the ICT process. In the above organic solvents, the fluorescence behavior of QN62 toward common metal ions, including Ag⁺, Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Fe²⁺, Fe³⁺, Hg²⁺, K⁺, Mg²⁺, Na⁺, Ni²⁺, Zn²⁺ and Pb²⁺ was measured. A dramatic enhancement in the fluorescence signals was observed only at 532 nm with the addition of Zn²⁺ in DMSO-H₂O (4:1, v/v) media, and at 527 nm with Mg²⁺ in ethanol-H₂O (9:1, v/v) solution (Fig. 2). With the addition of 1.0 equiv. Zn²⁺ and Mg²⁺ ions to the respective analytical systems, the enhancements in QN62 emission intensity were nearly 10-fold and 70-fold, respectively, and the fluorescence quantum yield also increased from 0.013 to 0.11 and 0.43. Other tested ions did not produce any obvious changes in the fluorescence emissions of QN62 under identical conditions. Interfering congeners Cd²⁺/Ca²⁺ exhibited minimal emission in the respective analyte system of Zn²⁺/Mg²⁺. Simultaneously, the colorless QN62 solution changed to yellowish-green/yellow in the presence of Zn²⁺/Mg²⁺ under irradiation of the 365 nm UV lamp, which was easily visible to the naked eye (Fig. 2. inset). These indicated that QN62 could serve as an excellent fluorescent probe for Zn²⁺ and Mg²⁺ by switching solvents. The enhanced fluorescence response of QN62 toward Zn²⁺/Mg²⁺ was possibly due to the interactions of the metal ions with the binding sites, which increased the electron withdrawal ability of the coordinating groups, and this was further beneficial for the ICT process (Sivakumar and Lee, 2021; Hossain, et al., 2017). The restriction of -C⚌N- isomerization also played an essential role in the fluorescence enhancement process (Wu, et al., 2018).

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Fig. 2

(a) Fluorescence responses of QN62 (20 μM) with different metal ions (20 μM) in DMSO-H₂O (4:1, v/v) (λex = 400 nm). (b) Fluorescence responses of QN62 (20 μM) with different metal ions (20 μM) in ethanol-H₂O (9:1, v/v) (λex = 385 nm). Inset: images of QN62 before and after addition of Zn²⁺/Mg²⁺ under 365 nm UV-light.

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The response time of QN62 to detect Zn²⁺/Mg²⁺ was studied. As shown in Fig. S6, the fluorescence intensity of QN62 treated with Zn²⁺/Mg²⁺ rapidly reached saturation within 2 min and then remained unchanged for 30 h. This performance could potentially provide a real-time detection method for Zn²⁺/Mg²⁺ ions.

Due to the high fluorescence sensing ability of QN62 toward Zn²⁺/Mg²⁺ in DMSO/ethanol solution, the UV absorption spectral response of QN62 before and after Zn²⁺/Mg²⁺ addition was further investigated (Fig. 3). The QN62 compound displayed a broad absorption band centered at 343 nm in DMSO-H₂O (4:1, v/v) and ethanol-H₂O (9:1, v/v) solutions, and the longer wavelength absorption band of QN62 could be assigned to π-π* and n-π* charge transfer (CT) transitions (Djouhra, et al., 2017; Xie, et al., 2018). Upon the addition of Zn²⁺/Mg²⁺, the absorption band at 343 slightly decreased, accompanied by new bands at 400 nm for Zn²⁺ and 386 nm for Mg²⁺, indicating the coordination reaction between QN62 and Zn²⁺/Mg²⁺. After adding Zn²⁺/Mg²⁺, the red shifts of the absorption peaks of compound QN62 were due to intramolecular charge transfer (Purkait, et al., 2019).

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Fig. 3

(a) Change in the absorption spectral of QN62 (20 μM) upon addition of Zn²⁺ (0–20 μM) in DMSO-H₂O (4:1, v/v). (b) Change in the absorption spectral of QN62 (20 μM) upon addition of Mg²⁺ (0–20 μM) in ethanol-H₂O (9:1, v/v).

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To extend the practical applicability of compound QN62, we investigated the fluorescence response of compound QN62 before and after Zn²⁺/Mg²⁺ addition at various pH values (Fig. S7). The experimental results showed that the fluorescence intensity of QN62 did not change under acidic, neutral, or alkaline conditions. However, after adding Zn²⁺/Mg²⁺, the chemosensor QN62 displayed notable emission enhancement under neutral and weakly alkaline conditions (pH 6–10 for Zn²⁺, pH 6–9 for Mg²⁺). In neutral and weakly alkaline conditions, the deprotonation process of QN62 enhanced the electron-donating ability of phenolic O⁻, producing a stronger binding affinity toward Zn²⁺/Mg²⁺ ions (Khan, et al., 2022). A pH value of 7.0 was selected as the experimental condition, which was within the biologically relevant pH range (5.5–7.5) (Shi, et al., 2013; Xu, et al., 2017).

To determine the sensitivity of the QN62 chemosensor for Zn²⁺/Mg²⁺, fluorometric titration was carried out (Fig. 4). The fluorescence intensity increased with a gradual increase in Zn²⁺/Mg²⁺ concentration and was saturated at 1.0 equiv. Zn²⁺/Mg²⁺. Moreover, the fluorescence intensities at 532 nm and 527 nm showed a good linear relationship with the Zn²⁺ and Mg²⁺ concentrations in the range of 0.0–1.0 equiv. (Fig. S8). These results indicated that the QN62 chemosensor could provide a potential method for the quantitative determination of Zn²⁺ and Mg²⁺. The binding constant (K) values of the complexes were calculated to be 8.29 × 10⁴ M⁻¹ for Zn²⁺ and 6.34 × 10⁴ M⁻¹ for Mg²⁺ (Fig. S9), which were comparable to previously reported probes for Zn²⁺ and Mg²⁺ (Dhara, et al., 2016; Sun, et al., 2020). The large binding constant values suggested QN62-Zn²⁺/QN62-Mg²⁺ complex formation (Jana, et al., 2016). The LOD values were calculated to be 32.3 nM for Zn²⁺ and 16.1 nM for Mg²⁺, which were low compared with the reported Zn²⁺/Mg²⁺ fluorescent probe (Table 1). Meanwhile, the LOQ values were also estimated to be 97.8 nM for Zn²⁺ and 48.9 nM for Mg²⁺. The values of LOD and LOQ were below the WHO recommended limit of Zn²⁺ (5 mg/L) and Mg²⁺ (30 mg/L) for drinking water and were lower than the total Zn²⁺ concentrations (200–300 μM) and Mg²⁺ concentrations (200–1000 μM) in human cells (Wang, et al., 2021; Kumar and Puri, 2012; Maret, 2015; Farruggia, et al., 2006).

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Fig. 4

(a) Emission spectral changes of compound QN62 (20 μM) with increasing concentration of Zn²⁺: 0–30 μM in DMSO-H₂O (4:1, v/v). (b) Emission spectral changes of compound QN62 (20 μM) with increasing concentration of Mg²⁺: 0–30 μM in ethanol-H₂O (9:1, v/v).

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Table 1 Comparison of the properties of some recently dual fluorescent probes for Zn²⁺ & Mg²⁺ and this work.

Structure of chemosensor	Tested Media	Excitation/ Emission(nm)	LOD (nM)	Ref.
	Acetonitrile/H2O tris-HCl, (7:3, v/v)	370/456 (Zn) 370/472 (Mg)	220 (Zn) 390 (Mg)	(Alam, et al., 2014)
	DMF/H2O(9:1, v/v, PBS buffer, pH7.4) (Zn) Acetonitrile (Mg)	460/536 (Zn) 350/560 (Mg)	307 (Zn) 297 (Mg)	(Dhara, et al., 2016)
	H2O	323/478 (Zn) 323/458 (Mg)	59.4 (Zn) 89.1 (Mg)	(Patil, et al., 2018)
	DMF/H2O (1:4, v/v) (Zn) DMF (Mg)	450/558 (Zn) 350/430 (Mg)	53 (Zn) 33 (Mg)	(Li, et al., 2016)
	DMSO-H2O (4:1, v/v) (Zn) ethanol-H2O (9:1, v/v) (Mg)	400/538 (Zn) 385/527 (Mg)	45.1 (Zn) 14.7 (Mg)	This work

The selectivity of QN62 for Zn²⁺/Mg²⁺ mixed with other metal ions was examined by fluorescent competition experiments. As illustrated in Fig. S10a, except for Cu²⁺, Co²⁺, Fe³⁺, Al³⁺, and Cr³⁺, the coexistence of other competitive ions showed minimal interference on the fluorescence intensity of the QN62-Zn²⁺ complex in DMSO-H₂O (4:1, v/v). The strong Lewis acidity of Al³⁺, Cr³⁺, and Fe³⁺ reduced the binding affinity of imine and phenolic hydroxyl groups towards the Zn²⁺ ion. In the case of Cu²⁺ and Co²⁺, the high coordination affinity and their inherent magnetic properties resulted in a decrease in fluorescence emission intensity at 532 nm (Gao, et al., 2020). Although Co²⁺, Fe³⁺, Al³⁺, and Cr³⁺ could cause interference with Zn²⁺ recognition, QN62 still displayed an obvious enhancement of fluorescence intensity, which could effectively sense the target ion. When QN62 acted as a magnesium ion in the fluorescent probe (Fig. S10b), the coexistence of Cu²⁺, Co²⁺, Ni²⁺, Al³⁺, Cr³⁺, and Fe³⁺ caused significant interference for the QN62-Mg²⁺ complex. Fluorescence quenching was possibly due to the strong complexation abilities or paramagnetic quenching properties of the interfering ions or high Lewis acidity (Wang, et al., 2021; Gao, et al., 2020; Zhou, et al., 2010; Komatsu, et al., 2007; Erdemir and Kocyigit, 2016). Additionally, we attempted to find the lowest concentration of interfering ions that would have a minimal influence on the determination of Zn²⁺/Mg²⁺. The experimental results were illustrated in Figs. S10c and Fig. S10d.

3.3 Binding studies

The sharp changes in the optical response of QN62 before and after Zn²⁺/Mg²⁺ addition revealed the formation of new species, QN62-Zn²⁺ and QN62-Mg²⁺ complexes. Because QN62 contained imine N, quinoline N, carbamoyl O, and phenolic hydroxyl O moieties, QN62 could utilize these binding sites to coordinate with Zn²⁺/Mg²⁺. To explore the binding stoichiometry of QN62 with Zn²⁺/Mg²⁺, the Job’s plot based on the emission intensity was studied (Fig. S11). When the value of [M²⁺]/[M²⁺ + QN62] was 0.5, the fluorescence intensity reached a maximum, indicating the formation of 1:1 stoichiometric complexes between QN62 and Zn²⁺/Mg²⁺. The stoichiometry of the complexes was further confirmed by the HR-MS spectral technique. The molecular ion peak m/z values of the QN62-Zn²⁺ and QN62-Mg²⁺ complexes were calculated to be 404.0377 and 364.0936, respectively, against experimental values of 404.0381 [QN62 + Zn²⁺ - H]⁺ and 364.1054 [QN62 + Mg²⁺ - H]⁺ (Fig. S12).

¹H NMR titration experiments were performed in DMSO‑d₆ to understand the binding mode of the QN62 compound toward Zn²⁺ (Fig. 5). With the gradual addition of Zn²⁺ ions, the amide –NH-C(=O) signal (H_b) of QN62 at 10.148 ppm slowly disappeared, while a new sharp signal at 9.372 ppm appeared, indicated that the amide converted to the enol-imine tautomeric form. With the addition of 1.0 equiv. of Zn²⁺, the proton signals of phenolic hydroxyl (H_a) at 12.202 ppm and enol (H_e) at 9.372 ppm disappeared, suggesting that deprotonation of the phenolic hydroxyl and enol in the amide groups occurred when the QN62 compound coordinated with Zn²⁺. The upfield signal shift of naphthalene hydrogens was possibly related to the increase in electron density caused by the deprotonation of the phenolic hydroxyl group (Dhara, et al., 2016). In addition, the peak of the imine (H_c) shifted upfield from 9.807 to 9.057 ppm, which could be possibly explained by the ICT effect (Kim and Kim, 2021). However, the signal at 9.017 ppm corresponding to the 2′-position of quinoline (H_d) shifted to 9.155 and the other protons of quinoline also underwent a different extent downfield shift, implying that quinoline N was involved in the coordination reaction between QN62 and Zn²⁺. These results indicated that QN62 could chelate with Zn²⁺ through expected binding sites. Considering that enolic O1 and phenolic hydroxyl O2 were located at the 2- and 6-positions of the naphthalene ring, deprotonated enolic O1 and phenolic hydroxyl O2 in one ligand molecule could not be coordinated to the same zinc ion center. The proposed binding mode of the QN62 probe with Zn²⁺ was shown in Scheme 2. Zinc ions was coordinated with two N atoms (quinoline N1, imine N2) and one O atom (deprotonated enolic O1) from one QN62 ligand molecule, and one deprotonated phenolic hydroxyl O2ⁱ of the adjacent other QN62 ligand.

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Fig. 5

¹H NMR titration of QN62 with Zn²⁺ in DMSO‑d₆.

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Scheme 2

Plausible binding mode of QN62 for Zn²⁺ and Mg²⁺.

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To further confirm the binding mode between QN62 and Zn²⁺/Mg²⁺, optimized structures of QN62-Zn²⁺ and QN62-Mg²⁺ were obtained by DFT calculations at the B3LYP/6-31G+ (d,p) level using the Gaussian 16 program (Becke, 1993; Frisch, et al., 2016). In the calculation process, the PCM model was adopted to consider the solvent effect of DMSO and ethanol (Tomasi, et al., 2005). As shown in Fig. 6a, the zinc or magnesium atoms in the optimized complexes coordinated with the deprotonated enol oxygen, imine nitrogen, and nitrogen of quinoline provided by one ligand molecule, and the oxygen of the deprotonated phenolic hydroxyl group of the adjacent ligand, resulting in a distorted tetrahedral coordination geometry. The distances of the coordination bond in the two complexes were in the range of 1.98–2.14 Å, which was comparable to previously reported coordination bonds (Withersby, et al., 1999; Bock, et al., 1999; Ezzayani, et al., 2022).

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Fig. 6

(a) Optimized structures of the complexes QN62-Zn²⁺ and QN62-Mg²⁺. (b) Frontier molecular orbitals of QN62, QN62-Zn²⁺ and QN62-Mg²⁺.

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As indicated by the frontier molecular orbital diagrams shown in Fig. 6b, the enol form of QN62 had nearly a planar conformation, which could provide π-conjugation and ICT from the donor part (naphthalene ring) to the acceptor part (quinoline unit and acylhydrazone unit) (Li, et al., 2016). In compound QN62, the π-electrons of the HOMO were localized over almost the entire molecule, while the π-electrons of the LUMO were restricted to the quinoline unit and the coordination sites. Thus, QN62 possessed ICT characteristics from the naphthalene ring to the coordination sites. In the energy-optimized structures of QN62-Zn²⁺ and QN62-Mg²⁺, the distribution of π-electrons of HOMO and LUMO were very similar. In the complexes, the π-electrons of HOMO and LUMO were distributed mainly over the donor moiety and the acceptor part, respectively, indicating that the complexes possessed more obvious ICT characteristics than the free QN62 probe. These results showed that the binding of Zn²⁺/Mg²⁺ to the ligand further encouraged the ICT process by increasing the electron withdrawal ability of the acceptor part (Hossain, et al., 2017). When Zn²⁺/Mg²⁺ coordinated with QN62, the energy gaps decreased, corresponding to the red shift of the UV–vis absorption bands, and the theoretical calculations supported the originally designed concept.

Based on the experimental results and theoretical calculations, we speculated on the response mechanism of QN62 for Zn²⁺/Mg²⁺ (Scheme 2). Before the addition of Zn²⁺/Mg²⁺, the weak fluorescence of the QN62 chemosensor was due to the synergistic contribution of the C⚌N isomerization and the ICT process. With the introduction of Zn²⁺/Mg²⁺ to the QN62 system, the binding sites participated in the coordination of QN62 with Zn²⁺/Mg²⁺, which further promoted the ICT process and prevented C⚌N isomerization, resulting in a significant fluorescence enhancement.

3.4 Reversibility of the chemosensor QN62

The reversibility of the detection process was further evaluated by alternating the addition of Zn²⁺/Mg²⁺ and ethylenediaminetetraacetic acid (EDTA) in the QN62 solutions (Fig. 7). Upon the addition of EDTA to the QN62-Zn²⁺/QN62-Mg²⁺ sensing solutions, the fluorescence intensity displayed a significant decrease and approached its original value, suggesting that EDTA could complex Zn²⁺/Mg²⁺ from the complexes and release free QN62. With the re-addition of Zn²⁺/Mg²⁺, the emission signals recovered, and this reversible cycle was repeated five times with a small emission signal decay. The results indicated that QN62 served as a reversible chemosensor for Zn²⁺/Mg²⁺ detection.

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Fig. 7

(a) Reversible changes in fluorescence intensity of QN62- (20 μM) at 532 nm upon alternate addition of Zn²⁺ and EDTA in DMSO-H₂O (4:1, v/v). (b) Reversible changes in fluorescence intensity of QN62 (20 μM) at 527 nm upon alternate addition of Mg²⁺ and EDTA in ethanol-H₂O (9:1, v/v).

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3.5 Application of chemosensor QN62 in test strip, water samples and cell imaging

To expand the potential practicability of QN62, we attempted to recognize Zn²⁺ and Mg²⁺ using silica-gel plates. Silica-gel plates were prepared by immersion in QN62 solution for a few seconds and then air-dried. Subsequently, the plates were successively treated with Zn²⁺ or Mg²⁺ by using the micro-capillary. We found that silica-gel plates immersed with QN62 displayed no fluorescence under UV light (Fig. 8). After micro-capillary treatment with Zn²⁺ or Mg²⁺, the silica-gel plates showed yellowish-green or yellow fluorescence. These findings suggested that the silica-gel plates made from QN62 could provide a convenient device for the on-site monitoring of Zn²⁺ and Mg²⁺.

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Fig. 8

Photograph of the silica-gel plates containing QN62 for the detection of Zn²⁺ and Mg²⁺ under UV light (365 nm).

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To evaluate the applicability of the QN62 compound in the samples, we carried out recovery tests using water samples from the campus area. Different concentrations of Zn²⁺/Mg²⁺ were added to water samples containing QN62. The experimental results showed that all the samples had acceptable recoveries with low standard deviation (SD) values, which illustrated the practical application of the QN62 chemosensor for detecting Zn²⁺ and Mg²⁺ in water samples (Table 2).

Cell imaging experiments were carried out to investigate whether QN62 could detect intracellular Zn²⁺ and Mg²⁺, and MCF-7 cells were selected. As shown in Fig. 9, the cells treated with free QN62 (10 μM) showed negligible fluorescence. After incubation with Zn²⁺/Mg²⁺ (10 μM), green fluorescence was observed, and the merged images showed that the fluorescence signal was located in the intracellular region. The cell imaging results suggested that the QN62 chemosensor could sense intracellular Zn²⁺ and Mg²⁺.

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Fig. 9

Bioimaging of exogenous Zn²⁺ and Mg²⁺ in MCF-7 cells with QN62 (10 μM). (a) Cells stained with QN62 for 30 min. (b) Cells treated with QN62 and then with 10 μM Zn²⁺ for 30 min. (c) Cells treated with QN62 and then with 10 μM Mg²⁺ for 30 min. a1-a3: Bright field; b1-b3: Green channel; c1-c3: Merged images. Green channel: λ_ex = 405 nm and collection: 500–550 nm.

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