ESIPT blocked CHEF based differential dual sensor for Zn²⁺ and Al³⁺in a pseudo-aqueous medium with intracellular bio-imaging applications and computational studies

Abstract

A novel 3-hydroxymethyl-5-methylsalicylaldehydenaphthyl-hydrazone (H₃SAL-NH) exhibits ESIPT behaviour due to proton transfer from the phenolic OH group to the azomethine N atom in the excited state. Through this ESIPT behaviour together with cis–trans isomerization of the azomethine group, the free ligand becomes very weakly fluorescent. However, in the presence of Zn²⁺ and Al³⁺ the ESIPT and isomerization are blocked due to coordination to the metal ions thereby causing turn on fluorescence for Al³⁺ and Zn²⁺. Moreover, Zn²⁺ can easily be displaced from the [H₂SAL-NH–Zn²⁺] complex by Al³⁺ thereby enhancing the differential selectivity for Al³⁺ over Zn²⁺. This probe was found to be selective for Al³⁺ over Zn²⁺ in the presence of Na₂H₂EDTA, under both intra- and extracellular conditions. The LODs for Zn²⁺ and Al³⁺ were determined by 3σ methods and were found to be 3.1 nM and 0.92 nM, respectively. Thus, the differentially selective turn-on fluorescence behaviour of H₃SAL-NH for Zn²⁺ and Al³⁺ is based on the combined blocking of ESIPT and C=N isomerization, and a chelation-enhanced fluorescence (CHEF) effect. The coordination modes of the complexes were investigated through spectroscopic and computational studies. H₃SAL-NH also exhibits good photostability and very low cytotoxicity and is useful for fluorescence imaging of Zn²⁺ and Al³⁺ ions in live HepG2 cells.

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Introduction

Molecular sensors are highly valuable tools for the selective recognition of chemical and biological species.¹ Though analyte selective molecular sensors are plentiful in the literature, probes with differential responses towards multiple analytes are highly desirable from the viewpoint of practical applications and are at the same time a challenge.^2,3

Aluminium is a nonessential element, found in the +3 oxidation state in most kinds of animal and plant tissues and in natural waters everywhere.^4–8 It has been shown clearly that aluminium accumulates in various mammalian tissues such as the brain, bone, liver and kidney^9,10 which is accompanied by renal failure¹¹ and associated with age.¹²

According to a WHO report, the average daily human intake of aluminium is approximately 3–10 mg per day.¹³ Thus the detection of Al³⁺ is attracting increasing interest in the areas of chemical, environmental and biological sciences, as the almost ubiquitous presence of this element has so heavily contaminated the environment that exposure to it is virtually inescapable.

However, compared to transition metals, the detection of Al³⁺ has always been a problematic task due to its lack of suitable spectroscopic characteristics and poor coordination ability¹⁴ and as result only a few fluorescent chemosensors are available in the literature.¹⁵

On the other hand, Zn²⁺ plays an important role in various fundamental biological processes, such as gene transcription, regulation of metalloenzymes, neural signal transmission and apoptosis. However, elevated levels of Zn²⁺ in humans have been implicated in neurodegenerative disorders.^16,17

A fluorescent probe is a molecular system that produces a detectable fluorescent signal upon interaction with a chemical species¹⁸ and a number of signaling mechanisms like photoinduced electron/energy transfer (PET),¹⁹ metal–ligand charge transfer (MLCT),²⁰ intramolecular charge transfer (ICT),²¹ excimer/exciplex formation,²² excited-state intra/intermolecular proton transfer (ESIPT),²³ and C=N isomerization have been developed and widely applied for the optical detection of different chemical species. As well as being good ligands for metal ions Schiff bases have antitumor,²⁴ anti-oxidant,²⁵ and attractive electronic and photophysical properties.²⁶

In this paper, a 3-hydroxymethyl-5-methylsalicylaldehyde-naphthylhydrazone based Schiff base (H₃SAL-NH) was found to be poorly fluorescent, in part due to isomerization through rotation around the C=N double bond in the excited state²⁷ and in part due to ESIPT involving the phenolic proton and the imine nitrogen of the ligand.²⁸ We reasonably anticipate that the ESIPT and C=N isomerization may be inhibited upon complexation with a metal ion which will render selective detection through spectral (UV-vis and/or fluorescence) changes.

Experimental section

Materials and methods

The starting materials 3-hydroxy-2-naphthyl hydrazide (Sigma-Aldrich) and 2-hydroxy-3-hydroxymethyl-5-methylbenzaldehyde (prepared in this laboratory) were used for the preparation of the ligands. Zn(NO₃)₂·6H₂O (Merck, Germany) and Al(NO₃)₃·9H₂O (Merck, Germany) were used to prepare Zn²⁺ and Al³⁺ complexes. The solvents, ethanol, diethyl ether and tetrahydrofuran (THF) (Merck, India), were of reagent grade and were dried before use.

Physical measurements

Elemental analyses were carried out using a Perkin-Elmer 240 elemental analyser. Infrared spectra (400–4000 cm⁻¹) were recorded from KBr pellets on a Nicolet Magna IR 750 Series-II FTIR spectrometer. ¹H-NMR was recorded in DMSO-d₆ on a Bruker 300 MHz NMR spectrometer using tetramethylsilane (δ = 0) as the internal standard. Lifetimes were measured in Horiba–Jobin–Yvon on a Hamamatsu MCP photomultiplier (R3809) and analysed using IBH DAS6 software. The pH of the solutions was recorded using a Systronics digital pH meter (Model 335, India) with the pH range 2–12. The pH meter was calibrated using standard buffer solutions (Acros Organics) of pH 4.0, 7.0 and 10.0. UV-vis spectra were recorded on an Agilent diode-array spectrophotometer (model: Agilent 8453) and steady-state fluorescence spectra were recorded on a Shimadzu spectrofluorimeter (model: RF-5301). ESI-MS⁺ (m/z) of the ligand and Zn(II)-complex were recorded on a Waters HRMS spectrometer (model: XEVOG2 QTOF).

Solution preparation for UV-vis and fluorescence studies

For both UV-vis and fluorescence titrations, a stock solution of 1.0 × 10⁻³ M of the probe H₃SAL-NH was prepared in THF. Similarly, 1.0 × 10⁻³ M stock solutions of the metal ions Al(NO₃)₃·9H₂O and Zn(NO₃)₂·6H₂O and other metal ions were prepared in THF : H₂O (6 : 4). A buffer solution in THF : H₂O (3 : 2, v/v) containing 10.0 mM HEPES and 10 mM LiCl was prepared and the pH was adjusted to 7.20 using dilute HCl and NaOH solutions. 2.5 ml of this solution was pipetted into the cuvette to which was added 20 μL of the probe and metal ions incrementally starting from 0 to 60 μL in a regular interval of volume and the UV-vis and fluorescence spectra were recorded for each solution.

Job plot

This method is based on the measurement of a series of solutions in which the molar concentrations of the two reactants vary but their sum remains constant. The absorbance of each solution was measured at 398 nm for Al³⁺ and at 414 nm for Zn²⁺ and plotted against the mole fraction of one reactant. A maximum in absorbance occurs at the mole ratio corresponding to the combining ratio of the reactants. The composition of the complex was determined by Job’s method and found to be (1 : 1) with respect to the ligand for both the Al³⁺ and Zn²⁺ complexes.

The preparation of 3-hydroxymethyl-5-methylsalicylaldehyde-naphthylhydrazone (H₃SAL-NH)

2-(Hydroxymethyl)-6-carbaldehyde-4-methylphenol was prepared according to the reported method.²⁹ 3-Hydroxy-2-naphthyl hydrazide (0.202 g, 1.0 mmol) was dissolved in 5 ml dry THF. To this solution, an ethanolic solution of 2-(hydroxymethyl)-6-carbaldehyde-4-methylphenol (0.166 g, 1.0 mmol) was added dropwise with continuous stirring. The resultant mixture was stirred for another 1 h maintaining a constant temperature at 40 °C. After the reaction was over, the white solid product was filtered and washed with cold diethyl ether and dried in air, then further recrystallized from EtOH (84% yield). Mp. 131 °C.

CHN analysis for C₂₀H₁₈N₂O₄: calculated (%): C, 68.56; H, 5.18; N, 8.00. Found (%): C, 68.54; H, 5.16; N, 7.97. ¹H-NMR (in DMSO-d₆) (δ, ppm): 2.27 (s, 3H); 4.56 (d, J = 4.9, 2H); 5.08 (t, J = 5.4, 1H); 7.14 (s, 1H); 7.26 (s, 1H); 7.35 (m, 1H); 7.51 (t, J = 7.6, 1H); 7.56 (d, J = 8.2, 1H); 7.91 (d, J = 8.1); 8.44 (s, 1H); 8.57 (s, 1H); 11.04 (s, 1H); 11.62 (s, 1H); 12.21 (s, 1H, NH), (please see Fig. S1† for the ¹H-NMR spectrum). ¹³C-NMR: (75 MHz, DMSO-d₆) δ_ppm: 20.60, 58.09, 111.07, 117.31, 120.45, 124.33, 126.32, 127.26, 127.92, 128.80, 129.15, 129.51, 130.08, 130.71, 130.91, 136.40, 151.08, 153.05, 154.37, 163.88 (Fig. S2†). ESI-MS⁺ (m/z): 373.1164 (H₃SAL-NH + Na⁺) (Fig. S3†).

Complex 1. Al(NO₃)₃·9H₂O (0.375 g, 1 mmol) was dissolved in 10 ml of a THF : ethanol mixture (2 : 8, v/v) and to this solution the ligand H₃SAL-NH (0.350 g, 1.0 mmol) in 3 ml THF was added. The color of the solution changed to yellow. The resulting mixture was stirred for 3 h. The volume of the solution was reduced to 5 ml under reduced pressure and diethyl ether (10 ml) was added. This was then kept at 0 °C for 12 h to afford complex 1 as a crystalline material. Yield: 0.356 g (∼52%). UV-vis (THF): λ_max 398 nm (Fig. S4†). CHN analyses for [Al(SAL-NH)(OH₂)]: C₂₀H₁₇N₂O₅Al (MW 392.34), calcd (%): C, 61.23; H 4.37; N, 7.14. Found (%): C, 61.29, H, 4.39, N, 7.10.

Complex 2. Zn(NO₃)₂·6H₂O (0.297 g 1.0 mmol) was dissolved in 10 ml of a THF : ethanol mixture (2 : 8, v/v) and to this solution, H₃SAL-NH (0.350 g, 1.0 mmol) in 3 ml THF was added. The color of the solution changed to bright yellow. The resulting mixture was stirred for 3 h. The volume of the solution was then reduced to 5 ml under reduced pressure and diethyl ether (10 ml) was added. This was then kept at 0 °C for 12 h to afford complex 2 as a crystalline material. Several trials to get single crystals were not successful. Yield: 0.398 g (∼70%). UV-vis (THF): λ_max, 414 nm (Fig. 1). CHN analyses for [Zn(H₂SAL-NH)(OH)]: C₂₀H₁₈N₂O₅Zn (MW 430.05), calcd (%): C, 55.64; H 4.20; N, 6.49. Found (%): C, 55.69, H, 4.23, N, 6.38.

Fig. 1 (A) Absorption titration spectra of H₃SAL-NH with Zn(NO₃)₂·6H₂O in HEPES buffer (10 mM) at pH 7.2 recorded in THF : H₂O (6 : 4, v/v); (B) Benesi–Hildebrand plot; (C) Job plot at 414 nm.

Computational details

Ground state electronic structure calculations in a THF solution of the ligand and complexes have been carried out using DFT³⁰ associated with the conductor-like polarizable continuum model (CPCM).³¹ Becke’s hybrid function³² with the Lee–Yang–Parr (LYP) correlation function³³ was used throughout the study. The geometries of the ligand and complexes were fully optimized in the solution phase without any symmetry constraints. On the basis of the optimized ground state geometry, the absorption spectral properties in a THF medium were calculated by time-dependent density functional theory (TDDFT)³⁴ associated with the conductor-like polarizable continuum model. We computed the lowest 40 singlet–singlet transitions and the results of the (TD) calculations were qualitatively very similar. The TDDFT (B3LYP) approach, a reliable method for calculating the spectral properties of many transition metal complexes,³⁵ has been adopted to get more accurate electronic excitation energies.

The effective core potential (ECP) approximation of Hay and Wadt was used to describe the core electrons for both aluminium and zinc whereas the associated “double-ξ” quality basis sets were used for the valence shell.³⁶ For H atoms we used the 6-31(g) basis set; for C, N and O atoms we employed 6-31+g as the basis set for all the calculations. The calculated electron density plots for the frontier molecular orbitals were prepared by using Gauss View 5.1 software. All the calculations were performed with the Gaussian 09W software package.³⁷ The GaussSum 2.1 program³⁸ was used to calculate the molecular orbital contributions from groups or atoms.

Cell culture

HepG2 cell line human hepatocellular liver carcinoma cells, were procured from the National Center for Cell Science, Pune, India, and used throughout the study. The cells were cultured in DMEM (Gibco BRL) supplemented with 10% FBS (Gibco BRL) and a 1% antibiotic mixture containing penicillin, streptomycin and gentamicin (Gibco BRL) at 37 °C in a humidified incubator with 5% CO₂. The cells were grown to 90% confluence, harvested with 0.025% trypsin (Gibco BRL) and 0.52 mM EDTA (Gibco BRL) in phosphate-buffered saline (PBS). The 35 mm plated was seeded in such way as to get a 60–70% confluent cell population for treatment after incubation.

Cell cytotoxicity assay

To test the cytotoxicity of H₃SAL-NH an assay was performed as per the procedure described earlier.³⁹ After treatment with H₃SAL-NH at different doses of 1, 10, 20, 50 and 100 μM, respectively, for 12 h, 10 μl of MTT solution (10 mg ml⁻¹ PBS) was added to each well of a 96-well culture plate and this was again incubated continuously at 37 °C for a period of 3 h. All media were removed from the wells and 100 μl acidic isopropyl alcohol was added into each well. The intracellular formazan crystals (blue-violet) formed were solubilized with 0.04 N acidic isopropyl alcohol and the absorbance of the solution was measured at 595 nm with a microplate reader (model: Thermo Scientific Multiskan EX). The cell viability was expressed as the optical density ratio of the treated cells to the control. Values are expressed as the mean ± standard error of three independent experiments. The cell cytotoxicity was calculated as % cell cytotoxicity = 100% − % cell viability.

Cell imaging study

HepG2 cells were incubated with 10 μM H₃SAL-NH. The stock solution (1 mM) was prepared by dissolving H₃SAL-NH in a mixed solvent (DMSO : water = 1 : 9 (v/v)) in the culture medium followed by incubation for 30 min at 37 °C. After incubation, the cells were washed twice with phosphate-buffered saline (PBS). Bright field and fluorescence images of the HepG2 cells were taken using a fluorescence microscope (Leica DM3000, Germany) with an objective lens of 40× magnification.

Results and discussion

UV-vis absorption studies

The electronic absorption properties of (H₃SAL-NH) were investigated in THF : H₂O (6 : 4, v/v) and HEPES buffer (10 mM) at pH 7.2. The band at 349 nm is assigned to the electronic transition involving the HO–C=C–C=N chromophore in the enol form.^40–43 The band at 313 nm is attributed to the azomethine C=N group.

It was anticipated that the nitrogen of the –C=N bond would take part in the coordination with Zn²⁺ to form a H₂SAL-NH–Zn²⁺ complex, thereby inhibiting the ESIPT (Scheme 1). Moreover, new absorption bands appear at 398 nm for Al³⁺ and at 414 nm for Zn²⁺, which is probably caused by an intraligand charge transfer (ILCT) transition of the π → π* type, which is certainly caused by a larger conjugation in the ligand system.

Scheme 1 Proposed ESIPT in the compound H₃SAL-NH (1).

The absorption titration spectra for a fixed concentration of H₃SAL-NH (20 μM) and a variable concentration of Zn²⁺ and Al³⁺ (0 to 26 μM) are shown in Fig. 1 and S4.† There is a gradual increase in the absorbance at 398 nm for Al³⁺ and 414 for Zn²⁺ with the increase in metal ion concentration, reaching a maximum at ∼1 : 1 metal to ligand concentration and then becoming saturated (Fig. 1A). All these data were fitted to the Benesi–Hildebrand equation to get the dissociation constant and stoichiometry of complexation.

where, A₀ and A_max are the absorbance of the pure ligand in the absence of the metal ion and in the presence of excess metal ions, respectively. A linear least square fit of (A_max − A₀)/(A − A₀) against 1/[M] clearly demonstrates n = 1, indicating a 1 : 1 complexation for both the metal ions. The linear fit also gives the apparent dissociation constants of the complexes as K_d = (7.17 ± 0.18) × 10⁻⁶ M for Al³⁺ and K_d = (1.31 ± 0.06) × 10⁻⁵ M, for Zn²⁺. The 1 : 1 L : M binding was further determined by Job’s method (Fig. 1C and S4†).

Fluorescence studies

In order to check the selectivity of the probe H₃SAL-NH for Zn²⁺ and Al³⁺ detection we have carried out fluorescence experiments with 20 μM H₂SAL-NH and 5 equivalents of different metal ions. It was interesting to note that the detection of Al³⁺ was not perturbed by biologically abundant Na⁺, K⁺, Ca²⁺, etc., metal ions. Several transition metal ions, namely Cr³⁺, Zn²⁺, Mn²⁺, Fe²⁺, Fe³⁺ and Hg²⁺, also caused no interference (Fig. 2A). However, the detection of Zn²⁺ is affected by Al³⁺ only so in absence of Al³⁺ the probe can selectively recognize Zn²⁺. In the presence of Zn²⁺ and Al³⁺ the probe is highly selective towards Al³⁺ only.

Fig. 2 (A) Histogram plot for the detection of Al³⁺ with different ions. (B) Histogram plot for the detection of Zn²⁺ in absence of Al³⁺ and with different ions.

The emission spectra of H₃SAL-NH (20 μM) and its fluorescence titration with Al(NO₃)₃·9H₂O and Zn(NO₃)₂·6H₂O (recorded in THF : H₂O (6 : 4, v/v)) in HEPES buffer (10 mM) at pH 7.2 are shown in Fig. 3 and 4. The ligand itself is very weakly fluorescent, showing a weak band centred around 543 nm, due to ESIPT and C=N isomerization. However, on addition of Al³⁺ or Zn²⁺ the fluorescence intensity increases enormously, giving turn-on fluorescence responses with values ∼30 fold higher for Al³⁺ at λ_em = 486 nm and ∼18 fold higher for Zn²⁺ at λ_em = 498 nm with respect to the free ligand. In addition, there is a gradual blue shift of λ_em from 543 nm for the pure ligand to 486 nm and 498 nm on complexation with Al³⁺ and Zn²⁺ respectively, which is accompanied with a large increase in the fluorescence lifetime (τ) of H₃SAL-NH (Fig. S5 and S6†). Thus, τ (0.0067 ns) for the free ligand (H₃SAL-NH) increases enormously [τ(SAL-NH-Al) = 5.04 ns (757 fold) and τ[H₂SAL-NH–Zn(OH)] = 3.2 ns (477 fold)] in the presence of 1.2 equivalents of the respective metal ions. The Benesi–Hildebrand equation was employed to analyze the fluorescence titration data to get the apparent dissociation constant values and the evaluated parameters are: K_d = (3.14 ± 0.08) × 10⁻⁶ M for Al³⁺ and K_d = (1.12 ± 0.04) × 10⁻⁵ M for Zn²⁺. It should be mentioned here that the K_d values evaluated from the absorbance and fluorescence titration data are in excellent agreement manifesting the self-consistency of our experimental results.

Fig. 3 (A) Fluorescence titration of H₃SAL-NH (20 μM) in HEPES buffer at pH 7.2 recorded in THF : H₂O (6 : 4, v/v) with the gradual addition of Al³⁺ with λ_ex = 398 nm and λ_em = 486 nm. Inset (B) B–H plot of F.I. vs. 1/[Al³⁺]; (C) UV-exposed image of H₃SAL-NH with Al³⁺.

Fig. 4 (A) Fluorescence titration of H₃SAL-NH (20 μM) in HEPES buffer at pH 7.2 recorded in THF : H₂O (6 : 4, v/v) with the gradual addition Zn(NO₃)₂·6H₂O with λ_ex = 398 nm and λ_em = 498 nm. (B) B–H plot of F.I. vs. 1/[Zn²⁺]; (C) UV-exposed emission image of H₃SAL-NH with Zn²⁺.

In the UV-vis spectrum of the free ligand, the absorption peak at 349 nm is assigned to the HO–C=C–C=N chromophore. When we generate the excitation spectrum of the ligand keeping the emission wavelength fixed at 557 nm, two excitation peaks appear at λ_max 348 and 460 nm manifesting the existence of both the enol and keto forms, the latter being assigned to the keto form, which arises due to the intramolecular proton transfer from the phenolic OH to the azomethine N resulting in C=O and –C–NH and thus increasing the conjugation (Fig. S7†). H₃SAL-NH is poorly fluorescent, partly due to isomerization around the C=N double bond (Scheme 2) and partly due to the intramolecular proton transfer in the excited state (ESIPT). Upon stable chelation with Al³⁺ or Zn²⁺, the C=N isomerization as well as the ESIPT are blocked (Scheme 1), leading to the increase in fluorescence intensity by chelation enhanced fluorescence (CHEF) effects.⁴⁴

Scheme 2 C=N isomerization.

Selectivity studies

Next, the selectivity of Al³⁺ over Zn²⁺ was examined by the addition of 1 equivalent of Al³⁺ to the [H₂SAL-NH–Zn²⁺] ensemble (Fig. 5) which showed an instantaneous decrease in the emission intensity at 498 nm due to the formation of an [Al³⁺–H₂SAL-NH–Zn²⁺] intermediate and then a gradual increase at 486 nm with time, clearly indicating the displacement of Zn²⁺ by Al³⁺. This also indicates a slow uptake of Al³⁺ by the receptor manifesting the fact that this complexation is kinetically slow but the formed complex is thermodynamically stable over the H₂SAL-NH–Zn²⁺ complex. Therefore, all the UV-vis and fluorescence spectra of the SAL-NH–Al³⁺ titrations were taken after half an hour of mixing the guest with the host molecule. However, when we used [SAL-NH–Al³⁺] and added an excess (2 to 100 equivalents) of Zn²⁺, we did not see any change in the spectral pattern (Fig. S8†). Thus the probe is highly selective towards only Al³⁺ even in the presence of Zn²⁺ [Fig. 2 and 5]. The ligand is also selective towards Zn²⁺ in the presence of other metal ions but in the absence of Al³⁺ [(Fig. 2)]. However, Na₂H₂EDTA extracts Zn²⁺ from the H₂SAL-NH–Zn²⁺ complex as [ZnEDTA]²⁻, thereby masking the fluorescence of the Zn–probe complex (Fig. 2), but Na₂H₂EDTA has no such effect on the [SAL-NH–Al³⁺] complex. Thus the probe is highly selective for Al³⁺ even in presence of excess Zn²⁺ ions (Fig. S9†).

Fig. 5 Selectivity for Al³⁺ over the H₂SAL-NH–Zn²⁺ complex.

The limits of detection (LODs) for Zn²⁺ and Al³⁺ were determined by 3σ methods which are found to be 3.1 nM and 0.92 nM, respectively (Fig. S10†). In the presence of 1 equivalent of Zn²⁺ and Na₂H₂EDTA the LOD of Al³⁺ for the [SAL-NH–Al³⁺] complex was calculated to be 3.2 nM (Fig. S10c†). The quantum yields of the Zn²⁺ and Al³⁺ complexes of H₃SAL-NH were determined to be 0.062 (Zn complex) and 0.0705 (Al complex), which are respectively 21 and 23 times higher than that of the pure ligand (0.003).

Different spectroscopic techniques were adopted to establish the mode of coordination of the H₃SAL-NH probe towards Al³⁺ and Zn²⁺ ions in solution. The mass spectrum of [M(SAL-NH)] in THF revealed that H₃SAL-NH : M = 1 : 1 with ESI-MS⁺ (m/z) = 437.20 for ([Zn(H₂SAL-NH)(OH)] + Li⁺) and ESI MS⁺ (m/z) = 407.17 for [Al(SAL-NH)(CH₃OH) + H⁺] (Fig. S3a and b†). A comparison of the FTIR spectra of the free H₃SAL-NH and its complexes revealed a shift in ν_C=N from 1643 cm⁻¹ for the free ligand to 1621 and 1635 cm⁻¹ for the Zn²⁺ and Al³⁺ complexes, respectively indicating a significant binding between them (Fig. S11†).

The coordination modes were further supported by ¹H-NMR studies (Fig. 6) which clearly showed a change in the chemical shifts of the azomethine proton of the free ligand from 8.57 ppm to 8.64 and 8.59 ppm in presence of 1 equivalent of Al³⁺ and Zn²⁺ respectively (Table S1†). The OH proton on the hydroxymethyl group of the free ligand vanishes due to its strong participation in bonding with Zn²⁺ but the other OH protons and the –NH proton remain unchanged due to their non-participating behavior but in the case of Al³⁺ all three OH protons vanish with the addition of 1 equivalent Al³⁺. This clearly signifies that the OH protons are engaged in binding with Al³⁺ but there is no shift of the –NH proton which suggests its non-participation in bonding with metal ions.

Fig. 6 ¹H-NMR titration of the free ligand (L = H₃SAL-NH) and with the addition of 1 equivalent of Zn²⁺ (a) and Al³⁺ (b) in DMSO-d₆ recorded on a 300 MHz Bruker NMR spectrometer.

Thus, we can conclude that two phenolate O atoms and one imine N atom and hydroxymethyl group of H₃SAL-NH are involved in binding with Al³⁺ but in case of the Zn²⁺ hydroxymethyl group, one imine N atom the hydroxo and amido O atoms are involved in binding but the two phenolate O atoms are not involved in binding. This was further supported by detailed DFT calculations on the free ligand and its Al³⁺ and Zn²⁺ complexes (vide infra). The non-participation of the phenoxo O41 and naphthoxo O17 in bonding with the Zn atom comes from the fact that they are not within bonding distance [Zn44–O41 = 2.93 Å and Zn44–O41 = 5.54 Å].

The binding affinity of the probe is greater for Al³⁺ than Zn²⁺ as evidenced from the formation constant values (K_Al³⁺ = 3.18 × 10⁵ M⁻¹ and K_Zn²⁺ = 8.93 × 10⁴ M⁻¹) and thus displacement of the latter by Al³⁺ can be explained by the fact that the present probe provides comparatively hard donor atoms for the hard acid Al³⁺.

Geometry optimization and electronic structure

The optimized geometries of H₃SAL-NH and its Zn²⁺ and Al³⁺ complexes are shown in Fig. 7. The compositions of the complexes as [Al(SAL-NH)(MeOH)] and [[Zn(H₂SAL-NH)(OH)] were adopted based on HRMS studies which indicated the presence of one MeOH and one hydroxyl group in their molecular fragments. H₃SAL-NH, and SAL-NH–Al³⁺ (1) and H₂SAL-NH–Zn²⁺ (2) complexes have the C1 point group. The important optimized geometrical parameters of the complexes are listed in Table S2.†

Fig. 7 Optimized molecular structures of the ligand, complex 1 and complex 2.

In the case of complex 1 the Al³⁺ center adopts a trigonal bipyramidal geometry. The calculated Al–N bond distance is 2.107 Å and the Al–O bond distance fall in the range 1.806–1.998 Å. Here the equatorial sites are occupied by one phenoxo oxygen (O48) one imime nitrogen (N4) and the oxygen (O22) of the hydroxymethyl group, while the axial sites are occupied by a phenoxo oxygen (O48) and an oxygen atom (O30) of the MeOH solvent. In the case of complex 2 the Zn²⁺ centre is tetracoordinated where all the Zn–O bond distances span the range 1.841–2.128 Å and the Zn–N distance is 2.247 Å.

In the case of H₃SAL-NH in the ground state, the electron density of the HOMO and HOMO−2 orbitals resides mainly on the phenyl ring and the phenoxo oxygen attached to the naphthyl moiety, whereas for the HOMO−3 orbital it remains at the naphthyl ring and for the HOMO−4 orbital it is at the imine nitrogen atom. The energy gap between the HOMO and LUMO is 3.78 eV (Fig. 8). In the case of the HOMO−3 orbital of complex 1 the electronic contribution comes mainly from the naphthyl moiety and in HOMO−2 it comes from the phenyl moiety. The HOMO–LUMO energy gap is 3.60 eV (Fig. 8). In the case of 2 the electronic contribution in the HOMO−5 and HOMO orbitals mainly originates from the phenyl moiety with a HOMO–LUMO energy gap of 3.09 eV (Fig. 8).

Fig. 8 Frontier molecular orbitals of H₃SAL-NH and its [Al(SAL-NH)(MeOH)] and [Zn(H₂SAL-NH)(OH)] complexes.

The UV-vis absorption spectra of the ligand used in the present work were calculated at room temperature in THF by the TDDFT method. The ligands show three well resolved peaks at 350, 313 and 302 nm and all have an ILCT character. These bands are assigned to the S₀ → S₄, S₀ → S₁₁ and S₀ → S₁₂ electronic transitions, respectively (Fig. 9). The absorption energies associated with their oscillator strengths are given in Table 1 and S3.†

Fig. 9 Frontier molecular orbitals involved in the UV-vis absorption of H₃SAL-NH in THF solution.

Table 1 The comparable calculated optical transitions with experimental UV-vis values for the ligand (H₃SAL-NH), complex (1) and complex 2

Ligand and complex (3)	Theoretical (nm)	Experimental (nm)	Electronic transition	f
Ligand	357.44	350	S0 → S4	0.7285
Ligand	311.13	313	S0 → S11	0.1418
Ligand	297.08	303	S0 → S12	0.4127
Complex 1	399.63	398	S0 → S2	0.0565
Complex 1	341.53	335	S0 → S5	0.0401
Complex 1	314.55	321	S0 → S6	0.2680
Complex 2	420.42	412	S0 → S1	0.1365
Complex 2	332.69	335	S0 → S6	0.4355

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The UV-vis spectrum of the [Al(SAL-NH)(MeOH)] complex shows three absorption bands at 398, 335 and 321 nm in THF at room temperature which correspond nicely to the TDDFT calculated absorption bands located at 399, 341 and 314 nm (Fig. 10) (Table S4† and 1). These three absorption bands can be assigned to the S₀ → S₂, S₀ → S₅ and S₀ → S₆ transitions, respectively (Fig. 10) originating from a mixture of MLCT and ILCT transitions (Table S4†).

Fig. 10 Frontier molecular orbitals involved in the UV-vis absorption of complex 1.

The [Zn(H₂SAL-NH)(OH)] complex shows two absorption bands at 412 and 335 nm (Fig. S12†) and the corresponding calculated absorption bands are located at 420 and 333 nm which are in excellent agreement with the experimental results (Tables S5† and 1). These two absorption bands can be assigned to the S₀ → S₁ and S₀ → S₆ transitions respectively originating from a mixture of MLCT and ILCT transitions (Table S5†).

pH studies

The dependence of the fluorescence intensity of the free ligand and its Al³⁺ and Zn²⁺ complexes on the pH of the medium was investigated in the pH range 2.0 to 12.0 at [H_3​SAL-NH] = 20 μM, [Mⁿ⁺] = 30 μM in 6 : 4 THF : H₂O v/v in HEPES buffer. It was observed that in the pH range 3 to 8 the F. I. of the free ligand remains almost constant. However, on addition of 1.2 equivalents of Al³⁺ the F. I. jumps to 275 ± 10 and remains constant in the pH range 7–9, but on a further increase in pH the F. I. gradually falls. Similar is the trend in the case of the Zn²⁺ complex but here the F. I. remains almost constant at ∼179 ± 10 in the pH range 7–11 and then the F. I. gradually falls. All these are shown in Fig. 11.

Fig. 11 pH dependence of fluorescence responses of H₃SAL-NH and its Zn²⁺ and Al³⁺-complexes in 6 : 4 (v/v) THF/water in HEPES buffer.

Cell imaging experiments

Taking into consideration the excellent sensing performance of H₃SAL-NH for Al³⁺ and Zn²⁺, we decided to utilize H₃SAL-NH for the fluorescence imaging of Al³⁺ and Zn²⁺ in living cells (Fig. 12). To determine whether H₃SAL-NH has any cytotoxic effect we have used HepG2 cells. H₃SAL-NH does not affect the cell viability, as confirmed by the MTT assay (Fig. S13†). There was no significant reduction in the tetrazolium salt, reflecting a decrease in formazan production for H₃SAL-NH up to 50 μM. More than 92% cell viability was observed for H₃SAL-NH at 10 μM after which the viability of the HepG2 cells decreases slightly (viability curve). Hence further experiments were carried out with 10 μM of H₃SAL-NH. The intracellular imaging of H₃SAL-NH on HepG2 cells under fluorescence microscopy displayed no or weak intracellular fluorescence when treated with 10 μM H₃SAL-NH (Fig. 12). An obvious fluorescence was observed inside the cells when the HepG2 cells were incubated with 10 μM H₃SAL-NH +10 μM Al³⁺ and 10 μM H₃SAL-NH +10 μM Zn²⁺ for 30 min at 37 °C. In order to validate the finding that Zn²⁺ and Al³⁺ bind with H₃SAL-NH, we have separately treated the HepG2 cells with 10 μM H₃SAL-NH +10 μM Al³⁺ and 10 μM H₃SAL-NH +10 μM Zn²⁺ for 30 min at 37 °C in another set of experiments followed by the addition of 100 μM Na₂H₂EDTA for another 30 min at 37 °C and fluorescence images were taken (Fig. 12). The HepG2 cells incubated with 10 μM H₃SAL-NH + 10 μM Zn²⁺ at 37 °C showed almost complete quenching of fluorescence due to the removal of Zn²⁺ from the H₂SAL-NH–Zn²⁺ complex by Na₂H₂EDTA, whereas there was no change in the fluorescence intensity after the addition of Na₂H₂EDTA to the SAL-NH–Al³⁺ complex (Fig. 12). This implies that the SAL-NH–Al³⁺ complex is more stable than the H₂SAL-NH–Zn²⁺ complex in the presence of Na₂H₂EDTA. We have presented a novel fluoroionophore for sensing Zn²⁺ and Al³⁺ ions. H₃SAL-NH also exhibits good photostability and very low cytotoxicity. The fluorescence images of the live cells reveal that it can easily diffuse into the cell and can easily sense the Zn²⁺ and Al³⁺ ions by forming a stable complex at low concentrations. However, unlike the Zn²⁺ ion, Al³⁺ forms a stable complex with H₃SAL-NH, particularly with respect to the reaction with H₂EDTA²⁻. This enhances the selectivity of the novel fluoroionophore for Al³⁺ over Zn²⁺.

Fig. 12 The phase contrast and fluorescence images of HepG2 cells captured after incubation with H₃SAL-NH, H₃SAL-NH + Zn²⁺ and H₃SAL-NH + Al³⁺ for 30 min at 37 °C, followed by the addition of 100 μM Na₂H₂EDTA after the cells were pre-incubated with (H₃SAL-NH + Zn²⁺) and (H₃SAL-NH + Al³⁺) for 30 min at 37 °C.

Conclusions

In summary, a novel 3-hydroxymethyl-5-methylsalicylaldehyde-naphthylhydrazone (H₃SAL-NH) fluoroionophore was synthesized and characterized by various spectroscopic techniques. Due to ESIPT behavior arising from proton transfer from the phenolic OH to the azomethine N atom in the excited state together with cis–trans isomerization along the C=N bond of the azomethine group, the free ligand becomes very weakly fluorescent. However, in the presence of selective metal ions like Zn²⁺ and Al³⁺ the ESIPT and isomerization are blocked through coordination to the metal ions, thereby exhibiting turn on fluorescence for Al³⁺ and Zn²⁺. Moreover, Zn²⁺ can easily be displaced from the [H₂SAL-NH–Zn²⁺] complex by Al³⁺ thereby enhancing the differential selectivity of Al³⁺ over Zn²⁺. Not only that, this probe could be made selective towards Al³⁺ over Zn²⁺ in the presence of Na₂H₂EDTA. All these properties can conveniently be demonstrated both in in vivo and in vitro conditions. Fluorescence and UV-vis titrations at a fixed concentration of the probe and a variable metal ion concentration gives us the conditional dissociation constants (K_d) and hence the K′_f (formation constants) values which were found to be in excellent agreement for the two techniques. The 1 : 1 stoichiometry of the reaction between H₃SAL-NH and Al³⁺ and Zn²⁺ were determined by Job’s method and were also in agreement with those obtained from the titration data. The LODs for Zn²⁺ and Al³⁺ were determined by 3σ methods and were found to be 3.1 nM and 0.92 nM, respectively. The coordination modes of the complexes were investigated by computational and spectroscopic studies. Thus, the differentially selective turn-on fluorescence behavior of H₃SAL-NH for Zn²⁺ and Al³⁺ is based on a combined blocking of ESIPT and C=N isomerization, and chelation-enhanced fluorescence CHEF effects. In vitro cell imaging experiments showed that it can easily diffuse into the cell and can easily sense the Zn²⁺ and Al³⁺ ions by forming stable complexes at low concentrations.

Acknowledgements

Financial supports from CSIR (Ref. 02(2490)/11/EMR-II) and DST (Ref. SR/S1/IC-20/2012) New Delhi are gratefully acknowledged.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra18424j

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