A multiple target chemosensor for the sequential fluorescence detection of Zn²⁺ and S²⁻ and the colorimetric detection of Fe^3+/2+ in aqueous media and living cells

Abstract

A novel multiple target sensor, (E)-5-((4-(diethylamino)-2-hydroxybenzyldene)amino)-1H-imidazole-4-carboxamide (DHIC), was synthesized for fluorescence detection of Zn²⁺ and S²⁻ and colorimetric detection of Fe^3+/2+ in aqueous media. DHIC can operate as a turn “on–off” sequential fluorescent sensor for Zn²⁺ and S²⁻. Detection limits (1.59 μM and 8.03 μM) for Zn²⁺ and S²⁻ are below the WHO standards (76.0 μM and 14.7 μM). The DHIC–Zn²⁺ complex could be reversibly reused with ethylenediaminetetraacetic acid. Importantly, DHIC could image sequentially Zn²⁺ and S²⁻ in living cells. Moreover, DHIC displayed a discriminatory color change from pale yellow to orange yellow to Fe^3+/2+. The detection limit of DHIC for Fe^3+/2+ (0.73 μM and 1.11 μM) is far below the EPA drinking water standard (5.37 μM). The sensor DHIC could be applied to analyze Fe³⁺ in real samples.

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1. Introduction

Zinc is one of the transition metal ions present in the body and has various roles in many physiological and biological processes like cell apoptosis, signal transduction, cellular metabolism, and DNA synthesis.^1–4 However, imbalance of Zn²⁺ in the human body can lead to fatal, serious diseases, including epilepsy, Alzheimer's disease, ischemic stroke, Parkinson's disease, and some types of carcinoma.^5–7 To control the Zn level in biological systems, therefore, fluorogenic chemosensors have been developed.⁸ Although many Zn²⁺ chemosensors have been reported, it's still a difficult task to discriminate Zn²⁺ and Cd²⁺,^9–11 since these elements have analogous photo-physical properties.^12,13 For these reasons, the development of easily synthesized, highly selective and sensitive zinc chemosensors has attracted considerable attention in biological and chemical areas.^14–18

Hydrogen sulfide (H₂S), the simplest biothiol, is generated by enzymes like mercaptopyruvate sulfurtransferase, cystathionine-β-synthase, and cysteine lyase.^19–22 H₂S belongs to the family of endogenous gasotransmitters such as CO and NO.^23–27 It contributes to a broad range of physiological processes, like oxygen sensing, reducing blood pressure, vasodilation, anti-inflammation and apoptosis.^28–31 However, excess of H₂S in cells causes various diseases like Alzheimer's disease, liver cirrhosis and Menkes disease.^28,32–36

Iron performs pivotal roles in many biological systems, including electron transfer, oxygen storage and oxygen carrier function in hemoglobin.^37–41 Its deficiency or superabundance not only causes some dysfunction of organs like the kidney, heart, and liver, but also leads to several diseases like anemia and diabetes.^42–44 Thus, the development of tools to analyze iron in biological and environmental fields has been of great interest.^45–48

Schiff bases that have donor atoms such as N and O form strong complexes with transition metal ions.^49–51 Thus, Schiff bases have often been used as chemosensors for detecting metal ions. The imidazole moiety with fluorogenic properties could be used as a part of a metal-ion sensor,^42,45,52 and the 4-diethylaminosalicylaldehyde moiety is also water-soluble and fluorogenic.^53,54 Thus, we designed and synthesized a novel Schiff base sensor, DHIC, on the basis of the condensation reaction of the imidazole and 4-diethylaminosalicylaldehude moieties; it is water-soluble and has the potential to detect multiple analytes. While many chemosensors for single analytes have been reported, single chemosensors targeting multiple analytes have been relatively less developed.^55–58 Since multi-functional chemosensors are cost-effective and convenient compared to single-response chemosensors, it is of great importance to develop multi-functional chemosensors.^59,60

Herein, we present a multiple target chemosensor, DHIC, (E)-5-((4-(diethylamino)-2-hydroxybenzyldene)amino)-1H-imidazole-4-carboxamide, which showed consecutive fluorescence turn “on–off” for Zn²⁺ and S²⁻. In addition, it could image consecutively Zn²⁺ and S²⁻ in living cells. The sensor DHIC could also detect Fe^3+/2+ by a change of color from pale yellow to orange yellow in a near-perfect aqueous medium (Bis–Tris buffer : DMF = 997 : 3). Importantly, among the chemosensors reported to date, DHIC is the first chemosensor that can recognize the four analytes, Zn²⁺, S²⁻, and Fe^3+/2+.

2. Experiment

Materials and instrumentation

Chemical reagents were obtained commercially. ESI-MS and NMR data were collected using a Thermo Finnigan ion trap machine and a Varian spectrometer. Spectral data of emission and absorption were obtained using spectrometers (LS45 and Lambda 2S UV/Vis) from PerkinElmer.

Synthesis of the receptor DHIC ((E)-5-((4-(diethylamino)-2-hydroxybenzyldene)amino)-1H-imidazole-4-carboxamide)

Triethylamine (1.2 mmol, 169 μL) and 5(4)-amino-4(5)-(aminocarbonyl)imidazole hydrochloride (1.0 mmol, 0.17 g) were dissolved in 15.0 mL of MeOH and stirred for 1.5 h. 4-Diethylaminosalicylaldehyde (1.0 mmol, 0.20 g) and a few drops of H₃PO₄ were added to the resulting solution, which was blended for an additional 7 h. A yellow powder was produced, which was gathered by filtration. Then, the yellow solid was washed twice with ether and dried under vacuum (0.19 g, 65.0%). ¹H NMR (400 MHz, DMSO-d₆): δ = 12.63 (s, 1H, NH, imidazole), 11.53 (s, 1H, OH), 9.12 (s, NCH, imine), 7.61 (s, 1H, CHNH), 7.51 (d, J = 8 Hz, 1H, m-phenyl), 7.41 (s, 2H, amide), 6.30 (d, J = 8 Hz, p-phenyl), 6.09 (s, o-phenyl), 3.36 (m, 4H, CH₂CH₃, ethyl), 2.41 (t, J = 12 Hz, 6H, CH₃CH₂, methyl); ¹³C NMR (100 MHz, DMSO-d₆): 162.07 (1C), 161.65 (1C), 157.65 (1C), 152.02 (1C), 148.16 (1C), 135.96 (1C), 132.10 (1C), 116.24 (1C), 109.68 (1C), 104.74 (1C), 97.28 (1C), 44.39 (2C), 13.00 (2C); Low Resolution Mass Spectrometry (LRMS) (ESI): m/z calcd for C₁₅H₁₉N₅O₂ + H⁺: 302.16; found: 302.10.

Fluorescence detection

Fluorescence and UV-visible titrations of DHIC with Zn²⁺. DMF (1.0 mL) was employed to dissolve the compound DHIC (0.9 mg, 3 × 10⁻³ mmol). 10.0 μL (3.0 mM) of DHIC was diluted with 2.99 mL of Bis–Tris buffer solution (10 mM, pH 7.0) to 10.0 μM. Bis–Tris buffer (5.0 mL) was employed to dissolve Zn(NO₃)₂ (0.1 mmol, 30.4 mg). 0–33 μL (2 × 10⁻² M) of the Zn²⁺ solution were added to each diluted DHIC (3.0 mL, 10.0 μM). Fluorescence measurements were executed after mixing them. For UV-vis titration, 0–21 μL (20.0 mM) of the Zn²⁺ solution were added to the DHIC (3.0 mL, 10.0 μM) prepared above. UV-vis measurements were executed after mixing them.

Job plot of DHIC with Zn²⁺. DMF (1.0 mL) was employed to dissolve the compound DHIC (3.01 mg, 1 × 10⁻² mmol). 0–12.0 μL of DHIC was added to fluorescence cells. They were diluted with Bis–Tris buffer for preparing a total volume of 2.988 mL. Bis–Tris buffer (1.0 mL) was employed to dissolve Zn(NO₃)₂ (3.0 mg, 0.01 mmol). 0–12.0 μL of the zinc solution were added to each diluted DHIC. The fluorescence cell contained a total volume of 3 mL. Fluorescence measurements were executed after mixing them.

Competition test. DMF (1.0 mL) was employed to dissolve the compound DHIC (0.9 mg, 3 × 10⁻³ mmol). 10.0 μL (3.0 mM) of DHIC was diluted with 2.990 mL of Bis–Tris buffer to 10 μM. Bis–Tris buffer (5.0 mL) was employed to dissolve 1 × 10⁻¹ mmol of KNO₃ or Fe(ClO₄)₂ or NaNO₃ or M(NO₃)₃ (M = Cr, Fe and Al) or M(NO₃)₂ (M = Pb, Zn, Cd, Mg, Co, Cu, Mn, Ni, and Ca). 33 μL (2 × 10⁻² M) of each cation solution was added to 3.0 mL each of DHIC (10 μM) to produce 21 equiv. 33.0 μL of Zn(NO₃)₂ was added to the mixture of DHIC and each cation solution to produce 21 equiv. Fluorescence measurements were executed after mixing them.

pH effect. By mixing HCl with Bis–Tris buffer and NaOH solution, buffers having different pH values (2–12) were made. With the desired pH solutions, DHIC (0.9 mg, 3 × 10⁻³ mmol) was dissolved in 1.0 mL of DMF, and 10.0 μL (3.0 mM) of DHIC was diluted with 2.99 mL Bis–Tris buffer to 10 μM. Bis–Tris buffer (5.0 mL) was employed to dissolve Zn(NO₃)₂ (30.4 mg, 10⁻¹ mmol). 33.0 μL (2 × 10⁻² M) of Zn²⁺ was transferred to each DHIC (10.0 μM). Fluorescence measurements were executed after mixing them.

NMR titration of DHIC with Zn²⁺. DMSO-d₆ (700 μL) was employed to dissolve DHIC (0.1 mmol, 3.0 mg) in three NMR tubes, respectively. Three different equiv. (1, 0.5, and 0 equiv.) of Zn(NO₃)₂ dissolved in DMSO-d₆ (300 μL) were transferred to DHIC. ¹H NMR measurements were executed after mixing them.

Fluorescence and UV-vis titrations of DHIC–Zn²⁺ with S²⁻. DMF (1.0 mL) was employed to dissolve DHIC (0.9 mg, 3 × 10⁻³ mmol). 10.0 μL of DHIC (3.0 mM) was diluted with 2.99 mL of Bis–Tris buffer to 10.0 μM. Bis–Tris buffer (5.0 mL) was employed to dissolve Zn(NO₃)₂ (0.1 mmol, 30.0 mg). 21.0 μL (20.0 mM) of Zn²⁺ solution was added to the DHIC solution (3.0 mL, 10.0 μM). Bis–Tris buffer (5.0 mL) was employed to dissolve Na₂S·9H₂O (5 × 10⁻¹ mmol, 130 mg). 0–11.4 μL (0.1 M) of S²⁻ was added to the complex solution. Fluorescence measurements were executed after mixing them. For UV-vis titration, 0–8.1 μL (0.1 M) of S²⁻ was added to the complex solution. UV-vis measurements were executed after mixing them.

Job plot of DHIC–Zn²⁺ with S²⁻. DMF (1.0 mL) was employed to dissolve DHIC (6.0 mg, 2 × 10⁻² mmol) and Zn(NO₃)₂ (6.1 mg, 2 × 10⁻² mmol), respectively. Two solutions were admixed to prepare the DHIC–Zn²⁺ complex. 0–30.0 μL of DHIC–Zn²⁺ were transferred to fluorescence cells. They were diluted with Bis–Tris buffer for preparing a total volume of 2.985 mL. Bis–Tris buffer (1.0 mL) was employed to dissolve Na₂S·9H₂O (4.8 mg, 2 × 10⁻² mmol). 0–15.0 μL of S²⁻ was added to diluted DHIC–Zn²⁺. The fluorescence cell contained a total volume of 3 mL. Fluorescence measurements were executed after mixing them.

Competition test. DMF (1.0 mL) was employed to dissolve DHIC (0.9 mg, 3 × 10⁻³ mmol). 10 μL (3.0 mM) of DHIC was diluted with 2.990 mL of Bis–Tris buffer to 10 μM. Bis–Tris buffer (5.0 mL) was employed to dissolve Zn(NO₃)₂ (0.1 mmol, 30.0 mg). 21.0 μL (20.0 mM) of Zn²⁺ was added to DHIC solution (3.0 mL, 10.0 μM). 0.5 mmol of Na₂S·9H₂O, TBAX (tetrabutylammonium salts) (X = N₃⁻, H₂PO₄⁻, OAc⁻, BzO⁻, SCN⁻ and CN⁻), NaNO₂, and TEAX (tetraethylammonium salts) (X = I⁻, F⁻, Br⁻ and Cl⁻) was dissolved in Bis–Tris buffer (5.0 mL). 10.2 μL (0.1 M) of each anion solution was added to 3.0 mL of each DHIC–Zn²⁺ (10.0 μM) to produce 34 equiv. 10.2 μL (0.1 M) of Na₂S was added to the mixture of DHIC–Zn²⁺ and each anion solution to produce 34 equiv. Fluorescence measurements were executed after mixing them.

pH effect on the DHIC–Zn²⁺ complex with S²⁻. By mixing HCl with Bis–Tris buffer and NaOH solution, buffers having different pH values (2–12) were made. With the desired pH solutions, DHIC (0.9 mg, 3 × 10⁻³ mmol) was dissolved in DMF (1.0 mL), and 10.0 μL (3.0 mM) of the DHIC was diluted with 2.99 mL Bis–Tris buffer to give 10 μM. Bis–Tris buffer (5.0 mL) was employed to dissolve Zn(NO₃)₂ (0.1 mmol, 30.0 mg). 21.0 μL (20.0 mM) of Zn²⁺ was added to DHIC (10.0 μM, 3.0 mL). Bis–Tris buffer (5.0 mL) was employed to dissolve Na₂S·9H₂O (0.5 mmol, 130 mg). 10.2 μL (0.1 M) of S²⁻ solution was transferred to each DHIC–Zn²⁺ (10.0 μM). Fluorescence measurements were executed after mixing them.

Imaging in living cells. HeLa cells were maintained in media having 0.1 g mL⁻¹ streptomycin, fetal bovine serum (10%), 10² U mL⁻¹ penicillin, and Dulbecco's modified Eagle's medium. The cells were bred under a humidified atmosphere containing 5% CO₂ at 37 °C. Cells were bred on a 12 well plate at a density of 10⁴ cells per 0.1 mL and then incubated at 37 °C for 19 h. Cells were treated with DHIC (2 × 10⁻² M) for 1 h before an additional incubation with Zn(NO₃)₂ (pH 7.43, 0.15 M NaCl; 600 μM) for 1 h. For the fluorescence quenching experiment, cells were treated with DHIC (20 μM) and Zn(NO₃)₂ (pH 7.43, 0.15 M NaCl; 600 μM) for 1 h each. Na₂S (pH 7.43, 0.15 M NaCl; 400 μM) was treated with the cells for 1 h. An EVOS FL fluorescence microscope was applied for imaging (λ_ex = 470 nm and λ_em = 510 nm).

MTT assay. HeLa cells grew and were maintained at 37 °C under a humidified atmosphere with 5% CO₂. Cell viability upon treatment with DHIC was determined by the MTT assay. Cells were seeded in a 96 well plate (2000 cells for 1 h incubation and 2000 cells for 24 h incubation in 100 μL per well). The cells were treated with DHIC at two concentrations (10 and 20 μM; 1% v/v final DMSO concentration). After 1 h or 24 h incubation, MTT [25 μL of 5 mg mL⁻¹ in PBS (pH 7.4, GIBCO)] was added to each well and the plate was incubated for 4 h at 37 °C. Formazan produced by cells was solubilized using an acidic solution of dimethylformamide (DMF; pH 4.5, 50% v/v, aq.) and sodium dodecyl sulfate (SDS; 20% w/v) overnight at room temperature in the dark. The absorbance was measured at 600 nm by using a microplate reader. Cell viability was calculated relative to cells containing an equivalent amount of DMSO. The measurements were conducted in triplicate.

Colorimetric detection

UV-vis titrations of DHIC with Fe^3+/2+. DMF (1.0 mL) was employed to dissolve DHIC (0.9 mg, 3 × 10⁻³ mmol). 20 μL of DHIC (3 mM) was diluted with 2.98 mL Bis–Tris buffer (10 mM, pH 7.0) to 20 μM. Bis–Tris buffer (5.0 mL) was employed to dissolve Fe(NO₃)₃ (or Fe(ClO₄)₂) (0.1 mmol). 0–3.3 μL (2 × 10⁻² M) of the Fe³⁺ solution (or 0–4.2 μL of the Fe²⁺) were transferred to DHIC (20.0 μM, 3.0 mL). UV-vis measurements were executed after mixing them.

Job plot measurements. DMF (1.0 mL) was employed to dissolve DHIC (3.01 mg, 10⁻² mmol). 6.0, 5.4, 4.8, 4.2, 3.6, 3.0, 2.4, 1.8, 1.2, 0.6 and 0 μL of DHIC were transferred to UV-visible cells. They were diluted with Bis–Tris buffer to produce a total volume of 2.994 mL. Bis–Tris buffer (1.0 mL) was employed to dissolve Fe(NO₃)₃ (or Fe(ClO₄)₂) (0.01 mmol). 0, 0.6, 1.2, 1.8, 2.4, 3.0, 3.6, 4.2, 4.8, 5.4 and 6.0 μL of iron were added to dilute DHIC. The UV-visible cell had a total volume of 3 mL. UV-vis measurements were executed after mixing them.

Competition test. DMF (1.0 mL) was employed to dissolve DHIC (0.9 mg, 3 × 10⁻³ mmol). 20.0 μL (3.0 mM) of DHIC was diluted with 2.98 mL of buffer to 20 μM. Bis–Tris buffer (5.0 mL) was employed to dissolve 0.1 mmol of KNO₃ or M(NO₃)₂ (M = Zn, Cd, Pb, Mg, Ca, Ni, Co, Mn and Cu) or Fe(ClO₄)₂ or NaNO₃ or M(NO₃)₃ (M = Cr, Fe and Al), respectively. 3.9 μL (2 × 10⁻² M) of each cation was added to 3.0 mL of DHIC (10 μM) to produce 1.3 equiv. 3.0 μL (2 × 10⁻² M) of Fe(NO₃)₃ (or 3.9 μL of the Fe(ClO₄)₂) was added to the mixture of DHIC and each cation solution to produce 1.3 equiv. (or 1.0 equiv.). UV-vis measurements were executed after mixing them.

pH effect. By mixing HCl with Bis–Tris buffer and NaOH solution, buffers having different pH values (2–12) were made. With the desired pH solutions, DHIC (0.9 mg, 3 × 10⁻³ mmol) was dissolved in DMF (1.0 mL), and 20.0 μL (3.0 mM) of DHIC was diluted with 2.98 mL Bis–Tris buffer to 20 μM. Bis–Tris buffer (5.0 mL) was employed to dissolve Fe(NO₃)₃ (or Fe(ClO₄)₂) (0.01 mmol). 3.0 μL (2 × 10⁻² M) of Fe³⁺ solution or 3.9 μL of Fe²⁺ was transferred to each DHIC solution (20 μM). UV-vis measurements were executed after mixing them.

3. Results and discussion

The multiple target compound DHIC was produced by reaction of 5(4)-amino-4(5)-(aminocarbonyl)imidazole hydrochloride and 4-diethylaminosalicylaldehyde in MeOH (Scheme 1), and analyzed by ¹H NMR, ¹³C NMR, and ESI-mass spectrometry.

Scheme 1 Synthesis procedure of DHIC.

Fluorescence sensing of DHIC for Zn²⁺

The fluorescence sensing capability of DHIC was checked with different metal ions (Mn²⁺, Cd²⁺, Ni²⁺, Fe³⁺, Pb²⁺, Cu²⁺, Fe²⁺, Co²⁺, Cr³⁺, Ca²⁺, Mg²⁺, Na⁺, Zn²⁺, Al³⁺ and K⁺) in Bis–Tris buffer (Fig. 1). The sensor DHIC showed a little fluorescence peak at 484 nm when excited at 420 nm. On the addition of 21 equiv. of the cations to DHIC, only zinc showed immediately a considerable fluorescence intensity change. Quantum yields (Φ) of 0.0041 and 0.0411 were calculated for DHIC and the DHIC–Zn²⁺ complex. These outcomes implied that the compound DHIC could operate as a “turn-on” fluorescent probe for Zn²⁺ and also definitely differentiate Zn²⁺ and Cd²⁺.

Fig. 1 (a) Fluorescence selectivity of DHIC (10.0 μM) with different cations (21 equiv.) with excitation at 420 nm. (b) Bar graph displaying the relative emission of DHIC at 484 nm upon treatment with different cations.

Fluorescence titration was executed to examine the spectroscopic properties of DHIC for Zn²⁺ (Fig. 2). With the addition of Zn²⁺, fluorescence emission at 484 nm continuously increased up to 21 equiv. of Zn²⁺. The binding properties were also inspected by UV-vis titration experiment (Fig. S1†). The absorbance at 410 nm decreased, and the bands at 270 and 460 nm increased with an isosbestic point (345 nm), which means the production of a species upon treatment of DHIC with Zn²⁺.

Fig. 2 Fluorescence titration of DHIC (10 μM) with different Zn²⁺ concentrations.

The 1 : 1 interaction ratio between DHIC and Zn²⁺ was obtained by Job plot analysis (Fig. S2†), which was affirmed by ESI mass spectrometry (Fig. S3†). A peak at m/z = 364.10 was indicative of [DHIC − H⁺ + Zn²⁺] [calc., 364.07]. The results of fluorescence titration of DHIC–Zn²⁺ species gave a binding constant of 1.7 × 10⁴ M⁻¹ by Benesi–Hildebrand analysis (Fig. S4†).⁶¹ The detection limit (3σ/K) of DHIC for Zn²⁺ was analyzed to be 1.59 μM (Fig. S5†). It was far below the WHO standard (76.0 μM).⁶²

The fluorescence variation of DHIC for Zn²⁺ was investigated to check the interference of other cations (Fig. S6†). Fe³⁺, Cu²⁺, Cr³⁺, Fe²⁺ and Co²⁺ caused fluorescence quenching (78%–100%), but other metal ions did not interfere. Their interference may be due to their inherent quenching character of fluorescence.^63,64 Meanwhile, Cd²⁺ did not inhibit the fluorescence emission of DHIC–Zn²⁺. These outcomes indicated that the compound DHIC could be a selective and effective sensor for Zn²⁺ and differentiate Zn²⁺ from Cd²⁺ having analogous properties. The pH effect on the sensing ability of DHIC for Zn²⁺ was examined in the wide pH range of 2–12 (Fig. S7†). The fluorescence intensity of DHIC with Zn²⁺ was kept between pH 7 and 11.

To comprehend the detection by DHIC of Zn²⁺, ¹H NMR titration was executed (Fig. 3). On the addition of Zn²⁺ to DHIC, the hydroxyl proton H₅ disappeared. The imidazole proton H₁ was shifted downfield, while the imine proton H₄ was a little shifted to upfield. These outcomes implied that the imine nitrogen atom and the hydroxyl oxygen atom may bind to Zn²⁺. Further addition (>1.0 equiv.) of Zn²⁺ showed no change of the proton positions, which suggested the ratio 1 : 1 of DHIC–Zn²⁺. Based on ESI-mass spectrometry, ¹H NMR titration and Job plot analysis, a possible structure of the zinc complex was suggested (Scheme 2).

Fig. 3 ¹H NMR titration of DHIC with Zn²⁺ in DMSO-d₆.

Scheme 2 Proposed structure of the DHIC–Zn²⁺ complex.

To study the reversibility of DHIC toward Zn²⁺, ethylenediaminetetraacetic acid (EDTA, 21 equiv.) was added to DHIC–Zn²⁺ (Fig. S8†). Addition of EDTA decreased the fluorescence intensity of DHIC–Zn²⁺, which is indicative of regeneration of free DHIC. On the addition of Zn²⁺ again, fluorescence emission returned to the original intensity of DHIC–Zn²⁺ species. These results showed that DHIC can be recycled with appropriate reagents like EDTA. The practical application of DHIC for sensing Zn²⁺ in real water samples was investigated by using a calibration curve. The curve showed a satisfying linearity between Zn²⁺ concentration and the fluorescence emission of DHIC (Fig. S9†). On the basis of the curve, DHIC was applied to the water samples (Table 1). Good recoveries and R.S.D. values were achieved for the samples.

Table 1 Determination of Zn(II) in water samples^a

Sample	Zn(II) added (μmol L^−1)	Zn(II) found (μmol L^−1)	Recovery (%)	R.S.D. (n = 3) (%)
a Conditions: [DHIC] = 10 μmol L^−1 in 10 mM Bis–Tris buffer–DMF solution (997 : 3, pH 7.0).
Drinking water	0.00	0.00	—	—
	5.00	4.96	99.2	3.53

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Fluorescence response of DHIC–Zn²⁺ to S²⁻

Based on the high affinity of Zn and sulfide,⁶⁴ a fluorescence spectral study of the DHIC–Zn²⁺ complex for a wide variety of anions (OAc⁻, I⁻, CN⁻, F⁻, S²⁻, SCN⁻, H₂PO₄⁻, Br⁻, B₂O⁻, N₃⁻, NO₂⁻ and Cl⁻) was performed in Bis–Tris buffer. With excitation at 420 nm, only S²⁻ reduced immediately the fluorescence intensity (Fig. 4). These observations indicated that DHIC–Zn²⁺ could be a great selective probe for S²⁻. Just in case, other sulfur-containing species, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), were also tested, but they did not show any change.

Fig. 4 Fluorescence selectivity of DHIC–Zn²⁺ (10.0 μM) with different anions (34 equiv.) with excitation at 420 nm.

We performed fluorescence titration of DHIC–Zn²⁺ with S²⁻ to study the sensing properties of DHIC–Zn²⁺ species (Fig. 5). On the incremental addition of S²⁻ to DHIC–Zn²⁺, the fluorescence emission was decreased constantly and saturated with 34 equiv. of S²⁻. To examine the binding properties of DHIC–Zn²⁺ with S²⁻, UV-vis titration was performed (Fig. S10†). On the addition of S²⁻ to DHIC–Zn²⁺, the absorbance at 300 and 500 nm increased, while the band at 450 nm declined with a distinct isosbestic point (472 nm).

Fig. 5 Fluorescence changes of DHIC–Zn²⁺ (10.0 μM) with different S²⁻ concentrations.

To inspect the binding interaction between S²⁻ and DHIC–Zn²⁺, a Job plot analysis was performed (Fig. S11†). It gave a 1 : 1 interaction ratio. In addition, their 1 : 1 ratio was affirmed by ESI-mass spectrometry. The positive-ion spectrum suggested that the peak at m/z = 302.10 was indicative of [DHIC + H⁺] (calc., 302.16) (Fig. S12†). The results assumed that the demetallation of the DHIC–Zn²⁺ complex occurred to form ZnS. With fluorescence titration, the association constant of 2.0 × 10³ M⁻¹ was afforded for DHIC–Zn²⁺ with sulfide by the Benesi–Hildebrand equation (Fig. S13†).⁶¹ The detection limit (3σ/K) was evaluated to be 8.03 μM, which is below the WHO standard (14.7 μM) (Fig. S14†).⁶⁵

To study the preferential selectivity for Zn²⁺, a competition test was carried out (Fig. 6). With various anions (34 equiv.; SCN⁻, I⁻, OAc⁻, F⁻, N₃⁻, Br⁻, H₂PO₄⁻, CN⁻, Cl⁻, B₂O⁻ and NO₂⁻), no interference was observed for detecting S²⁻. These outcomes demonstrated that the DHIC–Zn²⁺ complex could be an excellent fluorescence probe for detecting S²⁻ without the interference of other anions. To investigate the influence of pH, the fluorescence response to S²⁻ was measured at a broad range of pH (2–12; Fig. S15†). Upon the addition of S²⁻ to DHIC–Zn²⁺, the fluorescence emission showed a momentous decrease between pH 7 and 11, implying that DHIC–Zn²⁺ species could be used to detect S²⁻ in a biological range of pH.

Fig. 6 Competitive test of DHIC–Zn²⁺ (10.0 μM) to S²⁻ (34 equiv.) with other anions (34 equiv.) with excitation at 420 nm.

Cell imaging tests of DHIC with Zn²⁺ and S²⁻

For the applicability of DHIC in biological systems, its cytotoxicity was examined with HeLa cells by the MTT assay (Fig. S16†). HeLa cells were incubated with DHIC (10 and 20 μM) for 1 h and 24 h, respectively. After the incubation, cell viability turned out to be ca. 95% (1 h incubation) and 90% (24 h incubation) at both concentrations, indicating that DHIC was nontoxic under our experimental conditions. To evaluate the practical application of detecting Zn²⁺ and S²⁻ in living cells, fluorescence imaging was performed (Fig. 7). HeLa cells were bred with DHIC for 1 h and exposed to Zn²⁺ for another 1 h. Fluorescence in the cells did not appear without Zn²⁺. Contrastively, the presence of Zn²⁺ showed the increased fluorescence (Fig. 7(a)). Furthermore, the fluorescence of cells having DHIC–Zn²⁺ species exposed to S²⁻ for 1 h disappeared absolutely (Fig. 7(b)). These observations suggested that the compound DHIC could recognize consecutively Zn²⁺ and S²⁻ in living cells.

Fig. 7 (a) Fluorescence imaging of HeLa cells incubated with DHIC followed by the addition of Zn²⁺. Cells were preincubated with DHIC for 1 h prior to treatment with Zn²⁺. Conditions: [DHIC] = 20 μM; [Zn²⁺] = 600 μM; 37 °C; 5% CO₂. The scale bar is 50 μm. (b) Fluorescence imaging of HeLa cells incubated with DHIC and Zn²⁺ followed by the addition of S²⁻. Fluorescence of the cells treated with both DHIC and Zn²⁺ was quenched by introducing S²⁻. Conditions: [DHIC] = 20 μM; [Zn²⁺] = 600 μM; [S²⁻] = 400 μM; 37 °C; 5% CO₂. The scale bar is 50 μm.

Colorimetric sensing of DHIC for Fe^3+/2+

The colorimetric recognizing behavior of the compound DHIC with a wide range of metal ions (Pb²⁺, Mn²⁺, Fe²⁺, K⁺, Cu²⁺, Co²⁺, Mg²⁺, Cd²⁺, Cr³⁺, Na⁺, Ni²⁺, Zn²⁺, Ca²⁺, Al³⁺ and Fe³⁺) was also examined in Bis–Tris buffer (Fig. 8). DHIC showed immediate changes of color from pale yellow to orange yellow with an increase of the absorbance at 500 nm in the presence f Fe^3+/2+ ions, while no change was shown with other cations. This result illustrated that DHIC could be an efficient colorimetric detector for Fe^3+/2+ in aqueous media. Importantly, among the chemosensors reported to date, the compound DHIC is the first chemosensor that can recognize the four analytes, Zn²⁺, S²⁻, and Fe^3+/2+.

Fig. 8 (a) UV-vis changes of DHIC (20.0 μM) upon the addition of various cations. (b) The color changes of DHIC (20.0 μM) with different cations (22 equiv.).

UV-vis titrations of DHIC for Fe³⁺ and Fe²⁺ were performed to investigate their binding characteristics. Upon the addition of Fe³⁺ ions (Fig. 9), the UV-vis absorption spectrum showed that the absorption at 500 nm constantly increased and the absorption at 420 nm declined with an isosbestic point of 446 nm. The point demonstrated that a species was produced from the complexation of DHIC with Fe³⁺. UV-vis variation of DHIC upon the addition of Fe²⁺ was analogous to that of DHIC with Fe³⁺ (Fig. S17†).

Fig. 9 UV-vis absorption changes of DHIC (20.0 μM) with different Fe³⁺ concentrations (from 0 to 1.1 equiv.). Inset: Absorbance at 500 nm versus the number of equiv. of Fe³⁺ added.

Job plot analysis was performed to study the interaction modes between DHIC and Fe³⁺ and Fe²⁺. As shown in Fig. S18 and S19,† they showed 2 : 1 stoichiometries. Also, the interaction modes between DHIC and Fe^3+/2+ were affirmed with ESI mass spectrometry (Fig. S20†). Positive-ion mass data of DHIC upon the addition of Fe³⁺ and Fe²⁺ were indicative of the formation of 2·DHIC + Fe³⁺ − 2H⁺ [m/z: 656.20; calc.: 656.23]. These outcomes drove us to assume that Fe²⁺ of DHIC–Fe²⁺ produced from the interaction of DHIC with Fe²⁺ may be quickly oxidized to Fe³⁺ in air (Scheme 3). To demonstrate our suggestion, we investigated the UV-vis variations for the complexation of DHIC with Fe²⁺ under anaerobic and aerobic conditions (Fig. S21†). When DHIC and Fe²⁺ interacted in the absence of oxygen molecules, there was little spectral change of DHIC. However, the spectrum of the DHIC–Fe²⁺ complex exposed to air was significantly varied, and finally, analogous to the spectrum of the Fe³⁺-2·DHIC complex. Additionally, the time-dependent variations for the interactions of DHIC with Fe^3+/2+ displayed that the formation period (30 min) of the Fe³⁺-2·DHIC complex produced from the interaction of DHIC with Fe²⁺ was longer than that (12 min) afforded by the interaction of DHIC with Fe³⁺ (Fig. S22†). These observations indirectly proved our proposal. Based on the Job plots, time-dependence experiments, ESI-mass data, and degassing experiments, the possible structure of the iron complex is suggested in Scheme 3. On the other hand, the superoxide radical generated from the oxidation reaction of the DHIC–Fe²⁺ complex with air (O₂) might react with another Fe²⁺ complex to produce the Fe³⁺-2·DHIC complex.

Scheme 3 Proposed structure of the Fe³⁺-2·DHIC complex.

With the UV-vis titrations, the K values of DHIC with Fe^3+/2+ were calculated to be 5.0 × 10¹⁰ M⁻¹ and 1.0 × 10¹⁰ M⁻¹ by using Li's equation (Fig. S23 and S24†).⁶⁶ The detection limit (3σ/K) of DHIC for Fe³⁺ (or Fe²⁺) was calculated to be 0.73 μM (or 1.11 μM) (Fig. S25 and S26†). The detection limit of DHIC for iron was far below the EPA standard (5.37 μM).⁴²

To assess the influence of other cations on the Fe^3+/2+-2·DHIC, competitive tests were performed (Fig. 10 and S27†). There are no interferences with the detection of Fe^3+/2+, except for Cu²⁺ ions. Cu²⁺ inhibited ca. 50% of the absorption peak. These facts indicated that DHIC could be a valuable chemosensor capable of detecting Fe^3+/2+ without the interference of other cations. To investigate the influence of pH, pH tests of Fe^3+/2+ detection by DHIC were performed in a broad range of pH (2–12). As shown in Fig. S28 and S29,† the absorption of DHIC at 500 nm with Fe³⁺ increased between pH 4 and 10. For Fe²⁺, the absorption of DHIC at 500 nm also increased between pH 7 and 10. These outcomes indicated that DHIC could recognize iron ions in a broad pH range.

Fig. 10 UV-vis competitive test of DHIC (20.0 μM) toward Fe³⁺ with other cations.

To investigate the efficient application of DHIC to Fe³⁺ in real samples, a calibration plot was established for the determination of Fe³⁺. The plot displayed a satisfying linearity between Fe³⁺ concentration and the absorbance of DHIC (Fig. S30†). Based on the plots, DHIC was applied to water samples (Table 2). Suitable recoveries and R.S.D. values are given for the samples. These outcomes implied that DHIC may be efficient at detecting Fe³⁺ in real water samples.

Table 2 Determination of Fe(III) in water samples^a

Sample	Fe(III) added (μmol L^−1)	Fe(III) found (μmol L^−1)	Recovery (%)	R.S.D. (n = 3) (%)
a Conditions: [DHIC] = 20 μmol L^−1 in 10 mM Bis–Tris buffer–DMF solution (997 : 3, pH 7.0).
Drinking water	0.00	0.00	—	—
	5.00	5.03	100.6	3.53

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4. Conclusion

We presented a novel fluorescent and colorimetric probe, DHIC, which showed great sensitivity and selectivity to Zn²⁺, S²⁻, and Fe^3+/2+. DHIC could operate as a “turn on–off” fluorescent probe for the consecutive recognition of Zn²⁺ and S²⁻. The detection limit (1.59 μM) for Zn²⁺ ions was far below the WHO standard (76.0 μM). DHIC could be applied for drinking water samples for Zn²⁺ with satisfying R.S.D. values and recoveries. Significantly, DHIC can recognize consecutively Zn²⁺ and S²⁻ in living cells. In addition, DHIC could selectively detect Fe^3+/2+ by the naked-eye from pale yellow to orange yellow with very low detection limits (0.73 μM and 1.11 μM), which are far lower than the EPA standard (5.37 μM). The analytical methods such as the degassing test and ESI-mass spectrometry have illustrated that Fe²⁺ of the Fe²⁺-2·DHIC species was quickly converted to Fe³⁺ in air. Moreover, DHIC could quantify Fe³⁺ in real samples. Significantly, among the chemosensors reported to date, DHIC is the first chemosensor that can recognize the four analytes, Zn²⁺, S²⁻, and Fe^3+/2+. Thus, we believe that the compound DHIC will contribute to the design of a new kind of fluorescent and colorimetric probe for multiple targets in biological and environmental systems.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was kindly supported by the National Research Foundation of Korea (Grant No. NRF-2018R1A2B6001686 and NRF-2017R1A2B3002585).

References

1. J. Zhu, Y. Zhang, Y. Chen, T. Sun, Y. Tang, Y. Huang, Q. Yang, D. Ma, Y. Wang and M. Wang, A Schiff base fluorescence probe for highly selective turn-on recognition Zn²⁺, Tetrahedron Lett., 2017, 58, 365–370.
2. S. Yin, J. Zhang, H. Feng, Z. Zhao, L. Xu, H. Qiu and B. Tang, Zn²⁺-selective fluorescent turn-on chemosensor based on terpyridine-substituted siloles, Dyes Pigm., 2012, 95, 174–179.
3. D. Maity and T. Govindaraju, A differentially selective sensor with fluorescence turn-on response to Zn²⁺ and dual-mode ratiometric response to Al³⁺ in aqueous media, Chem. Commun., 2012, 48, 1039–1041.
4. A. Helal, M. Harun, O. Rashid, C. Choi and H. Kim, New regioisomeric naphthol-substituted thiazole based ratiometric fluorescence sensor for Zn²⁺ with a remarkable red shift in emission spectra, Tetrahedron, 2012, 68, 647–653.
5. J. M. Jung, S. Y. Lee, E. Nam, M. H. Lim and C. Kim, A highly selective turn-on chemosensor for Zn²⁺ in aqueous media and living cells, Sens. Actuators, B, 2017, 244, 1045–1053.
6. D. Jeyanthi, M. Iniya, K. Krishnaveni and D. Chellappa, Charge transfer based “turn-on” chemosensor for Zn²⁺ ion recognition using new triaryl pyrazoline derivative, Spectrochim. Acta, Part A, 2016, 159, 231–237.
7. K. T. Kim, S. A. Yoon, J. Ahn, Y. Choi, M. H. Lee, J. H. Jung and J. Park, Chemical Synthesis of fluorescent naphthalimide-functionalized Fe₃O₄ nanoparticles and their application for the selective detection of Zn²⁺ present in contaminated soil, Sens. Actuators, B, 2017, 243, 1034–1041.
8. X. Chen, S. Nam, G.-H. Kim, N. Song, Y. Jeong, I. Shin, S. K. Kim, J. Kim, S. Park and J. Yoon, A near-infrared fluorescent sensor for detection of cyanide in aqueous solution and its application for bioimaging, Chem. Commun., 2010, 46, 8953–8955.
9. N. Narayanaswamy, D. Maity and T. Govindaraju, Reversible fluorescence sensing of Zn²⁺ based on pyridine-constrained bis(triazole-linked hydroxyquinoline) sensor, Supramol. Chem., 2011, 23, 703–709.
10. A. Bhattacharyya, S. Ghosh, S. C. Makhal and N. Guchhait, Hydrazine bridged coumarin-pyrimidine conjugate as a highly selective and sensitive Zn²⁺ sensor: Spectroscopic unraveling of sensing mechanism with practical application, Spectrochim. Acta, Part A, 2017, 183, 306–311.
11. K. Wang, Z. Jin, H. Shang, C. Lv, Q. Zhang, S. Chen and Z. Hu, A highly selective fluorescent chemosensor for Zn²⁺ based on the rhodamine derivative incorporating coumarin group, J. Fluoresc., 2017, 27, 629–633.
12. R. Singh, A. Gogoi and G. Das, Benzothiazole based multi-analyte sensor for selective sensing of Zn²⁺ and Cd²⁺ and subsequent sensing of inorganic phosphates (Pi) in mixed aqueous medium, RSC Adv., 2016, 6, 112246–112252.
13. F. Yu, X. Guo, X. Tian and L. Jia, A ratiomeric fluorescent sensor for Zn²⁺ based on N,N′-Di(quinolin-8-yl)oxalamide, J. Fluoresc., 2017, 27, 723–728.
14. S. Goswami, A. Manna, S. Paul, A. K. Maity, P. Saha, C. K. Quah and H. Fun, FRET based ‘red-switch’ for Al³⁺ over ESIPT based ‘green-switch’ for Zn²⁺: dual channel detection with live-cell imaging on a dyad platform, RSC Adv., 2014, 4, 34572–34576.
15. Z. Xu, G. Kim, S. Jung, M. Jung, C. Lee, I. Shin and J. Yoon, An NBD-based colorimetric and fluorescent chemosensor for Zn²⁺ and its use for detection of intracellular zinc ions, Tetrahedron, 2009, 65, 2307–2312.
16. L. Li, S. Yun, Z. Yuan-Hui, M. Lan, Z. Xi, C. Redshaw and W. Gang, A single chemosensor for multiple analytes: Fluorogenic and ratiometric absorbance detection of Zn²⁺, Mg²⁺ and F⁻, and its cell imaging, Sens. Actuators, B, 2016, 226, 279–288.
17. Z. Xu, K. Baek, H. N. Kim, J. Cui, X. Qian, D. R. Spring, I. Shin and J. Yoon, Zn²⁺-Triggered Amide Tautomerization Produces a Highly Zn²⁺-Selective, Cell-Permeable, and Ratiometric Fluorescent Sensor, J. Am. Chem. Soc., 2010, 132, 601–610.
18. A. Helal, S. H. Kim and H. Kim, Thiazole sulfonamide based ratiometric fluorescent chemosensor with a large spectral shift for zinc sensing, Tetrahedron, 2010, 66, 9925–9932.
19. L. He, X. Yang, Y. Liu and W. Lin, Colorimetric and ratiometric fluorescent probe for hydrogen sulfide using a coumarin-pyronine FRET dyad with a large emission shift, Anal. Methods, 2016, 8, 8022–8027.
20. Y. Wang, X. Lv and W. Guo, A reaction-based and highly selective fl uorescent probe for hydrogen sulfide, Dyes Pigm., 2017, 139, 482–486.
21. B. Deng, M. Ren, J. Wang, K. Zhou and W. Lin, A mitochondrial-targeted two-photon fluorescent probe for imaging hydrogen sulfide in the living cells and mouse liver tissues, Sens. Actuators, B, 2017, 248, 50–56.
22. R. Zhang, X. Yu, Y. Yin, Z. Ye, G. Wang and J. Yuan, Development of a heterobimetallic Ru(II)-Cu(II) complex for highly selective and sensitive luminescence sensing of sulfide anions, Anal. Chim. Acta, 2011, 691, 83–88.
23. S. M. Kim, M. Kang, I. Choi, J. J. Lee and C. Kim, A highly selective colorimetric chemosensor for cyanide and sulfide in aqueous solution : experimental and theoretical studies, New J. Chem., 2016, 40, 7768–7778.
24. X. Feng, T. Zhang, J. Liu, J. Miao and B. Zhao, A new ratiometric fluorescent probe for rapid, sensitive and selective detection of endogenous hydrogen sulfide in mitochondria, Chem. Commun., 2016, 52, 3131–3134.
25. P. Wang, J. Wu, P. Su, C. Xu, Y. Ge, D. Liu, W. Liu and Y. Tang, Fluorescence ‘on-off-on’ peptide-based chemosensor for the selective detection of Cu²⁺ and S²⁻ and its application in living cell bioimaging, Dalton Trans., 2016, 45, 16246–16254.
26. S. Chen, H. Li and P. Hou, A novel imidazo[1,5-α]pyridine-based fluorescent probe with a large stokes shift for imaging hydrogen sulfide, Sens. Actuators, B, 2017, 1–7.
27. M. G. Choi, S. Cha, H. Lee, H. L. Jeon and S. K. Chang, Sulfide-selective chemosignaling by a Cu²⁺ complex of dipicolylamine appended fluorescein, Chem. Commun., 2009, 7390–7392.
28. K. Zheng, W. Lin, L. Tan and D. Cheng, A two-photon fluorescent probe with a large turn-on signal for imaging hydrogen sulfide in living tissues, Anal. Chim. Acta, 2015, 853, 548–554.
29. P. Zhang, J. Li, B. Li, J. Xu, F. Zeng, J. Lv and S. Wu, A logic gate-based fluorescent sensor for detecting H₂S and NO in aqueous media and inside live cells, Chem. Commun., 2015, 51, 4414–4416.
30. Y. Jiang, Q. Wu and X. Chang, A ratiometric fluorescent probe for hydrogen sulfide imaging in living cells, Talanta, 2014, 121, 122–126.
31. B. Gu, W. Su, L. Huang, C. Wu, X. Duan, Y. Li, H. Xu, Z. Huang, H. Li and S. Yao, Real-time tracking and selective visualization of exogenous and endogenous hydrogen sulfide by a near-infrared fluorescent probe, Sens. Actuators, B, 2018, 255, 2347–2355.
32. J. Zhang, R. Wang, Z. Zhu, L. Yi and Z. Xi, A FRET-based ratiometric fluorescent probe for visualizing H₂S in lysosomes, Tetrahedron, 2015, 71, 8572–8576.
33. Ş. N. Karuk Elmas, F. Ozen, K. Koran, I. Yilmaz, A. O. Gorgulu and S. Erdemir, Coumarin Based Highly Selective “off-on-off” Type Novel Fluorescent Sensor for Cu²⁺ and S²⁻ in Aqueous Solution, J. Fluoresc., 2017, 27, 463–471.
34. J. Li, C. Yin and F. Huo, Chromogenic and fluorogenic chemosensors for hydrogen sulfide: Review of detection mechanisms since the year 2009, RSC Adv., 2015, 5, 2191–2206.
35. Q. Meng, Y. Shi, C. Wang, H. Jia, X. Gao, R. Zhang, Y. Wang and Z. Zhang, NBD-based fluorescent chemosensor for the selective quantification of copper and sulfide in an aqueous solution and living cells, Org. Biomol. Chem., 2015, 13, 2918–2926.
36. Q. Meng, R. Zhang, H. Jia, X. Gao, C. Wang, Y. Shi, A. V. Everest-Dass and Z. Zhang, A reversible fluorescence chemosensor for sequentially quantitative monitoring copper and sulfide in living cells, Talanta, 2015, 143, 294–301.
37. K. B. Kim, H. Kim, E. J. Song, S. Kim, I. Noh and C. Kim, A cap-type Schiff base acting as a fluorescence sensor for zinc(II) and a colorimetric sensor for iron(II), copper(II), and zinc(II) in aqueous media, Dalton Trans., 2013, 42, 16569–16577.
38. H. Jung, N. Singh and D. Ok, Highly Fe³⁺ selective ratiometric fluorescent probe based on imine-linked benzimidazole, Tetrahedron Lett., 2008, 49, 2960–2964.
39. S. Das, K. Aich, S. Goswami, C. K. Quah and H. Fun, FRET-based fluorescence ratiometric and colorimetric sensor to discriminate Fe³⁺ from Fe²⁺, New J. Chem., 2016, 40, 6414–6420.
40. N. Narayanaswamy and T. Govindaraju, Aldazine-based colorimetric sensors for Cu²⁺ and Fe³⁺, Sens. Actuators, B, 2012, 161, 304–310.
41. H. Jia, X. Gao, Y. Shi, N. Sayyadi, Z. Zhang, Q. Zhao, Q. Meng and R. Zhang, Fluorescence detection of Fe³⁺ ions in aqueous solution and living cells based on a high selectivity and sensitivity chemosensor, Spectrochim. Acta, Part A, 2015, 149, 674–681.
42. US EPA, Secondary drinking water standards: Guidance for Nuisance Chemicals.
43. V. K. Bhardwaj, P. Saluja, G. Hundal, M. S. Hundal, N. Singh and D. O. Jang, Benzthiazole-based multifunctional chemosensor: Fluorescent recognition of Fe³⁺ and chromogenic recognition of HSO⁴⁻, Tetrahedron, 2013, 69, 1606–1610.
44. Z. Chi, X. Ran, L. Shi, J. Lou, Y. Kuang and L. Guo, Molecular characteristics of a fluorescent chemosensor for the recognition of ferric ion based on photoresponsive azobenzene derivative, Spectrochim. Acta, Part A, 2017, 171, 25–30.
45. Y. S. Kim, J. J. Lee, S. Y. Lee, T. G. Jo and C. Kim, A highly sensitive benzimidazole-based chemosensor for the colorimetric detection of Fe(II) and Fe(III) and the fluorometric detection of Zn(II) in aqueous media, RSC Adv., 2016, 6, 61505–61515.
46. S. Goswami, S. Das, K. Aich, D. Sarkar, T. K. Mondal, C. K. Quah and H. K. Fun, CHEF induced highly selective and sensitive turn-on fluorogenic and colorimetric sensor for Fe³⁺, Dalton Trans., 2013, 42, 15113–15119.
47. M. Zhao, X. Zhou, J. Tang, Z. Deng, X. Xu, Z. Chen, X. Li, L. Yang and L. Ma, Pyrene excimer-based fluorescent sensor for detection and removal of Fe³⁺ and Pb²⁺ from aqueous solutions, Spectrochim. Acta, Part A, 2017, 173, 235–240.
48. Y. Liu, R. Shen, J. Ru, X. Yao, Y. Yang, H. Liu, X. Tang, D. Bai, G. Zhang and W. Liu, A reversible rhodamine 6G-based fluorescence turn-on probe for Fe³⁺ in water and its application in living cell imaging, RSC Adv., 2016, 6, 111754–111759.
49. J. Jun Lee, G. Jin Park, Y. Sung Kim, S. Young Lee, H. Ji Lee, I. Noh and C. Kim, A water-soluble carboxylic-functionalized chemosensor for detecting Al³⁺ in aqueous media and living cells: Experimental and theoretical studies, Biosens. Bioelectron., 2015, 69, 226–229.
50. M. Hosseini, Z. Vaezi, M. R. Ganjali, F. Faridbod, S. D. Abkenar, K. Alizadeh and M. Salavati-Niasari, Fluorescence ‘turn-on’ chemosensor for the selective detection of zinc ion based on Schiff-base derivative, Spectrochim. Acta, Part A, 2010, 75, 978–982.
51. M. Hosseini, A. Ghafarloo, M. R. Ganjali, F. Faridbod, P. Norouzi and M. S. Niasari, A turn-on fluorescent sensor for Zn²⁺ based on new Schiff's base derivative in aqueous media, Sens. Actuators, B, 2014, 198, 411–415.
52. Y. W. Choi, G. R. You, M. M. Lee, J. Kim, K. D. Jung and C. Kim, Highly selective recognition of mercury ions through the ‘naked-eye’, Inorg. Chem. Commun., 2014, 46, 43–46.
53. Y. J. Na, Y. W. Choi, J. Y. Yun, K. M. Park, P. S. Chang and C. Kim, Dual-channel detection of Cu²⁺ and F⁻ with a simple Schiff-based colorimetric and fluorescent sensor, Spectrochim. Acta, Part A, 2015, 136, 1649–1657.
54. Y. Wang, H. Q. Wu, J. H. Sun, X. Y. Liu, J. Luo and M. Q. Chen, A novel chemosensor based on rhodamine derivative for colorimetric and fluorometric detection of Cu²⁺ in aqueous solution, J. Fluoresc., 2012, 22, 799–805.
55. S. Sarkar, T. Mondal, S. Roy, R. Saha, A. K. Ghosh and S. S. Panja, A multi-responsive thiosemicarbazone-based probe for detection and discrimination of group 12 metal ions and its application in logic gates, New J. Chem., 2018, 42, 15157–15169.
56. Y. K. La, J. A. Hong, Y. J. Jeong and J. Lee, A 1,8-naphthalimide-based chemosensor for dual-mode sensing: Colorimetric and fluorometric detection of multiple analytes, RSC Adv., 2016, 6, 84098–84105.
57. W. H. Ding, D. Wang, X. J. Zheng, W. J. Ding, J. Q. Zheng, W. H. Mu, W. Cao and L. P. Jin, A turn-on fluorescence chemosensor for Al³⁺, F⁻ and CN⁻ ions, and its application in cell imaging, Sens. Actuators, B, 2015, 209, 359–367.
58. K. Liu, X. Zhao, Q. Liu, J. Huo, Z. Li and X. Wang, A novel multifunctional BODIPY-derived probe for the sequential recognition of Hg²⁺ and I⁻, and the fluorometric detection of Cr³⁺, Sens. Actuators, B, 2017, 239, 883–889.
59. K. Krishnaveni, M. Iniya, D. Jeyanthi, A. Siva and D. Chellappa, A new multifunctional benzimidazole tagged coumarin as ratiometric fluorophore for the detection of Cd²⁺/F⁻ ions and imaging in live cells, Spectrochim. Acta, Part A, 2018, 205, 557–567.
60. M. Iniya, D. Jeyanthi, K. Krishnaveni and D. Chellappa, A bifunctional chromogenic and fluorogenic probe for F⁻ and Al³⁺ based on azo-benzimidazole conjugate, J. Lumin., 2015, 157, 383–389.
61. H. A. Benesi and J. H. Hildebrand, A Spectrophotometric Investigation of the Interaction of Iodine with Aromatic Hydrocarbons, J. Am. Chem. Soc., 1949, 71, 2703–2707.
62. Y. W. Choi, G. R. You, J. J. Lee and C. Kim, Turn-on fluorescent chemosensor for selective detection of Zn²⁺ in an aqueous solution: Experimental and theoretical studies, Inorg. Chem. Commun., 2016, 63, 35–38.
63. Y. Liu, Q. Fei, H. Shan, M. Cui, Q. Liu, G. Feng and Y. Huan, A novel fluorescent ‘off-on-off’ probe for relay recognition of Zn²⁺ and Cu²⁺ derived from N,N-bis(2-pyridylmethyl)amine, Analyst, 2014, 139, 1868–1875.
64. J. Y. Yun, T. G. Jo, J. Han, H. J. Jang, M. H. Lim and C. Kim, A highly sensitive and selective fluorescent chemosensor for the sequential recognition of Zn²⁺ and S²⁻ in living cells and aqueous media, Sens. Actuators, B, 2018, 255, 3108–3116.
65. Guidelines for drinking-water quality. Vol. 2, Health criteria and other supporting information: addendum, World Health Organization, Geneva, 2nd edn, 1998.
66. G. Grynkiewicz, M. Poenie and R. Y. Tsien, A new generation of Ca²⁺ indicators with greatly improved fluorescence properties, J. Biol. Chem., 1985, 260, 3440–3450.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8pp00408k

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